From Hazard to Risk

European Environmental Mutagen Society 33rd Annual Meeting

organised by the UK Environmental Mutagen Society

August 24-28, 2003

Aberdeen Exhibition and Conference Centre,

Aberdeen, Scotland, UK

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LIST OF CONTENTS P1 Scientific Programme P5 Presenters Abstracts P7 Special Lectures P9 Nutrition and Carcinogenesis P17 Germ and Cell Genotoxicity P24 DNA Damage and Repair P28 Genotoxic and Carcinogenic Effects of Mineral Fibres P34 Molecular Epidemiology of Cancer P39 Inducible Responses P42 Industrial Genotoxicity and Regulatory Guidelines P45 Ecogenotoxicology P50 Aneuploidy and Chromosome Stability P55 Closing Lecture P56 Poster Abstracts P138 Exhibition Stands SCIENTIFIC ORGANISING COMMITTEE Andrew Collins (Oslo, Norway) Angelo Carere (Rome, Italy) Ilse-Dore Adler (Neuherberg, Germany) Awadhesh Jha (Plymouth, UK) David Phillips (Sutton, UK) Chris Wild (Leeds, UK) Peter Jenkinson (Derby, UK) Brian Ratcliffe (Aberdeen, UK) Katrina Dunbar (Aberdeen, UK) CONFERENCE SPONSORS AsteaZeneca Bayer CropScience S.r.l. Covance Laboratories Istituto di Ricerche Biomediche Antoine Marxer (RBM) Movartis Pharma AG UKEMS SUSTAINING MEMBERS AstraZeneca CTL GlaxoSmithKline Huntingdon Life Sciences Kinetic Imaging Perceptive Instruments Pfizer Covance Laboratories Safepharm Laboratories Unilever

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Scientific Programme

Sunday 24 August 1500 Registration Opens 1600 Welcome and Introduction - Fleming Auditorium

Andrew Collins, Institute for Nutrition Research, University of Oslo 1625 Fritz Sobels Award Lecture - Fleming Auditorium

James Parry, University of Wales Swansea 1710 Special Lectures - Fleming Auditorium Nina Holland - Biomarkers in children and environmental exposure Miquel Borras – Modelling hazard and risk in the environment 1900 Welcome Reception - Main Entrance Concourse Monday 25 August 0900 Nutrition and Carcinogenesis (UKEMS Symposium) - Fleming Auditorium

Chaired by Brian Ratcliffe and Beatrice Pool-Zobel 0900 John Mathers - The biological revolution – towards a mechanistic understanding of the impact of diet on cancer risk 0930 Ian Johnson – New Approaches to the role of diet in the prevention of cancers of the alimentary tract 1000 Steffen Loft – Interventions with antioxidant and nutrients in relation to DNA damage and repair 1030 Rune Blomhoff – Nutrition and gene expression: In vivo imaging of oxidative-stress related genes 1100 Coffee Break – Crombie Suite & Gordon Suite, Aberdeen Exhibition and Conference Centre 1130 Parallel Sessions: Nutrition and Carcinogenesis (cont.) - Fleming Auditorium Chaired by Brian Ratcliffe and Beatrice Pool-Zobel 1130 Ian Rowland – Diet-gut microflora interactions in colon cancer 1200 Beatrice Pool-Zobel – In vitro/in vivo strategy to assess functional/antigenotoxic activities of plant foods 1230 Iris Benzie – Buccal cell model of comet assay for biomonitoring and nutritional studies Germ Cell Genotoxicity – Forbes Suite

Chaired by Martin Brinkworth and Ilse-Dore Adler 1130 Martin Brinkworth – Mechanisms of mutation induction in the male germ-line 1200 Ursula Eichenlaub-Ritter – In vitro approaches to the study of aneuploidy 1230 Francesca Pacchierotti – In vivo approaches to the study of aneuploidy in oocytes 1300 Lunch 1400 Parallel Sessions:

Nutrition and Carcinogenesis (cont.) - Fleming Auditorium 1400 Bernhard J. Majer – Use of human hepatoma cells for the detection of dietary mutagens and antimutagens 1430 Andrew Collins - Plant-derived foods and the modulation of oxidative stress 1445 Katarina Volkovova - Can antioxidant supplementation decrease the risk of cancer? 1500 Cyrille Krul – Intestinal health: evaluation of genotoxicity in cultured enterocytes induced by human intestinal

microbiota 1515 Wanda Baer-Dubowska – Modulation of cytochrome P450 1A and 2B activities by oral administration of cabbage and

sauerkraut juice in rat liver and kidney 1530 Lynne Ferguson – Is it possible to protect New Zealanders from colon cancer?

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Germ Cell Genotoxicity (cont.) – Forbes Suite 1400 Dr Yuri Dubrova – Transgenerational germline instability 1430 Maria Sierra – Female germ cell mutagenicity of model chemicals in Drosophila melanogaster: mechanistic

information and analysis of repair systems 1445 Diana Anderson – Oestrogenic compounds and oxidative stress (in human sperm and lymphocyte in the Comet

assay) 1500 Adolf Baumgartner – Parallel Comet and SKY evaluation of genetic damage in sperm and lymphocytes after

treatment with doxorubicin 1515 James Taylor – The Influence of Age on Responses to Genotoxins in Mouse Germ Cells 1530 Sabry Attia – Topoisomerase II-inhibitors amsacrine hydrochloride (m-AMSA), etoposide (VP-16) and merbarone

(MER) cause meiotic delay and aneuploidy in spermatocytes of mice 1545 Ilse-Dore Adler – Experimentally determined correction factor for translocations induced by intraperitoneal vs. dermal

exposure of male mice to acrylamide to improve genetic risk estimates 1600 Coffee Break – Crombie Suite and Gordon Suite 1630 Poster Session 1 – Gordon Suite 1830 Civic Reception – Main Entrance Concourse Tuesday 26 August 0900 DNA Damage and Repair (Joint Symposium with DNA Repair Network) - Fleming Auditorium

Chaired by David Phillips and Alan Lehmann 0900 Moon-shong Tang – Sequence selectivity of carcinogen-DNA binding and its role in determining p53 and ras

mutational spectrum in human cancer 0930 Kevin Hiom - BRCA1-A role in ubiquitination and the processing of DNA double-strand

breaks 1000 Jan Hoeijmakers – DNA damage and its impact on cancer and aging: lessons from

mouse repair mutants and human syndromes 1030 Sarah Trewick on behalf of Barbara Sedgewick -Repair of alkylated DNA bases by E. coli AlkB and human

dioxygenases 1100 Coffee Break – Crombie Suite and Gordon Suite 1130 Genotoxic and carcinogenic effects of mineral fibres – Forbes Suite

Chaired by Soterios Kyrtopoulos and Callum Searle 1130 Soterios Kyrtopoulos & Callum Searle - Introduction 1140 Ken Donaldson – Toxicology of asbestos 1210 Panagiotis Georgiadis – Oxidative stress and genotoxicity by mineral fibres: in vitro studies 1235 Jan Topinka – Induction of inflammation, oxidative stress and genotoxicity by mineral fibres in the respiratory system 1300 Lunch – Crombie Suite 1300 UKEMS General Assembly – Forbes Suite 1400 Parallel sessions:

DNA Damage and Repair (cont.) - Fleming Auditorium Chaired by David Phillips and Alan Lehmann 1400 Peter Moller – Oxidative DNA damage in lymphocytes and muscle cells of humans exposed to hypoxia 1430 Bernadette Doherty – Use of the comet-FISH assay to demonstrate DNA repair in selected gene regions 1500 Andreas Rothfuss – Repair kinetics of interstrand DNA crosslinks: Implications for the Fanconi Anemia/BRCA

pathway 1530 Jose Ferreiro – Nucleotide excision repair modulation by two components of the SAGA/ADA chromatin remodeling

complex, Gcn5 and Ada2 proteins, in the MET16 gene of Saccharomyces cerevisiae Genotoxic and carcinogenic effects of mineral fibres (cont.) – Forbes Suite

Chaired by Soterios Kyrtopoulos and Callum Searle 1400 Klaus Unfried – Induction of inflammation, oxidative stress and genotoxicity in mesothelial tissues 1430 Jana Tulinská - Mineral fibres and immunotoxicity 1455 Mária Dušinská – Biomarkers of effects of mineral fibres in exposed humans 1520 Robert Baan - Man-made mineral fibres: recent evaluations of cancer hazards by the International Agency for

Research on Cancer Monographs Programme 1540 Kyriakoula Ziegler-Skylakakis - Research needs for the development of Occupational Exposure Limits for MMMFs’

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1600 Coffee Break – Crombie Suite and Gordon Suite 1630 Poster Session 2 – Gordon Suite 1900 Traditional Scottish Evening – Crombie Suite, Aberdeen Exhibition and Conference Centre

Music by Itchy Fingers Wednesday 27 August 0900 Molecular Epidemiology of Cancer – Fleming Auditorium

(Joint Symposium with Molecular Epidemiology Group) Chaired by Chris Wild

0900 Mike Stratton – Mutational spectra of individual human cancers 0930 Paulo Vineis – Genetic testing: epidemiological and ethical issues 1000 Chris Wild – Molecular epidemiology of oesophageal cancer 1030 John Groopman – Molecular epidemiology of Liver Cancer: Aflatoxin, p53 and Hepatitis Viruses as a Paradigm 1100 Coffee Break - Crombie Suite and Gordon Suite 1130 Parallel Sessions: Molecular Epidemiology of Cancer (cont.) – Fleming Auditorium Chaired by Chris Wild 1130 Gillian Smith – Molecular epidemiology of colorectal cancer 1200 Odilia Popanda – Combinations of genetic variants in DNA repair genes enhance risk for non-small cell lung cancer 1215 Sandrine Roulland – Characterisation of BCL2-IGH translocation 1230 Hannu Norppa – Cytogenetic biomarkers and metabolic polymorphisms in occupational exposure to diisocyanates 1245 Karen Brown – DNA binding of tamoxifen in human colon after administration of a single 14C-labelled therapeutic

dose Inducible Responses – Forbes Suite

Chaired by Anthony Lynch 1130 Dr James Harvey (GSK, UK) on behalf of Dr Ron Newton (Eli Lilly, USA) – The Use of DNA Microarrays to

Characterize Genotoxicity 1150 Harry van Steeg – The effect of altered p53 functions on mutagenesis and carcinogenesis in DNA repair-deficient Xpa

mice 1210 Richard Walmsley – DNA damage induced GFP expression in yeast identifies most direct acting mutagens detected

by the Ames test in addition to clastogens previously identified only in mammalian cell tests 1230 Anthony Lynch – Biophotonics using transgenic promoter-reporter models and their application to predictive

genotoxicity 1300 Lunch – Crombie Suite 1400 Choice of 3 Conference Tours

Buses for all excursions will leave at 1400 from the Aberdeen Exhibition and Conference Centre and drop delegates at conference hotels. Enquiries and Bookings – directly to the tour operator, Pam Wells, by email or website Email: pamwellspj@aol.com, Website: www.pamwells.com Aberdeen to Crathes Castle Cost: £23.00 (including castle entry) Aberdeen to Royal Lochnagar Distillery Cost: £30.00 (including entry and a £3 voucher towards whisky purchase) Aberdeen City Tour by Coach Cost: £10.00

1900 Conference Dinner – Ballroom, Ardoe House Hotel (to the rear of the main hotel) After dinner entertainment by Dr Jazz

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Thursday 28 August 0830 Special Interest Groups: Parallel Sessions Industrial Genotoxicity & Regulatory Guidelines – Fleming Auditorium

(Joint Session with the Industrial Genotoxicology Group) Chaired by David Kirkland and Julie Clements 0830 Daniel Marzin – Validation of an in vitro model to demonstrate the interference of apoptosis

in in vitro aberration assays 0900 Peter Kasper – Current developments in regulatory control of genotoxic impurities in drug substances: An interim

report 0930 Andrew Smith – The new EU technical guidance document for mutagenicity testing and risk assessment of industrial

chemicals and biocides Ecogenotoxicology – Forbes Suite Chaired by Awadhesh N. Jha 0830 Lee Shugart – Issues Pertinent to Research Agenda in Genetic Ecotoxicology 0848 Awadhesh Jha – Relative Sensitivity of two bivalve mollusc species 0906 David Dixon – Stress responses in the haemocytes and gill cells of the deep-sea vent mussel Bathymodiolus

azoricus 0924 Giada Frenzilli – Time course evaluation of DNA damage in mussels 0942 Claudia Bolognesi – The micronucleus test in marine animals, as a biomarker for genotoxic environmental

contamination Aneuploidy and Chromosome Stability – Rooms 18-20 Chaired by James Parry 0830 Tracy Warr – Comparative genomic hybridisation: principles and application to analysis of brain tumours 0848 Ilse Decordier – The frequencies of micronuclei induced by aneugens are dependant on caspase-3 activity 0906 Lucia Migliore – Chromosome malsegregation: a link between Alzheimer disease and Down’s syndrome? 0924 Antonio Antoccia - Alterated inhibition of Diazepam-mediated centrosome splitting leads to mitotic checkpoint

activation and apoptosis-like death in tumour cells‘ 0942 Elizabeth Parry – Analysis of chromosome changes associated with the progression of Barrett’s oesophagus 1000 Coffee Break – Crombie and Gordon Suites 1015 Special Interest Groups: Parallel Sessions (continued) Industrial Genotoxicity & Regulatory Guidelines – Fleming Auditorium 10.15 Lutz Muller – Strategy for genotoxicity testing and classification of genotoxicity test results – report on initial activities

of the IWGT expert group 10.45 Nicola Loprieno – A strategy for the testing of hair dye cosmetic ingredients for their potential

mutagenicity/genotoxicity in the European Union Ecogenotoxicology – Forbes Suite 1015 Paola Venier – Traditional biomarkers and new indexes for detecting the exposure to genotoxins in Mytilus

galloprovincialis 1033 Brett P. Lyons – Biomarkers of carcinogenic exposure in estuarine fish species for the assessment of biological

effects of contaminants 1051 Argelia Castano – Fish cells to evaluate genotoxicity in environmental risk assessment procedures 1109 Siegfried Knasmüller – Use of plant bioassays for the detection of genotoxins in soils and in the aquatic environment Aneuploidy and Chromosome Stability – Rooms 18-20 1015 Diana Anderson – Male – mediated development defects and resulting chromosomal abnormalities 1033 Aleksandra Fucic – Micronucleus induction in mouse peripheral reticulocytes by 5-nitrofurantoin 1051 Francesca Degrassi – Persistence of chromatid cohesion at anaphase results in aberrant chromosome segregation 1109 Elisabetta Borotoletto – Combined effects of modeled microgravity and ionising radiation on human primary

lymphocytes 1130 EEMS General Assembly – Fleming Auditorium 1230 Lunch – Crombie Suite 1330 Closing Lecture – Fleming Auditorium

Stephen West – DNA repair and genome instability: from bacteria to man 1430 Close of Conference

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Special Lectures Biomarkers in children and environmental exposure N. Holland1, M.Bastaki1, K.Birch1, P.Duramad1, C.Furlong2, A.Bradman1, R.Richter2, B.Eskenazi1 1University of California, Berkeley, USA; 2University of Washington, Seattle, USA. A pledge of an international community of scientists “to promote the protection of Children’s Environmental Health” was initially signed in March 2002 in Thailand and is known as “Bangkok statement”. This statement recognized that a growing number of diseases in children have been linked to environmental exposures. It further emphasized that the principle “children are not little adults” requires full acknowledgement and a preventive approach. This pledge has since been reaffirmed through many venues around the world. Children’s exposure to environmental pollution is likely to be greater than that of adults due to differences in metabolism, sensitivity, and outdoor activity. A number of available studies indicate that in some cases children’s environmental exposures may be comparable to occupational exposures of adults. The main risk factors include pesticides, air and water pollution, lead, environmental tobacco smoke, infections, and inadequate diet. While all children are vulnerable to environmental hazards, minority children and children of lower socioeconomic status are particularly at risk because they tend to live in less healthful environments. Molecular epidemiology employs biological markers to assess exposure, early adverse health effects, and differential susceptibility of individuals in exposed populations. The main advantage of biomarker studies is the ability to detect effects long before clinical manifestation of disease, allowing for intervention and prevention. Biological and environmental samples, including maternal blood, cord blood, urine, and dust from homes can be effectively separated into multiple fractions and analysed by various methods to assess biomarkers of exposure and effect. Development of non-invasive methods is particularly important for community-based biomarker studies involving children. Exfoliated epithelial cells from the mouth and bladder can be easily collected and analysed for cytogenetic markers as well as analysis of DNA-polymorphisms. A functional genomics approach can be used to establish whether the activity of specific genes modifies the effects of exposure and developmental health outcomes. These data will help to determine if there are subpopulations that are more susceptible to pesticides or air pollution, and thus will help to inform future policy decisions regarding allowable exposure to children and pregnant women. Using examples from our studies of inner city African-American mothers and children, and a Latina farmworker population, we will describe the advantages of employing biomarkers in children's studies, as well as identify areas requiring additional work and clarification.

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Modelling hazard and risk in the environment M. Borràs1 and J. Nadal2

1 Experimental Toxicology and Ecotoxicology Unit, Barcelona Science Park, Barcelona, Spain; 2 Animal Biology Department, Faculty of Biology, University of Barcelona, Barcelona, Spain. Risk is defined as the probability for a given toxicological hazard of being materialised in actual biological harm. This involves some form of mathematical relationship between exposure and toxic effects. Present regulatory issues are based on the ratio PEC/PNEC, where PEC is the predicted exposure concentration (derived from the analysis of chemicals in the environment) and PNEC represents the predicted no-effect concentration, estimated from laboratory tests. Such simplified models neglect some potentially very important factors acting in real-life situations. Our own approach to the study of atmospheric and edaphic pollution, focused on realism, includes the use of sentinel species (animals as prospectors and integrators of information, both along the spatial and the temporal axis) and selected biomarkers. We aim to: 1.- consider pollution as a complex mixture; 2.- take into account the homeostasis of the environment and of the living organisms; 3.- realism (all data obtained in the field; calculations based on actual effects, rather than on hazard; exposure measured as internal dose). A first proposal for a test battery is:

blocks of information

sentinel species

biomarkers serum biochemistry

systemic effects (specially respiratory in air pollution)

Apodemus sylvaticus

histopathology

Fertility

Apodemus sylvaticus

histopathology (testes, ovary)

reproduction

teratogeny

Xenopus larvae

malformations (adapted FETAX) Micronucleus Test, mice Comet Test, mice

Genotoxicity

Apodemus sylvaticus, Eisenia foetida

Comet Test, earthworms abundance

Populations

arthropods, birds

biodiversity

Each one of these blocks is then represented by the weighed sum of the results of the tests performed within the block. A final value is obtained to represent the integrated toxicological harm (ITH) occurred in a given location. To assess exposure taking into account bioavailability, we propose: a.- in soil contamination studies, analysis of metals in viscera and in fanera. b.- for atmospheric pollution, the gaseous fraction, fully available, is directly taken from immission gases analysis; the solid fraction is assessed through metals in the sentinel organisms. Values of both fractions are then combined. Finally, a regression line is established, confronting exposure vs. ITH in four or five locations with decreasing exposure levels, ranging from the immediate neighbourhood of the pollution focus to controls, following the main dissemination line. In that model we may interpolate new exposure data, to find the corresponding predicted ITH. Such a prediction may be directly interpreted as a form of risk assessment, or, alternatively, these pairs (toxicological harm/exposure) could then be related to a conventional scale of ecotoxicological risk.

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Nutrition and Carcinogenesis (UKEMS Symposium) The biological revolution – towards a mechanistic understanding of the impact of diet on cancer risk John Mathers Human Nutrition Research Centre, School of Clinical Medical Sciences, University of Newcastle, UK There is strong epidemiological evidence to show that differences in diet explain a significant proportion of the variation in cancer incidence worldwide. However, because of the complex nature of eating behaviour and the chemical heterogeneity of foods, it remains very difficult to ascertain which aspects of diet, in what quantities and over what time-frames are responsible for modifying risk. In addition, there are few dietary intervention studies demonstrating reduction in cancer risk. Much faster progress has been made in understanding the biological basis of cancer. It is now clear that damage to the genome resulting in aberrant expression of genes (principally suppression of tumour suppressor genes and inappropriate expression of oncogenes) is fundamental to tumorigenesis. It is also becoming clear that much of the inter-individual variation in cancer experience is due to differences in the amount of damage experienced and/or the capacity to repair that damage. Both of these processes are influenced strongly by dietary factors and by genetic predisposition (polymorphisms in the requisite genes). Indeed we hypothesise that understanding diet:gene interactions in DNA damage and in repair will not only explain much of the inert-individual variation in risk but also offer opportunities to design better dietary intervention studies aimed at chemoprevention. The Human Genome maps and the SNPs databases, together with the rapid development of tools suitable for investigating genetic and epigenetic changes in small tissue biopsies provide the means to begin to test hypotheses about the mechanisms by which diet influences cancer risk directly in human subjects. This is likely to form a significant component of the emerging science of nutrigenomics. New approaches to the role of diet in the prevention of cancers of the alimentary tract I.T. Johnson Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK. Cancers of the alimentary tract are, collectively, amongst the major causes of morbidity and deaths from cancer across the world today. Of the ten million new cases of cancer diagnosed in 2000, about 2.3 million were cancers of the pharynx, oesophagus, stomach or colorectum. In spite of their major contribution to the overall burden of disease, epidemiological studies have repeatedly shown that cancers of the digestive organs are also amongst the most susceptible to modification by dietary factors. Thus around three quarters of sporadic colorectal cancers in industrialised societies are probably caused in some way by diet, and even within the narrower confines of the European Union, there are variations in the incidence of colorectal and oesophageal cancers of about two-fold and five-fold respectively. The problem is that there is no evidence to suggest that diet exerts its effects primarily via food-borne carcinogens, which could in principle be eliminated from the food-chain. It is far more probable that the adverse effects of diet are caused largely by over-consumption of energy coupled with lack of exercise, and the absence of a complex variety of protective factors. Gastrointestinal cancers arise from mucosal epithelial cells through progressive carcinogenic processes that usually require a large fraction of the individual’s lifespan. Colorectal carcinoma develops via the adenoma-carcinoma sequence, in which progressive loss of differentiation and normal morphology in a growing focal lesion is associated with the acquisition of somatic mutations, and of epigenetic changes that lead to gene-silencing via aberrant methylation of CpG-islands. These molecular events are accompanied by functional changes in the expanding clone, including evasion of apoptosis and up-regulation of cyclooxygenase-2 (COX-2). Although there are important differences in both morphology and pathology, carcinomas of the oesophagus, stomach and colon have all been shown to be susceptible to chemoprevention by NSAIDS, and to the protective effects of diets rich in fruits and vegetables. Amongst the latter, brassica vegetables seem to be particularly effective. Plant foods contain a variety of components including polyunsaturated fatty acids, and secondary metabolites such as glucosinolates and flavonoids, which modulate phase 1 and 2 enzymes, inhibit COX-2, and induce apoptosis. The future challenge is to fully characterise and evaluate these effects at the cellular and molecular level, and to maximise their value as protective mechanisms for the population as a whole.

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Interventions with antioxidants and nutrients in relation to DNA damage and repair S Loft and P Møller Inst. of Public Health, Panum, 3 Blegdamsvej, DK-2200 Copenhagen N, Denmark. Cells are constantly exposed to oxidants from metabolic and other biochemical reactions as well as external factors. Failure of the enzymatic antioxidative system, or low levels of endogenous and nutritional antioxidants may lead to mutagenic oxidative DNA damage as well as dysregulation of cell cycle control, resulting in carcinogenesis. Elevated levels of oxidative DNA damage have been reported in many groups of subjects, including smokers, diabetics, HIV patients, and after physical activity. It is suggested that nutritional antioxidants can reduce DNA damage and thus cancer risk. In human cells oxidatively modified nucleobases and strand breaks can be measured in the DNA from cells and tissues. Oxidized bases and nucleosides from DNA repair, the nucleotide pool and cell turnover can be measured in urine, representing the average rate of damage in the body, whereas the level of oxidized bases in DNA reflects the balance between damage and repair at steady state. DNA damage is considered a marker of biologically effective exposure. DNA repair capacity can be assessed from mRNA or protein levels or enzyme activity. The effects of single nutrients as well as various vegetables, fruit and carotenoid and polyphenolic rich products, have been assessed with biomarkers, mainly including damage in white blood cell DNA, urinary excretion of oxidized bases and nucleosides and DNA repair capacity. The basal levels as well as the reported effects of the interventions have been variable, possibly reflecting differences in the populations, regimens, functional correlates of the biomarkers as well as between laboratories and assays. It should be noted that all the biomarkers are subject to substantial seasonal variation. In general, single dose antioxidant interventions have shown protective effects with respect to lymphocyte DNA oxidation. With respect to published studies involving effects of multiple doses of antioxidants or nutrients on oxidative DNA damage in white blood cells, about half turned out with some significant effect on one or more outcomes. It appeared that the positive studies had less robust design in comparison with the studies not showing significant effects. In particular, proper (placebo) control periods or, preferably, parallel (placebo) group designs are crucial in the interpretation. On the other hand, such designs have relatively low statistical power. Also, intervention studies reporting high levels of oxidized bases, possibly due to artifactual oxidation, and apparent preventive effects, may need reevaluation, considering the recent progress to agreement on cellular levels of oxidative DNA damage. It is possible that significant effects, particularly in terms of DNA base oxidation, are more likely to be detected in subjects under oxidative stress, e.g. smokers, diabetics, HIV infected or poorly nourished individuals. Indeed, we have found no effects of depletion or supplementation with large quantities fruits and vegetables, fruit extracts or antioxidants in well-nourished young subjects, whereas vitamin C supplementation in smokers appeared to reduce oxidation of DNA bases. Similar effects have been shown e.g. in HIV patients. Moreover, subjects with particular genotypes of metabolism and other defense enzymes may also be important for the effect of nutrient interventions. In general, intervention involving antioxidant supplements or fruit and vegetable products have not shown effect on 8-oxodG excretion. With respect to DNA repair capacity, oxidized purine incision activity have been reported elevated in lymphocytes from subjects after kiwi fruits intake, whereas in our hands mRNA levels of DNa repair enzymes have been unchanged by various fruits and vegetables as well as pure supplements.

So far, studies of oxidative DNA damage in humans provide support for direct protective effects of antioxidants or nutrients with respect to oxidative DNA damage in short-term and in subjects who are poorly nourished or under oxidative stress. In the future care should be taken with respect to design of such intervention studies and considerations of genotypes of defense enzymes as well as DNA repair capacity. P Møller, S Loft. Oxidative DNA damage in white blood cells of humans in dietary antioxidant supplementation studies. Am J Clin Nutr 76: 303-310, 2002 Møller P, Vogel U, Pedersen A, Dragsted LO, Sandström B, Loft S. No effect of 600 g fruit and vegetables per day on oxidative DNA damage and repair in healthy non-smokers. Cancer Epidemiol Biomarkers Prev, in press Jaruga P, Jaruga B, Gackowski D, Olczak A, Halota W, Pawlowska M, Olinski R. Supplementation with antioxidant vitamins prevents oxidative modification of DNA in lymphocytes of HIV-infected patients. Free Radical Biol Med 32: 414-20, 2002 Collins AR, Harrington V, Drew J, Melvin R. Nutritional modulation of DNA repair in a human intervention study. Carcinogenesis 24: 511-5, 2003 ESCODD, Measurement of DNA oxidation in human cells by chromatographic and enzymic methods. Free Radicals Biol Med 34: 1089-1099, 2003

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Nutrition and gene expression: in vivo imaging of oxidative-stress related genes Rune Blomhoff, Institute for Nutrition Research, University of Oslo, Norway Diet plays a major role in the development of several chronic degenerative disease. Although it has often been assumed that antioxidants in fruits and vegetables protect against oxidative stress-related diseases, results from intervention trials with single compounds such as vitamins E and C or beta-carotene have not been conclusive. We have assessed systematically the content of total antioxidants in a variety of foods used worldwide. Our results demonstrate that there is more than a thousand fold difference between total antioxidants in various dietary plants. Plants that contain most antioxidants include many berries, walnuts, sunflower seed and pomegranates (1) and several herbs (2). To test the effect of various plants in vivo we have produced transgenic mice with NF-kB, AP1 and/or ARE regulated promoters linked to the luciferase gene. By using a VIM camera we use a bioluminescent approaches to study gene expression of the reporter-gene non-invasively in living mice (3). In initial experiments we fed transgenic mice that express luciferase controlled by the GCSh promoter (i.e. the rate limiting enzyme in GSH production) antioxidant-rich extracts of berries, and found significant increased luciferase activity in brain and skeletal muscle (4). The same overall pattern was also found in mice given ellagic acid, a phenolic acid found in many berries. The effects of various antioxidant-rich foods or extracts on expression of oxidative stress-related genes will be presented based on this molecular in vivo imaging technology. 1. Halvorsen et al. (2002). A systematic screening of total antioxidants in dietary plants. Journal of Nutrition. 132(3):461-471. 2. Dragland et al. (2003). Several Culinary and Medicinal Herbs are important sources of dietary antioxidants Journal of Nutrition. 133(5):1286-90. 3. Carlsen et al. (2002). In vivo imaging of NF-kappa B activity. Journal of Immunology 168(3):1441-6. 4. Carlsen et al. (2003). Berry intake increases the activity of the gamma-glutamylcysteine synthetase promoter in transgenic reporter mice. Journal of Nutrition. 133(7):2137-40. Diet-gut microflora interactions in colon cancer Ian Rowland Northern Ireland Centre for Food and Health, School of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA, Northern Ireland. Colorectal cancer is one of the major causes of death from malignant disease in Western Europe, USA, Australia and New Zealand. It constitutes around 14% of new cases of cancer in these countries and the incidence has risen slightly over the last 20 years. There is considerable evidence from laboratory animals and human studies that the colonic microflora is involved in the aetiology of colon cancer. For example, germ-free rats treated with the carcinogen 1,2-dimethylhydrazine have a lower incidence of colon tumours than similarly treated rats having a normal microflora. We have demonstrated potent DNA-damaging activity in faecal extracts from a proportion of healthy human subjects. Intestinal bacteria possess a range of xenobiotic metabolizing enzymes such as nitrate reductase and β-glucuronidase which enable them to produce, from dietary components, substances with genotoxic, carcinogenic and tumour-promoting activity. They can synthesize carcinogens such as N-nitroso compounds, activate carcinogens to reactive DNA-damaging metabolites and deconjugate detoxified carcinogens in the colon releasing the active carcinogen. In addition they can produce tumour promoters such as ammonia and bile acids. It follows from the above that dietary regimes that modify the gut microflora may alter colon cancer risk. Probiotics, such as lactobacilli and bifidobacteria, and non-digestible oligosaccharides (NDO) such as fructo-oligosaccharides, galacto-oligosaccharides, lactulose and inulin, have considerable potential in this regard. Consumption of probiotics or NDO can significantly increase numbers of lactic acid producing bacteria in the gut, and there is evidence that they modulate certain biomarkers of colon cancer risk in humans. Furthermore, in studies in laboratory animals, they have been shown to inhibit DNA damage and suppress pre-cancerous lesions in the colon, and to reduce tumour incidence in both carcinogen-treated rats and transgenic apc-Min mice. Two studies suggest that feeding a combination of probiotic and prebiotic has a more potent inhibitory effect on preneoplastic lesions than either product alone.

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In vitro/in vivo strategy to assess functional/antigenotoxic activities of plant foods Beatrice L. Pool-Zobel1, Gabriele Beyer-Sehlmeyer1, Michael Glei1, Nina Habermann1, Esther Hartmann1, Rosin Hughes2, Christoph Persin3, Volker Böhm1, Ian Rowland2, Rainer Schubert1, Gerhard Jahreis1, 1Institute for Nutrition, Friedrich Schiller University, Dornburger Str. 25, D-07743 Jena, Germany; 2Northern Ireland Centre for Diet and Health (NICHE) School of Biomedical Sciences, University of Ulster, Colerain, BT52 1SA, UK; 3Kampffmeyer Food Service GmbH, Trettaustr. 32-34, 21107 Hamburg, Germany; Introduction: There is obviously a continuous need to assess how plant foods contribute to reducing risks of colorectal cancer development and to better understand putative mechanisms. Dietary fibre causes stool bulking, decreases passage time of faeces, modulates metabolism of carcinogens, scavenges reactive intermediates and is fermented by the gut flora to yield the short chain fatty acids (SCFA) butyrate, acetate and propionate. Butyrate is potentially chemoprotective by suppressing growth, inducing different patterns of glutathione S-transferases (GST) and enhancing chemoresistance. Dietary fibre sources (vegetables, fruits, grains) also contain a number of pharmacologically active phytoprotectants, which are converted to aglycons and further breakdown products in vivo. Aim: Aim of this work was to assess relative functional activities arising from SCFA and phytoprotectants for mediating blocking and suppressing agent activities in HT29 human colon cells, and to design dietary intervention with respective foods, based on the effects determined in vitro. Methods: Dietary fibre from Soya, wheat, linseed, fruits, and other sources were fermented with human faecal slurries in vitro, using an anaerobic protocol. The supernatants were analysed for SCFA, and corresponding SCFA mixtures were prepared. HT29 colon tumour cells were treated for 72 h with individual SCFA or complex samples. Growth of cells, activities of GST, and chemoresistance towards 4-hydroxynonenal (HNE) were determined. Selected fibre sources were used to produce bread, which was studied in a placebo, controlled, cross-over study in healthy human subjects, smokers and non smokers. In addition to standard physiological and immunological parameters, the detection of DNA damage and chemoresistance by faecal water was utilised as a biomarker to study local effects. DNA damage in peripheral lymphocytes was studied to assess systemic effects. Results: Fermentation products inhibited cell growth more than corresponding SCFA-mixtures, and the SCFA mixtures were more active than butyrate, probably due to phytoprotectants and to propionate, respectively, which both also inhibit cell growth. Only butyrate induced GST, whereas chemoresistance was caused by SCFA mixtures, but not by all corresponding fermentation samples. Faecal water from subjects fed prebiotic bread supplemented with additional dietary sources was less genotoxic than faecal water from subjects fed control bread (smokers). Systemic genotoxicity was reduced by bread enriched with prebiotic/antioxidative ingredients (in smokers). Conclusions: This study is one of the first of its kind comparing biological activities by complex mixtures produced from fermentation of different dietary fibres. It demonstrates clearly that fermentation supernatants contain compounds which (1) enhance antiproliferative properties of butyrate (propionate, phytochemicals) (2) do not alter its capacity to induce GST, and (3) prevent chemoresistance in tumour cells in vitro. The in vivo ingestion of food containing the fibre sources resulted in local and systemic antigenotoxicity, which was especially apparent in smokers. 1Department of Human Nutrition, Institute for Nutrition, Friedrich Schiller University, Dornburger Str. 29, D-07743 Jena, Germany DNA damage in buccal cells using the comet assay: Method development and preliminary findings on inter-relationships between lymphocytic and buccal cell DNA damage, and plasma and saliva antioxidant defence 1Szeto YT, 1Yow CMN, 1Tse M, 2Collins AR, 1Benzie IFF. 1The Hong Kong Polytechnic University and 2University of Oslo. Background and aims: The comet assay is well-validated test for DNA damage, and has been used successfully in biomonitoring and nutritional studies1-4. Lymphocytes are the most readily accessible human nucleated cells, and hence are the most commonly used cell model. It would be useful, however, if another type of nucleated cell could be used in human intervention trials. Genoprotective or genotoxic effects of dietary agents could then be more comprehensively assessed. There are two reports of buccal cells being used successfully in the comet assay5,6. Buccal cells are easily collected by a gentle scraping of the lining of the cheek of the mouth. Disappointingly, however, our early trials following the published protocol5 gave poor results, and buccal cells were highly resistant to lysis, preventing satisfactory performance of the comet assay. The aims of this study, therefore, were to overcome this problem, to test the feasibility of using the buccal cell model in human dietary supplementation trials, and to perform a pilot study exploring inter-relationships between comet assay results in buccal cells and lymphocytes, and between DNA damage and plasma and saliva antioxidant status. Method: Comet assay experiments were performed based on published reports1-4, but with different conditions of lysis, including pH, buffer type, duration of lysis treatment, concentration of proteinase K and presence of trypsin. Baseline DNA damage and response to standard oxidant challenge (with H2O2,) was measured as %DNA in the tail in buccal cells and lymphocytes collected from seven healthy, fasting, consenting subjects were measured, along with plasma and saliva antioxidant status (as the FRAP value and ascorbic acid concentration) in the same subjects7. All samples were collected at the same time from fasting subjects, and tests were performed on the day of collection.

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Results: The cell membrane and teflon-like cell envelope of buccal cells could be removed by treatment with trypsin (0.25%) for 30min followed by proteinase K (1mg/ml) treatment for 60min. Lower concentrations of proteinase K and absence of trypsin left the cell envelope largely intact, while high concentration of proteinase K completely dissolved the cell contents, including DNA. Using this refined procedure, plus 60 min in standard lysis solution, and electrophoresis (in 0.01M NaOH, 1mM Na2EDTA, pH 9.1, 18min at 12V), a satisfactory comet image was obtained. However, baseline DNA damage in buccal cells was high: Mean (SD) 42.7(3.4)% DNA in the tail, compared with 4.4(0.3)% for lymphocytes (n=7). Owing to the high background damage no dose response to H2O2 treatment was seen in buccal cells, but a clear dose-response was seen in lymphocytes. No significant correlation was seen between DNA baseline damage in buccal cells and lymphocytes. Plasma FRAP and ascorbic acid levels in plasma were 1101(146) and 67(14) µM respectively, while in saliva the FRAP value was 577(282) µM. There was no significant correlation between plasma and saliva FRAP (p>0.05). A significant inverse correlation (r= -0.7900: P<0.05) was seen between fasting plasma ascorbic acid concentration and %DNA in the comet tail of lymphocytes after treatment with 45µM H2O2. Conclusions: The buccal cell comet assay model is feasible in analytical terms, and offers a potentially useful minimally invasive method to assess response to dietary change. The half-life of buccal cells is 14 days, and further study is planned to investigate the effect of >14 days dietary antioxidant supplementation on buccal cell DNA damage. However, as background damage to these cells is high, they are unlikely to offer a useful model for ex vivo testing of genoprotective or genotoxic effects of dietary agents. The significant direct association seen between fasting plasma ascorbic acid and resistance of lymphocytic DNA to oxidative challenge is interesting, and supports a role for vitamin C, or something very closely associated with it in the diet, in human defence against oxidative stress at the DNA level. 1. Singh NP, Danner DB, Tice RR, Brant L & Schneider EL. DNA damage and repair with age in individual human lymphocytes, Mut Res. 1990;237:123-130. 2. Duthie SJ, Ma A, Ross MA & Collins AR. Antioxidant supplementation decreases oxidative DNA damage in human lymphocytes, Cancer Res. 1996;56:1291-1295. 3. Szeto YT & Benzie IFF. Effects of dietary antioxidants on human DNA ex vivo. Free Rad Res 2002; 36:113-118. 4. Szeto YT, Collins AR & Benzie IFF. Effects of dietary antioxidants on DNA damage in lysed cells using a modified comet assay procedure. Mut Res 2002;500:31-38. 5. Rojas E. Valverde M. Sordo M & Ostrosky-Wegman P. DNA damage in exfoliated buccal cells of smokers assessed by the single cell gel electrophoresis assay. Mut Res 1996;370:115-120. 6. Valverde M. del Carmen Lopez M. Lopez I. Sanchez I. Fortoul TI. Ostrosky-Wegman P & Rojas E. DNA damage in leukocytes and buccal and nasal epithelial cells of individuals exposed to air pollution in Mexico City. Environ Mol Mut 1997:30:147-152. 7. Benzie IFF & Strain JJ. Ferric reducing (antioxidant) power as a measure of antioxidant capacity: the FRAP assay. Methods in Enzymology, L. Packer, ed. Academic Press, Orlando 1999, pp 15-27.Germ Cell Genotoxicity Use of human hepatoma cells for the detection of dietary mutagens and antimutagens B.J. Majer1, V. Ehrlich1, M. Uhl1, B. Laky1, E. Hofer1, F. Darroudi2, S. Knasmüller1

1 Institute of Cancer Research, University of Vienna, Austria, 2 Department of Radiation Genetics and Chemical Mutagenesis, Leiden University, The Netherlands. We investigated the usefulness of two human derived hepatoma cell lines (HepG2, Hep3B) for the detection of dietary carcinogens. We showed that both cell lines retained the activities of xenobiotic drug metabolizing enzymes involved in the activation and detoxification of dietary compounds such as CYP1A, GST, UDPGT, NAT1, SULT. Subsequently we tested a panel of different groups of dietary carcinogens and clear-cut positive results were obtained in micronucleus (MN) assays in HepG2 cells with representatives of different groups such as heterocyclic amines, polycyclic aromatic hydrocarbons, spice ingredients such as safrol, mycotoxins (including aflatoxin B1, ochratoxin, trichothecenes), ethanol and its metabolite acetaldehyde, and heavy metals found in food and drinking water (e.g. As, Cd). With dietary nitrosamines clear-cut dose effects were obtained only with NDMA, but not with NPYR and NDEA. On the contrary, only a few of the above mentioned compounds induced MN in Hep3B cells under identical experimental conditions. The lack of sensitivity of this cell line is probably due to low activities of drug metabolizing enzymes. We also developed a protocol for SCGE tests (comet assay) with the hepatoma cells. Comparative investigations with a panel of different classes of dietary carcinogens showed that the sensitivity of this endpoint is similar to that of the MN test. Only with one compound, namely the mycotoxin citrinin, a negative result was obtained in the comet assay but a positive effect was obtained in the MN test. We could demonstrate with pan-centromeric probes that this discrepancy is due to the fact that citrinin acts as an aneugene. HepG2 cells were also succesfully used in a number of antimutagenicity studies with different dietary constituents: typical examples are the inhibition of the activity of benzo[a]pyrene and PhIP by chrysin, a flavonoid, and by the coffee constituents kahweol and cafestol, as well as protective effects of cruciferous vegetables and their constituents. In some cases, the effects could be attributed to induction of detoxifying (phase II) enzymes. We could clearly demonstrate that the protective mechanisms seen in the human derived cells are different from those operative in in vitro experiments with exogenous enzyme homogenates. Taken together, the present results suggest that genotoxicity tests with HepG2 are a unique tool for the detection of diet specific genotoxic carcinogens and also for the identification of protective compounds. The experimental work was supported by a EU grant (to SK and FD).

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Plant-derived foods and the modulation of oxidative stress Andrew Collins1, Božena Smolkovová, Alena Kažimirová, Magdaléná Barančoková, Alexandra Horská, Viera Spustová, Pavel Blaziček, Katarína Volkovová, Marta Staruchová, Marica Krajčovičová-Kudláčková, Ladislava Wsólová and Mária Dušinská Institute of Preventive and Clinical Medicine, Limbová 14, 833 01 Bratislava, Slovakia; and 1Institute for Nutrition Research, University of Oslo, POB 1046 Blindern, 0316 Oslo, Norway. Fruit and vegetables in the human diet are generally considered to protect against various chronic degenerative diseases, including cancer. The mechanism of this protection is likely to include antioxidant action against endogenous and exogenous oxidative damage to biomolecules. Biomarkers of oxidative damage should reflect differences in fruit and vegetable consumption, for instance in vegetarians compared with non-vegetarians, or between the seasons in countries where seasonal availability of different foods is still a factor determining the normal pattern of consumption. In a comparison of 24 vegetarians and 24 omnivores, total oxidative DNA damage (measured with the comet assay in lymphocytes) was slightly and significantly higher in the omnivores. The sub-group of lactovegetarians had the lowest level of DNA damage. Frequencies of micronuclei and of chromosome aberrations did not differ between groups. Seasonal variation in diet and biomarkers was studied in three population groups in Slovakia: a group of survivors of myocardial infarction; a control group of healthy, normal lipidaemics, living in Bratislava; and a rural control group. In rural Slovakia, home production of food is still an important determinant of diet. Blood samples were taken in two consecutive winters and summers. Serum malondialdehyde (MDA) was monitored as an index of oxidative stress (lipid peroxidation). Folate and antioxidants were measured in plasma. Folate and MDA changed in a reciprocal way with season (folate being at its highest in summer and MDA being at lowest levels). Linear regression analysis showed a strong negative association between MDA and plasma folate. Consumption of dietary antioxidants, folic acid, and wine were estimated from food frequency questionnaires. Vegetable consumption was strongly seasonal (higher in summer). Base oxidation of lymphocyte DNA was previously shown to be higher in winter months than in summer months. It seems clear that oxidative damage to biomolecules is determined to a significant degree by the individual level of consumption of plant-derived foods, and that plasma folate may be valuable as an indicator of vegetable consumption. Research supported by EC contract Inco-Copernicus 15CT961012 Can antioxidant supplementation decrease the risk of cancer? K. Volkovová1, M. Barančoková1, A. Kažimírová1, M. Staruchová1, A. Horská1, A.R. Collins2, B. Smolková1, L. Wsólová1, M. Dušinská1 1 Institute of Preventive and Clinical Medicine, Bratislava, Slovakia 2 Institute for Nutrition Research, University of Oslo, POB 1046 Blindern, N-0316 Oslo, Norway. The aim of this study was to examine the effect of antioxidant supplementation on oxidative damage and genetic instability in middle aged men, smokers and non-smokers. A total of 124 men from Bratislava and from the rural population near Bratislava were investigated in the age range 48±6 years; 64 men (22 smokers and 42 non-smokers) were supplemented with antioxidants, 60 (25 smokers and 35 non-smokers) were given placebo for 12 weeks. The antioxidant supplement consisted of vitamin C (100 mg), vitamin E (100 mg), β-carotene (6 mg), and selenium (50 µg). Samples of blood were taken on two occasions: at the beginning and at the end of the supplementation trial. Concentrations of dietary antioxidants, ferric reducing ability (FRAP), malondialdehyde (MDA) as an indicator of lipid peroxidation in plasma and parameters of genomic stability such as micronuclei, chromosome aberrations and oxidative DNA damage in isolated lymphocytes were measured. Antioxidant supplementation significantly increased the levels of vitamin C, β carotene, α-tocopherol and selenium in plasma. The index of combined antioxidant effect of the non-enzymatic defenses in plasma measured as FRAP increased significantly (P=0.000) after antioxidant supplementation as well. The increases in antioxidant parameters after supplementation were more pronounced in the group of non-smokers then in the group of smokers. Consistently, there was a significant decrease of MDA concentration in the group of non-smokers, while in smokers the decrease of MDA concentration was not significant. Antioxidant supplementation did not affect the number of cells with micronuclei or the number of micronuclei in lymphocytes; however there was a significant positive correlation (p<0.001) between the MDA concentration at the beginning of the supplementation trial and the change in the number of cells with micronuclei after supplementation compared with before supplementation. The decrease in oxidated purines and pyrimidines in DNA was significant in the group of smokers. The % of aberrant cells decreased significantly after antioxidant supplementation in both smokers and non-smokers but significantly only in smokers. These results indicate that antioxidant supplementation a) has a normalizing effect on oxidative damage e.g. diminishes oxidative DNA and lipid damage significantly when they are high initially, and b) antioxidant supplementation is effective in decreasing the genetic instability in lymphocytes of middle aged men. This study was supported by EU contract IC15-CT96-1012.

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Intestinal health: evaluation of genotoxicity in cultured enterocytes induced by human intestinal microbiota Cyrille Krul, Jet van der Zijden, Marleen van Nuenen, Johannes van de Sandt, Koen Venema TNO Nutrition and Food Research, Zeist, The Netherlands. The large intestine contains numerous mutagenic and carcinogenic metabolites, and research has revealed that the human colonic microbiota probably plays a major role in colon cancer. It is demonstrated that, on the one hand, microbiota contain enzymatic activities that convert procarcinogens into carcinogens, and on the other hand pre- and probiotics may reduce colon cancer. However, the genotoxic activity of the human microbiota and the putative preventive effects of (food) compounds (or its metabolites) on colon cancer are largely unknown. Since the use of animal studies is only limited for this propose, we developed an in vitro method to investigate the genotoxicity of the large intestinal content and the effects of (food) compounds and its metabolites on the genotoxic activity of the human microbiota. A metabolic active, faecal microbiota of human origin with physiological density of microorganisms, can be established in the TNO in vitro large Intestinal Model (TIM-2), which closely simulates the colonic environment (pH, anaerobic), peristaltic mixing and removal of fermentative metabolites by dialysis. The genotoxicity of the large intestinal content was evaluated in the Comet assay (single cell gel electrophoresis). This is a suitable method to detect DNA strand breaks and alkali labile sites. Sub confluent Caco-2 and HT29 cells (human colonic cell lines) were exposed for 1-3 hours to samples taken from the lumen and dialysis fluid of TIM-2. After exposure, cells were collected and toxicity was determined by trypan blue exclusion. Comets were quantified with an image analysis system and DNA damage was expressed as mean tail moment of at least 300 cells. Our results showed that the lumen content as well as dialysis fluid samples of TIM-2 were able to increase the mean tail moment of Caco-2 and HT29 cells. The lumen samples were more toxic compared to the dialysis fluid. Genotoxicity was evaluated at viability over 75%. The mean tail moment was dose related increased. In Caco-2 cells, the mean tail moment induced by the lumen content and dialysis fluid was approximately 6- and 2-fold increased, compared to the control (unexposed) cells, respectively. In HT29 cells, the mean tail moment induced by the lumen content and dialysis fluid was approximately 8.5- and 2.4-fold increased, compared to the control (unexposed) cells, respectively. Experiments are ongoing to investigate the effect of various compounds on the genotoxicity of the large intestinal content in TIM-2. The selected compounds positively (such as inulin and resistance starch) or negatively (such as antibiotics (clindamycine) or Clostridium difficile infection) modulate the microbiota. The results of these experiments will be presented. Furthermore, pattern-recognition techniques will be used to obtain more insight in the correlations between genotoxicity and different metabolites produced (metabolomes) in the large intestine. Modulation of cytochrome P450 1A and 2B activities by oral administration of cabbage and sauerkraut juice in rat liver and kidney. Szaefer Hanna, Krajka-Kuźniak Violetta, Bartoszek Agnieszka, Baer-Dubowska Wanda Department of Pharmaceutical Biochemistry University of Medical Sciences, Poznań, Poland Cruciferous vegetables contain several organosulphur compounds, which have been shown to possess anticarcinogenic activity in animal models. Organosulphur substances include isothiocyanates and their biosynthetic precursors, glucosinolates and indole-3 carbinol. The specific characters, their amount and proportion depend on the Brassica species. While anticarcinogenic activities of broccoli and water cress were the subject of intensive studies, cabbage and sauerkraut were not although they represent important ingredients of Eastern and Middle European diet. In this study the effect of cabbage and sauerkraut juice on the activities of CYP1A1, 1A2, 2B, and the expression of CYP1A1 was investigated in Wistar rats liver and kidney. Animals were treated by gavage with cabbage or sauerkraut juice for 4, 10 and 30 days. In rat liver the significant increase of EROD (the marker of CYP1A1) and MROD (the marker of CYP1A2) activities were observed after 30 days of treatment with cabbage or sauerkraut juice in comparison with the control group of animals receiving water only. At the same time the activity of PROD (the marker of CYP2B) was diminished in both tissues. At the earlier time points the activities of these enzymes were not changed or decreased. The increased activities of EROD and MROD were observed in kidney already after 4 days of treatment. Western blot analysis with CYP1A1 specific antibody showed significant increase in the levels of hepatic CYP1A1 in cabbage and sauerkraut juice treated animals. These results indicate that both cabbage and sauerkraut juice may affect selectively the cytochrome P450 isoforms involved in the activation of specific carcinogen. This effect depends however on treatment period and might be tissue specific.

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Is it possible to protect New Zealanders from colon cancer? L. R. Ferguson, N. Karunasinghe and M Philpott Discipline of Nutrition and ACSRC, Faculty of Medical & Health Sciences,The University of Auckland, Auckland, New Zealand. New Zealand has one the highest incidences of colon cancer in the world, and there is good reason to believe that this relates to the eating habits of the population. For many years, New Zealand has had an agriculture-dominated economy, and this has led to a national diet that is high in meat consumption but somewhat low in fruits and vegetables. Controversy over Maori fishing rights have reduced fish from a frequent dietary item to more of an expensive luxury. Despite an intensive and continuing “5-a-day fruits and vegetables” campaign, a national nutrition survey published in 1999 revealed that by far the majority of New Zealanders are eating somewhere between 2-3 total servings, with the most commonly eaten fruit being the banana, and vegetable the potato. An extensive international database would suggest that none of these dietary trends is beneficial for reduction of colon cancer incidence. However, the local data are not out of line with trends in other countries, and do not explain why New Zealand is apparently worse off than these. Our own studies have focused on differences in trends within New Zealand as important clues to what is happening. The 2001 colon cancer registry data have been localised into regions and into racial groups, and compared with other demographic trends. Multiple regression analysis suggest that race and soil selenium status are the two major factors that predict colon cancer occurrence, with the interaction between the two appearing as highly statistically significant (p<0.005). New Zealand’s low soil selenium has been well-recognised for many years. Most plants will not accumulate selenium against a gradient, so that locally grown fruits and vegetables, as well as animals which graze on local pastures that are not selenium-supplemented in some way, all contribute to a low selenium status in certain regions. In a study in Auckland that involves a group of middle-aged men, we have found that blood selenium levels of approximately half of these men appear inadequate for repair or surveillance of DNA damage. In that the Auckland soil selenium levels are higher than in many other parts of the country, this raises a considerable concern about these other members of the New Zealand population. As well as soil selenium, the other key factor appears as racial group, and it has been known for some years that Maori and Pacific Island groups have substantially lower levels of colon cancer than Europeans. This does not appear to be genetic but may well relate to dietary habits. Chemopreventive factors in some of the South Pacific fruits and vegetables are a plausible factor in this protection.

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Germ Cell Genotoxicity Mechanisms of mutation induction in the male germ-line M.H. Brinkworth1, T.E. Schmid2, J.D. Taylor1, A. Baumgartner1, H. Bollwein3 and I.-D. Adler4 1 Department of Biomedical Sciences, University of Bradford, Bradford BD7 1DP, UK, 2 School of Public Health, University of California in Berkeley, USA, 3 Clinic of Veterinary Medicine of the Ludwig-Maximilian-University, Munich, Germany, 4 GSF-Institute of Experimental Genetics, D-85764 Neuherberg, Germany. It is now recognised that there are considerable potential sources of genetic damage to male gametes that could result in infertility or heritable effects on offspring. An increasing number of men survive cancer treatments at reproductive age having been exposed to very high doses of mutagens. Advances in techniques of assisted reproduction allow men with genetic infertility to father children. Similarly, it is increasingly common for men of advanced age to become fathers although the germ-line mutation rate is higher than in women and increases with age. Environmental and occupational mutagen exposures are also of interest, as is their interaction with any of the foregoing risk factors. However, as yet, the mechanisms influencing the appearance of mutations in the germ-line are obscure. High, acute doses of the mutagenic, chemotherapeutic agent cyclophosphamide in rodents are not generally thought to cause heritable, developmental mutations leading to morphological abnormalities in the foetuses. Sub-chronic or chronic, low-dose exposure by contrast results in a significant elevation of developmental defects in the offspring of animals mated after the end of dosing (Trasler et al., 1985, Nature, 316:144-6; Jenkinson et al., 1987, Mutat Res 188:57-62). Examination of the testes of sub-chronically treated rats revealed a significant decrease in the proportion of apoptotic germ-cells at the same dose level that yielded abnormal foetuses (Brinkworth and Nieschlag, 2000, Mutat Res 316:144-6). This suggests that genetically damaged cells could survive to participate in fertilisation, which may have relevance for environmental or occupational exposure. At high, acute doses, which mimic therapeutic exposure more closely, apoptosis presumably deletes the damaged cells that could cause these specific heritable effects. The limited epidemiological studies so far conducted indicate that the offspring of cancer-treatment survivors do not have an elevated risk of heritable disease. Molecular studies have failed to show an increase in minisatellite mutations in sperm produced by men following recovery of sperm production after therapy (e.g., Armour et al., 1999, Mutat Res 445:73-80), perhaps indicating that this sort of damage is normally eliminated during spermatogenesis. Infertility caused by low sperm counts and other sperm defects have long been known to be associated with various types of genetic damage including aneuploidy and chromosome damage but few studies have examined multiple end-points or used well-characterised study populations. Recently, we have shown that elevated proportions of the sperm of infertile men with idiopathic oligozoospermia are genetically damaged. They have significant gonosomal aneuploidy, strand breaks detected by the comet assay and chromatin damage as measured by the sperm chromatin structure assay (SCSA) (Schmid et al., 2003, Hum Reprod 18:1474-80). This condition is often also associated with mixed testicular atrophy, in which some seminiferous tubules produce sperm normally, others show germ cell maturation arrest, while others have no germ cells at all. We hypothesised that these phenomena could be explained by the induction of a range of types of genetic damage very early in germ cell development. To test this hypothesis, five pregnant female (102/ElxC3H/El)F1 mice per group were dosed on day 9 of gestation with 0, 5, 10, 20 or 40 mg/kg N-ethyl-N-nitroso-urea (ENU). At this stage of pregnancy, the primordial germ cells migrate from the yolk sac to the genital ridge. The animals were allowed to deliver and raise the offspring to maturity. The male F1's were examined at 2, 4 and 6 months of age for effects on sperm production parameters, and COMET assay and SCSA damage. (Other genetic end-points including aneuploidy are currently under investigation.) Statistically significant effects were found on all end-points, especially, but not exclusively, at the top two doses. However, there was no clear difference between the extent of the effects induced by 20 and 40 mg/kg ENU, which could be because of the death of the most severely damaged primordial germ cells at the higher dose. There was no consistent trend towards an amelioration or a worsening of the different effects through time, though this needs confirmation at further time points. Particularly striking is the finding of comet and SCSA damage in mature sperm that had been exposed months earlier as primordial germ cells. Presumably, the genotoxin induced mutations in genes that were needed for the normal subsequent development of the sperm nucleus. Testis cross-sections of treated animals showed mixed tubular atrophy, also indicating that mutations had been induced that only expressed their effects during spermatogenesis, long after their induction. The ENU studies represent the first establishment of an animal model that mimics human idiopathic, male infertility. Since mutagenic events appear to be able to produce the phenotype of this disease, it is possible that this form of male infertility has its origin in genotoxin exposure. This could also have implications for the genetic health of the children if such men are eventually able to become fathers.

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In vitro approaches to the study of aneuploidy in oocytes U. Eichenlaub-Ritter, F. Sun, U. Winterscheid, Y. Shen and I. Betzendahl Universität Bielefeld, Fakultät für Biologie, Genotechnologie/Mikrobiologie, D-33501 Bielefeld, Germany. After completion of pachytene stage in the embryonal ovary, primary mammalian oocytes meiotically arrest for up to decades (e.g. in the human) within a primordial follicle. The continuous hormone-independent regulatory processes initiating follicular recruitment to the growing pool of a cohort of primordial follicles before and after puberty are still largely unknown. Most of the follicles within the ovary undergo atresia, and die prior to or after birth. Accordingly, the primordial follicle pool with immature oocytes becomes continuously depleted with increasing maternal age. Hormones are required for the late, antral stages of folliculogenesis and oocyte growth in the sexually mature female. We know now that complex bi-directional signalling events between the oocyte and the somatic compartment are required for follicular proliferation and differentiation, and for formation of a large antral follicle with a fully-grown, meiotically competent oocyte. The immature oocyte acquires the competence to re-initiate meiosis, and to complete both meiotic divisions and support early embryogenesis in a sequential fashion during the growth phase. Most epigenetic processes and synthesis of proteins and RNA for resumption of meiosis and early embryogenesis are taking place prior to the surge by lutenizing hormone (LH), which finally triggers chromosome condensation, spindle formation and progression through first meiosis up to metaphase II in the follicle-enclosed oocyte in vivo. Second anaphase with segregation of sister chromatids is subsequently triggered by fertilization. Cytogenetic and molecular studies collectively suggest that human oogenesis is highly susceptible to meiotic errors, especially at first division. The major aetiological factor in oocyte aneuploidy is maternal age and depletion of the follicle pool. It is still unknown to what extent alterations in hormonal signalling e.g. during the oocyte growth phase, ageing processes affecting integrity of cell organelles, and environmental exposures contribute to the high rate of meiotic nondisjunction in female meiosis. In view of the complexity of oocyte meiosis, the longevity of the female germ cell, constitutive atresia, and the para- and autocrine regulation of oogenesis, it has been difficult to devise in vivo assays to detect aneugens and define thresholds of exposure in experimental studies. Classical aneugens, which disturb microtubule dynamics are assumed to primarily affect the latest stages of meiosis when spindles are formed and chromosomes segregate at anaphase I and II. Accordingly, many in vivo studies comprise administration of drugs at around the time of hormonally-stimulated resumption of meiosis, and chromosomal analysis of metaphase II oocytes or 1-cell blocked embryos. Fully grown meiotically arrested mammalian oocytes of some species like the mouse are able to spontaneously resume maturation in vitro and develop to metaphase II after their isolation from the follicle. In order to directly compare the LOAEL of chemicals inducing aneuploidy in cultured somatic cells with that in mammalian oocytes possessing unusual anastral spindles we exposed mouse oocytes maturing spontaneously in vitro to known aneugens and chromosomally analysed them for meiotic errors. With this in vitro approach concentration, most effective metabolite and sensitive stages of exposure can be analysed. Furthermore, oocytes can be processed for immunofluorescence or in vitro assays to identify the cellular target and mechanism of drug action, as well as the influences on cell cycle and chromosome behaviour. We found that displacement of chromosomes from the spindle equator (congression failure) was a good indicator for conditions inducing aneuploidy. LOAEL of classical aneugens causing nondisjunction in mouse oocytes appeared similar to that in cultured human lymphocytes. However, concentrations of spindle proteins in primate oocytes are more limited, and this may contribute to the species-specific, exceptionally high sensitivity of human oocytes to meiotic nondisjunction. Recently, we employed novel enhanced polarizing microscopy (Polscope) to detect spindle aberrations in living oocytes. Since it is non-invasive this method can provide information on adverse exposures in experimental animals but also in humans, since living human oocytes isolated for assisted reproduction can be analysed for presence of a spindle. In view of the potential significance of perturbations in hormonal control and in gap-junctional communication between the oocyte and somatic cells within the follicle for induction of meiotic aneuploidy, we also developed a novel in vitro assay. Pre-antral follicles are isolated mechanically from the ovary of prepubertal mice. During 12 days of culture they develop to the antral stage in vitro in the presence of recombinant hormone. Resumption of maturation is initiated by LH at day 12, and mature oocytes in metaphase II are obtained on the next day. In vitro grown oocytes have normal spindles and chromosomes segregate with high fidelity. Data on the influences of aneugenic exposures of oocytes in this novel in vitro assay will be presented and discussed, especially with regard to recent reports suggesting that exposures to endocrine disrupting substances in food may pose a risk to faithful chromosome segregation in oocytes, and may contribute to congenital abnormality and trisomy in humans. Supported by EU (QCRT-2000-00058).

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In vivo approaches to the study of aneuploidy in oocytes Francesca Pacchierotti Section of Toxicology and Biomedical Sciences, ENEA, CR Casaccia, Roma, Italy. The experimental study of chemically induced aneuploidy in mammalian oocytes has evolved in the last 20 years. This evolution has been stimulated by the increasing understanding of the molecular events that regulate oocyte maturation and chromosome segregation at meiosis, by the development of sophisticated techniques for culturing oocytes in vitro and by the expanding application of assisted reproductive technologies in humans. Initially, the largest effort was invested in testing chemicals affecting the integrity of the spindle as the most obvious target for interfering with chromosome segregation. Compounds such as colchicine, vinblastine, taxol, griseofulvin, benomyl were demonstrated to induce very high frequencies of aneuploidy after acute treatment in vivo before meiosis I, usually with increases over spontaneous level much larger than those observed in somatic and male germ cells. However, studies testing variation of sensitivity as a function of treatment time showed that this was a critical variable for the quantitative outcome of the experiments and suggested that preovulatory oocytes passed in a short time through phases of variable sensitivity to the action of spindle poisons and that a rapid decrease of the effective (target cell) chemical concentration might occur a short time after a single acute dose. Although these studies unequivocally demonstrated the possibility to experimentally induce high levels of aneuploidy in mouse oocytes and strongly validated the cytogenetic analysis of mouse oocytes and zygotes as a reliable test system for the detection of meiotic aneuploidy, they did not contribute greatly to our understanding of the possible environmental causes of constitutive aneuploidy in humans, because of the limited human exposure to the specific chemicals and the acute high doses tested. More recently, experiments have been carried out on chemicals more relevant for human exposure, such as nicotine, tamoxifen or clomiphene citrate, encompassing a wider range of possible molecular targets. In addition, building upon the increasing knowledge of the molecular mechanisms underlying the meiotic processes, the attention has been focused on the consequences of chemically induced inhibition of biochemical reactions involved in the regulation of oocyte maturation and chromosome segregation. Protein phosphorylation-dephosphorylation reactions and proteasome-mediated proteolysis ensure a timely separation of centromeres and regulate the cascade of events required for orderly metaphase to anaphase transition. Transient (6 h) in vitro exposure of mouse oocytes to inhibitors of proteasomes or phosphatases were shown to induce untimely separation of centromeres and aneuploidy in meiosis I. These studies, which uncouple damage to structures (spindle) from cell cycle alterations, offer new support to the classic hypothesis that temporal perturbations during mammalian oocyte maturation predisposes oocytes to aneuploidy. What is the relevance of these findings for understanding causes and mechanisms of human aneuploidy, including the possible impact of environmental agents, and for designing a “new generation” of in vivo studies? Some hints come from a comparison between observations from in vitro and in vivo exposures to the same chemicals (diazepam, trichlorphon) which suggest that exposures sustained for longer times, such as those achieved under continuous in vitro culture with chemical-added medium, are more effective than single acute in vivo administered doses. In vivo studies have indeed tried to circumvent this problem with fractionated administrations. In the more recent in vivo experimental studies on chemically induced aneuploidy in mouse oocytes, small but significant increases of the frequency of hyperploid metaphases were often detected over matched and historical controls. These observations might indicate that the first meiotic oocyte exposed in vivo is not an “all or none” target to chemical-induced aneuploidy as the first studies on spindle poisons might have suggested, but that also small significant effects might occur and should be carefully investigated by large enough sample sizes and the best possible harvesting techniques. A recent paper (Hunt et al., Curr Biol 13, 546, 2003) showed that accidental exposure of mice to bisphenol A leaked from damaged plastic cages and drinking bottles induced alterations of chromosome congression at metaphase and meiotic aneuploidy in the oocytes. This might be the first strong case of aneuploidy induced by an environmental source of exposure, which, in addition, might be relevant for human populations. Experimental treatments consisting of 7 daily oral administrations by gavage in the week preceding ovulation confirmed the association between bisphenol A exposure and aneuploidy, but the magnitude of effect was lower than that observed during the accidental leakage. Either a too short exposure period or a too rapid clearance of the daily dose with respect to the dose slowly uptaken from drinking water might explain the quantitative difference: both hypotheses open new perspectives to the design of in vivo testing of aneuploidy in mammalian oocytes.

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Transgenerational germline instability Y.E. Dubrova Department of Genetics, University of Leicester, Leicester LE1 7RH. One of the major challenges of modern genetics is to explore the recent advances in mutation research and integrate them into our current approaches for the estimation of genetic risks in humans. For example, it has recently been recognised that ionising radiation can not only induce mutations seen in the directly exposed somatic cells, but can also lead to delayed effects with new mutations arising many cell divisions after the initial irradiation damage. However, if genomic instability can also be induced in the germline of exposed parents the delayed transgenerational effects may be manifested in their offspring, therefore presenting even more delayed risk in humans. We have recently obtained the first experimental evidence that germline mutation rates in unexposed offspring of irradiated male mice did not return to normal unexposed mutation rates, but were very similar to that of the irradiated males. These data were generated using our approach for monitoring germline mutation in mice, which is based on a set of hypervariable expanded simple tandem repeat (ESTR) DNA loci. Our results show that radiation-induced instability can be transmitted for at least two generations after initial paternal exposure. Paternal exposure to either X-rays or fission neutrons resulted in increased mutation rates in the germline of both generations. Pre- and post-meiotic paternal exposure resulted in similar increases in mutation rate in the germline of both generations, suggesting that radiation-induced ESTR instability is manifested in diploid cells following fertilization. The extent of transgenerational increase clearly varied with the different strains (BALB/c>CBA/H>C57BL/6). Our data suggest that germline instability is caused by some DNA-dependent signal transmitted from the irradiated father and implicate an epigenetic mechanism for the transgenerational instability. These results, together with the data on radiation-induced genome instability in somatic cells raise the important issue of delayed effects of ionising radiation. Female germ cell mutagenicity of model chemicals in Drosophila melanogaster: mechanistic information and analysis of repair systems L.M. Sierra, J. Hernando, L. Álvarez, J.A. Ferreiro, I. Sancho and M.A. Comendador. Área de Genética. Dpto. Biología Funcional e Instituto Universitario de Oncología del Principado de Asturias (IUOPA). University of Oviedo. 33006 Oviedo. Spain. Female and male germ cells differ on gametogenesis mechanisms, cytoplasmic composition and DNA repair activity, and both of them contribute to the gene pool of future generations. However, most germ cell mutagenicity studies in higher eukaryotic organisms have been carried out on males. To study the response of female germ cells to mutagen/carcinogen exposure, contributing at the same time to the equilibrium between mutagenicity database of males and females, and checking also the validity of D. melanogaster for this kind of analysis on females, the mutagenicity of three model chemicals like N-ethyl-N-nitrosourea (ENU), diethyl sulfate (DES) and hexamethylphosphoramide (HMPA), and the monofunctional methylating chemotherapeutic drug streptozotocin (STZ), is analysed on repair efficient females of D. melanogaster, using a modified sex linked recessive lethal (RL) test. DES and ENU are also analysed on nucleotide excision repair (NER) and bypass tolerance mechanism (BTM) deficient females, to check the activity of these systems on the repair of oxygen and nitrogen ethylation damage in these cells. Results indicate that: (1) ENU, HMPA, DES and STZ are mutagenic on efficient repair female germ cells of D. melanogaster, which are all pre-meiotic cells. Among the three cell stages analysed, oogonia was not so well studied as oocytes, mature or immature. Oogonia are cells difficult to reach, although probably not so much as spermatogonia stem cells. However, oocytes both mature and immature are easily analysed, and provide enough information about chemical mutagenicity and influence of repair systems to consider that, even when oogonia could not be reached, their analysis could be a model for pre-meiotic germ cells. (2) A measure of mutagenic activity, estimated as % RL frequency per molar concentration per treatment time, indicates that DES is the less active chemical, followed by STZ, HMPA and ENU. This order correlates with the reparability of the respective induced DNA damages, and with the mutagenic and carcinogenic potency of the analysed chemicals, considering the toxicity of cross-linking agents. This result indicates that, although the effect of chemicals is higher in male than in female germ cells, the sensitivity of female germ cells is highly dependent on the reparability of the induced damages. Because of this, female germ cells seem to be less susceptible to chemicals inducing easily repaired DNA damages, than postmeiotic male germ cells. Consequently, when enough data about mutagenicity on females will be achieved, the response of female germ cells to a chemical might give an indication about the efficient reparability of the induced DNA damages, and therefore about its mutagenic activity. (3) NER efficiently repairs nitrogen ethylation damage and seems to contribute to the processing of oxygen damage in female germ cells. (4) BTM processes oxygen ethylation damage, whereas for ethylation of nitrogen atoms results are not clear. Since female germ cells are repair active cells, the influence of repair systems on the removal of induced DNA damages is more easily detected than when treating male germ cells, specifically postmeiotic ones because then only the maternal repair activity is analysed. Finally, the results of this work indicate that the differences between male and female germ cells affect the response to chemical exposure and therefore demonstrate the necessity of analysing female cells in germ cell mutagenicity studies.

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Oestrogenic compounds and oxidative stress (in human sperm and lymphocytes in the Comet assay) 1D. Anderson, 1T.E Schmid, 1,2A. Baumgartner , 1E. Cemeli, 1M.H. Brinkworth and 1J. Wood. 1Department of Biomedical Sciences, University of Bradford, Bradford, UK 2GSF- Institute of Molecular Radiobiology, Neuherberg, Germany. Reactive oxygen species (ROS) are produced by a wide variety of chemicals and physiological processes in which enzymes catalyse the transfer of electrons from a substrate to molecular oxygen. The immediate products of such reactions, superoxide anion radicals and hydrogen peroxide can be metabolised by enzymes such as superoxide dismutase (SOD) and catalase (CAT) respectively and depending on its concentration by Vitamin C (Vit C). Under certain circumstances the ROS form highly reactive hydroxyl radicals. We examined human sperm and lymphocytes after treatment with six oestrogenic compounds in the Comet assay, which measures DNA damage, and observed that all caused damage in both cell types. The damage was diminished in nearly all cases by catalase, and in some instances by SOD and Vit C. This response pattern was also seen with hydrogen peroxide. This similarity suggests that the oestrogen-mediated effects could be acting via the production of hydrogen peroxide since catalase always markedly reduced the response. The variable responses with SOD indicate a lesser involvement of superoxide anion radicals due to SOD - mediated conversion of superoxide to hydrogen peroxide generally causing a lower level of DNA damage than other ROS. The variable Vit C responses are explained by a reduction of hydrogen peroxide at low Vit C concentrations and a pro-oxidant activity at higher concentrations. Together these data provide evidence that inappropriate exposure to oestrogenic compounds could lead to free - radical mediated damage. Parallel Comet and SKY evaluation of genetic damage in sperm and lymphocytes after treatment with doxorubicin A. Baumgartner1,2, T.E. Schmid1,3, E. Cemeli1, I.-D. Adler4,and D. Anderson1 1 University of Bradford, Dept. of Biomedical Sciences, Bradford, UK, 2 GSF Research Center, Institute of Molecular Radiobiology, Neuherberg, Germany, 3 Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, USA 4 GSF Research Center, Institute of Experimental Genetics, Neuherberg, Germany. In recent years two different techniques have gained importance: the alkaline single cell gel electrophoresis assay (Comet assay) to detect DNA damage such as DNA strand breaks within nuclei and a multicolour fluorescence in situ hybridisation method, spectral karytotyping (SKY), to detect chromosomal aberrations in all human metaphase chromosomes simultaneously (23 pairs of autosomes, X and Y). In the present study, the induction of DNA strand breaks in human sperm and in human lymphocytes in vitro has been studied after treatment with the widely used anti-cancer drug, doxorubicin. Doxorubicin effectively binds DNA by intercalation causing DNA damage via the production of reactive oxygen radicals (ROS) and by stabilizing the topoisomerase II cleavage complex. These multiple effects caused a dose-related increase in DNA strand breaks in human sperm and human lymphocytes, which was observed in the Comet assay. The SKY technique has been used to detect chromosomal aberrations in human lymphocyte metaphases after treatment with doxorubicin identifying doxorubicin-induced chromatid breaks and chromatid exchanges. The SKY results correlate very well with the findings of the Comet assay. These data are the first reporting the use of the Comet assay and SKY analysis in one study after chemical treatment. In a parallelogram approach, the results suggest that doxorubicin induce DNA strand breaks, which may lead to a range of chromosomal aberrations in lymphocytes and in human sperm. Increased genetic damage in sperm after gestational mutagen exposure J. Taylor1, T.E. Schmid1, H. Bollwein2, A. Baumgartner1, I.-D. Adler3, M.H. Brinkworth1 1 Department of Biomedical Sciences, University of Bradford, UK 2 Department of Animal Reproduction, Ludwig-Maximilian-University, Munich, Germany 3 GSF-Institute of Experimental Genetics, Neuherberg, Germany.

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The aim of this project was to model the testicular and seminal defects by exposure of mice to lower doses of genotoxins. There are many routes by which damage could lead to idiopathic male infertility. However, genetic damage that is induced very early in male germ cell development may have the most widespread effects on subsequent gametes. Therefore, this project examined mutagenic effects on primordial germ cells. Five pregnant female mice per dose group were treated with a single intra-peritoneal injection of vehicle (0.9% Saline), 5, 10, 20 and 40 mg/kg ENU on day 9 of gestation. They were allowed to deliver their litters and the male offspring were raised to maturity. Two males were randomly selected from each litter for further study. A significant decrease in testis weight, sperm motility and count was found in the 40 mg/kg and 20 mg/kg ENU group compared with the control group in the seminology analysis. The SCSA was performed on 10,000 sperm from each of 5 animals per dose group including the control. A significant increase (p<0.01) in COMP αT (representing the amount of denatured, single-stranded DNA over the total cellular DNA) was found in the 40 mg/kg and 20 mg/kg ENU group (18.6% and 38.9%) compared with the control group (7.6%). DNA strand breaks were measured by the Comet assay. A significant increase (p<0.01) in tail moment in the Comet assay was found in the 40 mg/kg and 20 mg/kg ENU dose group (14.2 and 9.4) compared with the control group (6.7). This study is the first demonstration that mutagenic damage to male germ-cells in the mouse yields a similar spectrum of effects to those seen in oligozoospermic, infertile men. This could be an indication of similar mechanisms operating in both situations. The results will be of great value, especially to clinicians caring for patients with idiopathic male infertility but also in basic research as they point to a novel mechanism that may explain the aetiology of idiopathic male infertility. Topoisomerase II-inhibitors amsacrine hydrochloride (m-AMSA), etoposide (VP-16) and merbarone (MER) cause meiotic delay and aneuploidy in spermatocytes of mice S.M. Attia 1, 3, T.E. Schmid 1, 2, O.A. Badary 3, F.M. Hamada 3, I.-D. Adler 1 1 GSF-Institute of Experimental Genetics, D-85764 Neuherberg, Germany, 2 School of Public Health, University of California, USA, 3 Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt. The nuclear enzyme topoisomerase II (topo II) is a ubiquitous enzyme that relaxes supercoiled DNA and decatenates intertwined DNA by breakage and reunion of DNA double strands. Topo II is a specific target for some of the most active drugs used in the treatment of human malignancies. The topo II-targeted agents currently in clinical use, etoposide and

amsacrine, act by increasing the levels of topo II-mediated DNA cleavage and are referred to as topo II "poisons". Recently, catalytic inhibitors that display high activity against eukaryotic type II topoisomerases have been described, such as merbarone (MER). In contrast to topo II poisons, these agents act by inhibiting the catalytic activity of the enzyme and display no ability to stimulate DNA cleavage. Due to their mode of action, topo II inhibitors are suspected to have aneugenic potential. The ability of the three topo II inhibitors, amsacine hydrochloride (mAMSA), etoposide (VP-16) and merbarone (MER), to induce meiotic delay and non-disjunction in mouse spermatocytes was investigated. The progression from meiotic divisions to epididymal sperm was determined by injecting young adult male mice with BrdU to label the last round of DNA synthesis before meiosis. The animals were treated 13 days later with the test chemicals, when the BrdU-labelled cell was undergoing meiotic divisions. At daily intervals, 20–24/25 d after treatment, sperm were collected from the epididymes. BrdU-containing sperm were identified with a FITC-labelled anti-BrdU antibody and green fluorescent sperm were scored automatically with a laser scanning cytometer. In the controls and the MER-treated group, the maximum frequencies of BrdU-labelled sperm appeared 22 d after treatment in the epididymes. It was found that m-AMSA (15 mg/kg) and VP-16 (50 mg/kg) induced a meiotic delay of 24 and 48 hrs, respectively. The timing for sperm sampling in the aneuploidy assay was adjusted accordingly. In the aneuploidy assay, hyper-haploid and diploid sperm were determined by fluorescence-in situ-hybridisation (FISH) with fluorochrome-labelled DNA-probes specific for chromosomes 8 (CY3, red), X (CY3+FITC, white) and Y (FITC, green). From each of 5 animals in treatment and control groups, 10,000 sperm were analysed for the numbers and colours of chromosome signals by fluorescence microscopy. In these experiments, male mice were treated with 5, 10, 15 and 20 mg/kg m-AMSA, 12.5, 25 and 50 mg/kg VP-16 or 15, 30 and 60 mg/kg MER. Sperm were sampled from the Caudae epididymes 24 d after VP-16 treatment, 23 d after m-AMSA treatment or 22 days after MER treatment. Significant increases above the concurrent controls in the frequencies of hyperhaploid sperm were found after treatment with 10, 15 and 20 mg/kg of m-AMSA (0.07%, 0.096% and 0.10%, respectively; solvent controls 0.058%), with 25, 50 mg/kg of VP-16 (0.074% and 0.122%; solvent controls 0.052%) and with 60 mg/kg MER (0.098%; solvent controls 0.044%). Furthermore, significant increases in the frequencies of diploid sperm were found with 15 and 20 mg/kg of m-AMSA (0.014% and 0.028%, respectively; solvent control 0.004%), all three doses of VP-16 (0.024%, 0.032% and 0.056%, respectively; concurrent solvent controls 0.004 and 0.0%, respectively) and with 30 and 60 mg/kg of MER (0.022% and 0.050%, respectively; concurrent solvent controls 0.004% and 0.002%, respectively). The prevalence of sperm with two identical sex chromosomes (hyper haploids XX8 and YY8 or diploids XX88 and YY88) over sperm with the two different sex chromosomes (hyper haploids XY8 or diploids XY88) indicates that the second meiotic division was more sensitive than the first meiotic division. Despite their different modes of action, the topo II-poisons amsacrine and etoposide and the catalytic topo II-inhibitor merbarone induced non-disjuntion of chromosomes during meiosis in spermatocytes of male mice. The results suggest that cancer patients may stand transient risk for siring chromosomally abnormal offspring after chemotherapy with these topo II inhibitors. Research supported by EU-contract QLK-CT 2000-00058 PEPFAC

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Experimentally determined correction factor for translocations induced by intraperitoneal vs. dermal exposure of male mice to acrylamide to improve genetic risk estimates I.-D. Adler, H. Gonda, I.S. Otten GSF-Institute of Experimental Genetics, D-85764 Neuherberg, Germany. Acrylamide (AA) is an important industrial chemical used mainly in the production of polymers. It is neurotoxic and was shown to be a clastogen in mammalian cell cultures, a rodent carcinogen and a germ-cell mutagen (Dearfield et al., 1988, Mutat. Res. 195, 45-77). Dose-dependent induction of dominant lethal mutations and heritable translocations by AA was demonstrated after intraperitoneal (ip) exposure of male mice (Adler et al., 1994, Mutat. Res. 309, 285-291). However, humans are exposed by dermal contact and, to a lesser extent, by ingestion. Therefore, it was important to study the effects of AA after dermal exposure in mouse experiments in order to derive a more reliable quantification of genetic risk for humans. In mice, AA is readily absorbed through the skin and binds to DNA in the testes (Carlson and Weaver, 1985, Toxicol. Appl. Pharmacol. 79, 307-313). The first indication that dermal application of AA induces dominant lethal mutations in male mice was published by Gutierrez-Espeleta et al. (1992, Fundam. Appl. Toxicol. 18, 189-192). The authors found a dose-related response to dermal exposure on five consecutive days in the dose range from 25 mg/kg/d to 100 mg/kg/d that did not deviate from linearity. The effect was higher in females fertilized 7-8 d than 9-10 d after the end of exposure. In order to compare the magnitude of dominant lethal induction between ip and dermal exposure, 5 daily doses of 50 mg/kg AA were applied every morning to 50 (102/ElxC3H/El)F1 males by both routes, the solvent control group received saline only. For ip treatment, AA was dissolved in physiological saline. For dermal application on the shaved backs of the animals, AA was dissolved in a small amount of saline mixed with corn oil (1:19). The applied volumes were 0.1 ml per 10 g of body weight. All males were mated at a 1:1 ratio with virgin females of the same stock starting on the afternoon of the day after the last exposure. Females were replaced four times at four-day intervals with a total of five mating intervals. Percent-induced dominant lethals was calculated by the formula [1 - (live implants per female in the treated group/live implants per female in the control group)] x 100. Significantly increased rates of dominant lethals were observed during the first three mating intervals. For ip exposure these values were 81.7%, 85.7% and 45.4%, respectively; for dermal exposure the corresponding values were 22.1%, 30.6% and 16.5%, respectively. A heritable translocation assay was performed with the same regimen of dermal exposure and 100 treated C3H/El males were mated to 102/El females 1.5 to 8.5 d after the end of exposure. The choice of the mating interval was based on the sensitivity pattern seen in the dominant lethal experiment. Pregnant females were allowed to come to term and all offspring were raised to maturity. The identification of translocation carriers was performed according to the protocol of Adler 1980 (Teratogen. Carcinogen. Mutagen. 1, 75-86). All translocation heterozygotes were karyotyped by G-banding. A total of 475 offspring (258 males and 217 females) were screened and 41 translocation carriers were identified (28 males and 13 females). Thus, total translocation frequency after oral exposure was 8.6% as compared to 21.9% after ip exposures to 5x50 mg/kg AA (Adler 1990, Banbury Report 34, 115-131). The calculated ratio of ip vs. dermal of 1:0.39 can be applied to derive a more realistic genetic risk for human dermal exposures. However, there are two caveats: 1) The ip treated males were mated 7-11 d after the end of treatment while the dermal treated males were mated 1.5-8.5 d after the end of treatment, i.e. the exposed germ cell stages were overlapping and not identical, and 2) the dermal exposed males were not prevented by collars to lick some of the solutions applied to their backs and, therefore, may have been exposed dermal as well as orally, the latter to an unknown extent. Yet, AA-workers in Chinese family-run factories without any hygienic measures are exposed also through the skin and by ingestion. The extrapolation from linear dose response at high short-term experimental exposure to chronic human exposure with low doses is a matter of debate and ideally should be verified experimentally. Unfortunately, no animal facility is large enough to allow the appropriate experiments. Thus, using the correction factor for ip vs. dermal exposure will provide as close a risk estimate as we can presently supply.

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DNA Damage and Repair (Joint Symposium with DNA Repair Network) Sequence selectivity of carcinogen-DNA binding and its role in determining p53 and ras mutational spectrum in human cancer Tang, Moon-shong Department of Environmental Medicine, New York University School of Medicine, Tuxedo, New York 10987, USA.

Tumor suppressor gene p53 and protooncogene K-ras are two of the most frequently mutated genes in human cancer. Although more than two hundred mutation sites in the p53 gene have been found in human cancer it appears that different cancers have different p53 mutational hotspots. For example, codons 157, 158, 175, 245, 248, 273 and 282 are the mutational hotspots for tobacco related lung cancer, codon 249 is the sole mutational hotspot for liver cancer, and codons 280 and 285 are the two unique mutational hotspot for bladder cancer. Similar situation was also found in ras gene family. Although mutations at codons 12, 13, and 61 in any of ras genes can induce their oncogenic function, mutation at codon 12 of K-ras gene is the predominant ras mutations found in human cancer. These results suggest that targeted DNA damage may determine p53 and ras mutational spectrum in human cancer. To test this hypothesis, using UvrABC incision method in combination with ligation mediated PCR, we have mapped the DNA adduct distribution at nucleotide level in p53 and ras genes in human cells treated with well established cancer etiological agents for lung, liver and bladder cancers. We have found that cancer etiological agents preferentially form DNA adducts at p53 and K-ras mutational hotspots. Furthermore, DNA adduct formed at these mutational hotspots are poorly repaired. These results provide evidence directly linking DNA damage to human cancer. The Role of BRCA1 in ubiquitination, Fanconi’s anaemia and the repair of DNA damage Kevin Hiom1, Cassandra J. Vandenberg1, Fanni Gergely2, Chong Yi Ong1, Paul Pace1, Donna L. Mallery1 & K.J. Patel1 1MRC Laboratory of Molecular Biology, Cambridge, UK. 2Wellcome/Cancer Research UK Institute, Cambridge, UK. Although loss of the tumour suppressor BRCA1 results in profound chromosomal instability the fundamental defect underlying this catastrophic phenotype is not yet known. In vivo BRCA1 exists in a heterodimeric complex with the BARD1 protein. Interestingly both these proteins contain a RING domain, which has recently been implicated for a role in the ubiquitination pathway. We have now purified BRCA1 and BARD1 as a complex and demonstrated that together these proteins have E3 ubiquitin ligase activity, catalysing two distinct types of ubiquitin modification. Firstly BRCA1/BARD1 mediates the monoubiquitination of nucleosome core histones in vitro, including the variant histone H2AX that co-localises with BRCA1 at sites of DNA damage. Secondly, it catalyses the formation of multiple polyubiquitin chains on itself. Strikingly a single point mutation in the RING domain of BRCA1, which is known to cause a predisposition to breast cancer, abolishes the E3 ligase activity of BRCA1/BARD1. Fanconi anaemia is an autosomal recessive cancer susceptibility syndrome characterized by congenital abnormalities, progressive bone marrow failure and cells that are extremely sensitive to DNA crosslinking agents such as mitomycin C. Monoubiquitination of the FANCD2 protein is a key step in the Fanconi anaemia (FA) tumour suppressor pathway, coinciding with this molecule¹s accumulation at sites of genome damage. Strong circumstantial evidence points to a requirement for BRCA1 in this step. The role of BRCA1 in the repair of DNA damage and particularly the role of its E3 ubiquitin ligase activity in the FA damage response pathway will be addressed.

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DNA damage and its impact on cancer and aging: lessons from mouse repair mutants and human syndromes Jan Hoeijmakers Repair of alkylated DNA bases by E. coli AlkB and human DNA-dioxygenases B. Sedgwick, P. Koivisto, S.C. Trewick, T. Duncan, and T. Lindahl Cancer Research UK London Research Institute, Clare Hall Laboratories, Potters Bar EN6 3LD, UK.

Diverse DNA repair mechanisms have evolved that remove cytotoxic and mutagenic DNA damage induced by alkylating agents. Methylpurine-DNA glycosylases excise 3-methyladenine in the first step of base excision repair, whereas suicidal O6-methylguanine-DNA methyltransferases directly demethylate O6-methylguanine by methyl transfer on to themselves. We have recently resolved a third type of DNA repair mechanism specific for alkylated bases. The E. coli AlkB protein, and its human homologues, ABH2 and ABH3, oxidise the methyl groups of 1-methyladenine and 3-methylcytosine in DNA to directly regenerate adenine and cytosine. The methyl adduct is released as formaldehyde. These enzymes are members of the α-ketoglutarate Fe(II)-dependent dioxygenase superfamily, and couple oxidative demethylation of the damaged bases to decarboxylation of α-ketoglutarate. The lesions processed by AlkB are formed mainly in single stranded DNA, and are consequently predicted to arise in replication forks and transcription bubbles where, if not repaired, they could block DNA and RNA polymerases. In E. coli, all three types of activities that repair alkylated DNA bases are induced up to 1000-fold as part of the adaptive response to alkylating agents. Short oligonucleotides containing defined single adducts are useful as substrates in kinetic, mechanistic and structural studies of DNA repair enzymes. We have examined the ability of AlkB, and human ABH2 and ABH3, to repair 1-meA in short oligodeoxynucleotides. We have defined DNA substrate characteristics that influence recognition and efficiency of repair, and have identified the minimal substrate that is demethylated by AlkB. To extend the substrate range of AlkB to other alkyl adducts induced by environmental agents, we have also examined the ability of AlkB to counteract the adverse effects of small epoxides and larger alkyl halides.

Oxidative DNA damage in lymphocytes and muscle cells of humans exposed to hypoxia Peter Møller and Steffen Loft Inst. of Public Health, Panum, 3 Blegdamsvej, DK-2200 Copenhagen N, Denmark. Lack of oxygen supply relative to the metabolic need (hypoxia) appears paradoxically to be associated with increased production of reactive oxygen species (ROS) and oxidative stress. We have shown that high altitude hypoxia conferred by a stay at 4559 m above sea level was associated with increased steady state level of oxidative DNA damage in circulating mononuclear blood cells and higher urinary excretion of the specific oxidative DNA base damage, 7-hydro-8-oxo-2’deoxyguanine (8-oxodG) (Møller et al., FASEB J 15:1181-1186, 2001). In recent experiments we have investigated the effect of prolonged high altitude hypoxia in human skeletal muscles as a major oxygen-consuming organ, and found increased levels of strand breaks and endonuclease III sensitive sites after two weeks of hypoxia, whereas oxidative DNA damage detected by formamidopyrimidine DNA glycosylase (FPG protein) was unaltered. The expression of 8-oxoguanine DNA glycosylase 1 (OGG1), determined by quantitative RT-PCR of mRNA levels, was not significantly changed during high altitude hypoxia, although the data could not exclude a minor upregulation. These data indicate that oxidative DNA damage can be induced in muscle nuclei similarly to e.g. in lymphocytes and this may be relevant for loss of muscle function during aging. Moreover, high altitude hypoxia may serve as a good model for oxidative stress. Antioxidant genes are not upregulated in muscle tissue by prolonged hypoxia despite increased generation of oxidative DNA damage. Use of the comet-FISH assay to demonstrate DNA repair in selected gene regions B. Doherty 1, D.McKenna1, N. F. Rajab1, S. McKeown2, S. Downes1, and V. McKelvey-Martin1 1 Cancer and Ageing Research Group, School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, UK, 2 School of Applied Medical Science And Sports Studies, University of Ulster, Jordanstown, Northern Ireland, UK. The alkaline Comet assay (ACA) can be used to quantify DNA damage and repair in individual cells. In order to observe DNA damage and repair in transcriptionally active gene domains, this technique can be combined with fluorescent in situ hybridization (FISH). The Comet- FISH assay uses fluorescently labelled DNA probes which hybridize to specific gene sequences. In this way, the location of hybridization spots within the comet head or tail indicates whether or not the gene of interest lies within, or near to, a region of damaged DNA. In this study, we have used the Comet-FISH assay to demonstrate preferential repair in the actively transcribed p53 gene domain relative to the overall genome following

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gamma irradiation. Bladder cancer cell lines were processed for the ACA using standard protocols. For all cell lines, cells were irradiated at 5Gy and allowed to repair for 0, 15, 30 and 60 minutes. Slides were then dried for FISH. At each time point, the number of hybridization spots and their location within the comet head or tail was recorded in order to ascertain if the region of interest was damaged. Irradiated cells showed more spots than control cells with the majority in the tail. As repair time increased, the number of cells showing a high number of spots was reduced with the majority of spots moving back into the comet head by 15 min repair suggesting preferential repair in the transcriptionally active p53 domain relative to overall genomic DNA repair. To examine this further, cells known to be deficient in preferential repair are also being evaluated using the Comet-FISH assay for evidence of a deficiency in this process. Repair kinetics of interstrand DNA crosslinks: Implications for the Fanconi anemia/BRCA pathway Andreas Rothfuss and Markus Grompe Department of Molecular and Medical Genetics; Oregon Health & Science University, 3181 SW Sam Jackson Park Rd., Portland OR 97239; USA. The detailed mechanisms of DNA interstrand crosslink (ICL) repair in human cells are not known, but current models suggest that recognition and repair of ICL occur primarily during the S-phase of the cell cycle. Here we provide evidence for a refined model in which ICL are recognized and rapidly incised independent of DNA replication. The use of a modified comet assay protocol enabled us to investigate the fate of ICL under physiologic conditions, which are compatible with cell survival and repair. Our results show that primary human fibroblasts efficiently incise photoactivated psoralen-induced ICL even after arrest in G1 of the cell cycle. Quantitative analyses of γH2AX foci formation indicated that DNA-double strand breaks (DSB) form as intermediates during ICL repair in primary human cells. Our data show that DSB occur exclusively in S-phase, possibly when a replication fork becomes arrested at an incised ICL. The efficient formation of DSB is dependent on a preceding incision event, since ERCC1 mutant cells show a significantly reduced and delayed formation of DSB. In contrast, Fanconi Anemia (FA) primary fibroblasts from different complementation groups are fully proficient in the sensing and incision of ICL as well as in the subsequent formation of DSB. We therefore conclude that the FA/BRCA pathway acts downstream in ICL repair, most likely during the resolution of DSB in S-phase. Supported by a grant of the Deutsche Forschungsgemeinschaft (DFG) and NHLBI Program Project Grant 1PO1HL48546. Nucleotide excision repair modulation by two components of the SAGA/ADA chromatin remodeling complex, Gcn5 and Ada2 proteins, in the MET16 gene of Saccharomyces cerevisiae J.A. Ferreiro1,2, N.G. Powell1, N. Karabetsou3, J. Mellor3 and R. Waters4. 1) School of Biological Sciences, University of Wales Swansea, Swansea SA2 8PP, UK. 2) Present Address: Area de Genética. Dpto Biología Funcional e Instituto Universitario de Oncología del Principado de Asturias (IUOPA). University of Oviedo. 33006 Oviedo. Spain. 3) Microbiology Unit, Department of Biochemistry, Oxford University, Oxford OX1 3QU, UK. 4) Department of Pathology, University of Wales College of Medicine, Cardiff CF14 4XN, UK. Eukaryotic genomes are folded into nucleosomes and higher order chromatin structures. This compaction reduces the accessibility of enzymatic activities to the DNA, and alters the activity of important cellular processes like transcription, replication and DNA repair. Accessibility to DNA folded into nucleosomes is regulated by ATP-dependent remodeling complexes and by histone acetyltransferases (HATs) and deacetylases. Among the different histone acetyltransferases found in Saccharomyces cerevisiae, Gcn5p has been shown to be required for normal expression of 5 to 20% of the genes. Gcn5p was initially identified as a transcriptional activator necessary to promote maximum levels of Gcn4p-dependent transcription and has been found to be part of two large chromatin remodelling complexes, ADA and SAGA. The transcriptional adaptor Ada2p, another component of the ADA and SAGA complexes, has been shown to interact with Gcn5p and to be required to recruit TATA Box-binding Protein to Gcn5 dependent promoters. Recently, yeast cells deficient for the histone acetyltransferase Gcn5p were found to be impaired in nucleotide excision repair (NER) of CPDs present in the transcribed and non-transcribed strands of the yeast MFA2 gene, but not in repair of the genome overall. We have taken advantage of the dependence on Gcn4p for transcription of MET16 gene to study how the rate of DNA repair by NER at nucleotide resolution is influenced by the absence of Gcn5 or Ada2 proteins. This was studied in both strands of the MET16 promoter and the transcribed region in relation to the transcriptional activity of MET16 (repressed and derepressed). Finally, we also determined how deletion of the GCN5 or ADA2 genes modifies MET16 chromatin structure in either transcription condition.

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Our results indicate that, although MET16 transcription is induced in both gcn5∆ and ada2∆ strains when cells are grown in derepressing conditions, both proteins are required to obtain the full induction of MET16. On the contrary, deletion of the GCN5 or ADA2 genes showed little or no influence on MET16 chromatin structure. In relation to DNA repair, we found that the MET16 transcription level has an important influence on the rate of CPD removal. This influence was dependent on the strain, but also on the DNA strand and region analyzed. In general, most CPDs induced in the transcribed strand of the wild type and ada2∆ strains were repaired faster in derepressing than in repressing conditions, while in the gcn5∆ strain the reverse trend was found, most CPDs were repaired faster in repressing conditions. Our experiments also demonstrate that Gcn5 and Ada2 proteins are required in the two transcription conditions for efficient repair of CPDs present in both DNA strands. In derepressing conditions, CPDs in the transcribed strand were less efficiently repaired in both mutant strains. This deficiency was even more evident in the non-transcribed strand, where most CPDs were poorly repaired. In repressing conditions repair in the transcribed strand, downstream of the TATA-box, was delayed by 1.5 to 2 hours in the gcn5∆ and ada2∆ strains. In the three strains analyzed, the rate of repair along the non-transcribed strand was characterized by a wave pattern with three regions of fast repair corresponding approximately to internucleosomal regions. In this strand deletion of GCN5 or ADA2 had less influence than in derepressing conditions. The data will be discussed in light of current models relating nucleotide excision repair and chromatin structure.

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Genotoxic and Carcinogenic Effects of Mineral Fibres Genotoxicity and carcinogenesis of mineral fibres S.A. Kyrtopoulos1 and C. Searle2 1 National Hellenic Research Foundation, Athens; 2 European Commission, Directorate General Research, Brussels. Despite the now almost complete asbestos ban, concerns over the health consequences of asbestos fibres continue, given such fibres are likely to remain present in the human environment for many years in the future. An additional cause for the continued public health concern is related to the increasing use of asbestos-substitute fibres, whose toxicity is still not fully understood. The Workshop on Genotoxicity and Carcinogenesis of Mineral Fibres aims to review recent progress regarding the mechanisms of fibre toxicity, focussing in particular on aspects of relevance to genotoxicity and carcinogenesis. Both direct and indirect pathways to fibre genotoxicity are recognised. Direct genotoxicity can arise via Fenton-type reactions of fibre-associated metals (leading to the production of reactive oxygen species which in turn initiate genetic damage), as well as via direct interaction of fibres with the mitotic apparatus. On the other hand, the well-recognised ability of biopersistent fibres to induce chronic inflammation can serve as an indirect pathway to the induction of oxidative stress, genotoxicity and cell proliferation. While both mechanisms are known to play a role in fibre toxicity, their relative contribution to the in vivo effects of different fibres and at different doses is still inadequately understood, an uncertainty which has important consequences for low-dose extrapolation and risk assessment. Resolution of this uncertainty can come from improved mechanistic understanding based on in vitro and in vivo studies, and the Workshop will focus on recent progress in this front. Another focus of the Workshop is fibre-induced immune toxicity, which may play an important role not only in the progression of carcinogenesis but also in the induction of other fibre-related disease. In addition to the need for increased understanding of the molecuar mechanisms of fibre toxicity, in view of the continued human exposure to fibres there is need for improved biological monitoring of exposed populations. As in other areas of molecular epidemiology, the use of biomarker methodologies holds a promise of refined assessment of individual exposure, early detection of biological effects and the recognition of genetically susceptible individuals. In contrast to other types of environmental genotoxins, past work on the molecular epidemiology biomarkers of mineral fibre toxicity has been relatively limited, and recent progress in this area will also be reviewed in the Workshop. Finally, the current state of affairs regarding the classification of mineral fibres for carcinogenicity, and the setting of occupational exposure limits, will be reviewed. Perhaps most importantly, the qualitative and quantitative uncertainties affecting these important activities, and the knowledge gaps from which they result, will be discussed and relevant research priorities identified. The Workshop on Genotoxicity and Carcinogenesis of Mineral Fibres is organised in the context of a Research Project funded by the European Union ("Mechanisms of toxicity of asbestos-substitute mineral fibres: New approaches to hazard and risk assessment" – FIBRETOX). It is partly funded by the European Union (contract no. QLK4-CT-99-01629). The toxicology of fibres: an overview K. Donaldson ELEGI Colt Laboratory, Centre for Inflammation Research, Medical School, University of Edinburgh. Fibres comprise a chemically and structurally heterogeneous group of materials that are defined on a regulatory basis by their shape. This criterion is not health-based and takes no account of composition, which is key to determining heterogeneity in the pathogenicity of fibres. The broad range of fibre types in use and the continued development of new fibre types with new properties is a challenge for the regulatory and toxicology communities. Crocidolite, chrysotile, tremolite, amosite, actinolite and anthophyllite asbestos have been evaluated by IARC as proven (Group 1) carcinogens. IARC has also evaluated glasswool, continuous glass filament rockwool/stonewool and slagwool, para-aramid fibres and wollastonite fibres as having inadequate evidence of carcinogenicity in humans but have classified refractory ceramic fibres and special purpose glass fibres (such as E glass) as possible human carcinogens. Glass wools and rock/slag wools show no carcinogenic effects in humans or rats but inflammatory and fibrotic effects have been documented at high exposure. Refractory ceramic fibres have produced pathogenic effects in rats and mesotheliomas in hamsters; there are reports of pleural plaques in some human populations exposed to RCFs, but no mesotheliomas as yet. There also have been isolated reports of tumours with various fibres, even non-biopersistent ones following high-dose instillation into the rat peritoneal cavity. However this assay has been criticised as not being relevant to the evaluation of risk. The dominant ‘long/respirable/biopersistent’ paradigm of fibre pathogenicity is based, almost exclusively, on data derived from silicate fibres. It is important therefore to highlight that non-silicate fibres such as organic fibres may not fit under this paradigm. Fibres have an aerodynamic diameter that is 1.5 to 3 times their actual diameter depending on their density and length and very long fibres can penetrate deeply into the lungs, provided that they are thin. Such fibres can penetrate beyond the ciliated airways to where they present the alveolar macrophage system with problems of effective

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phagocytosis and clearance. Slow clearance of long biopersistent fibres is well documented. Short fibres can be effectively phagocytosed but macrophages have difficulty in completely enclosing long fibres and frustrated or partial phagocytosis can result in chronic stimulation of the macrophages leading to inflammation and failure of clearance. However, under high exposure conditions, short biopersistent fibres can overload clearance mechanisms and may be pathogenic. Asbestos fibres can act as direct or indirect carcinogens. There is abundant evidence that asbestos fibres and some man-made fibre types can generate free radicals by different mechanisms, including hydroxyl radical by Fenton chemistry. It is hypothesised that these radicals can form DNA adducts such as 8-OH-DG that are mis-repaired giving rise to mutations. Macrophages and polymorphonuclear leukocytes (PMN) recruited to the lungs can release an array of oxidants and mitogens that could cause proliferation and produce adducts, oxidised bases, single- and double-strand breaks and DNA cross-links; a combination that can lead to carcinogenesis. Cigarette smoke acts as a co-factor in asbestos carcinogenesis and SV40 may act similarly. Oxidative stress and genotoxicity by mineral fibres: In vitro studies P. Georgiadis National Hellenic Research Foundation, Athens. Inflammation is believed to play an important role in the mechanism of fibre-induced lung disease, including cancer, where it may be an indirect pathway to genetic damage via the production of reactive oxygen species and the induction of cellular proliferation. On the other hand, direct interaction of fibres with epithelial cells may constitute a second pathway to genetic damage and oncogenesis. Hence the examination of the ability of fibres to modulate inflammation- and oxidative defence-related genes in lung cells may provide useful information for understanding the mechanistic basis of fibre genotoxicity and carcinogenicity. In this context, the effects of for 4 mineral fibres (amosite, rockwool, glasswool, wollastonite) on expression of several inflammation-related genes (TNF-α, IL-1α and IL-6), and oxidative-stress related genes (MnSOD, iNOS and COX-2), were studied in vitro using primary cells from Fisher 344 rats: a) Alveolar macrophages (AM). These cells give the initial signal for induction of inflammation, and the effect of different fibers in them should reflect their ability to initiate the inflammation process. b) Type II alveolar epithelial cells. The effect of fibers in this system should reflect alterations of relevance to the direct mechanism of fiber carcinogenesis. c) Type II epithelial cells, in no-contact co-culture with AM stimulated with fibers. The effects in this system should reflect the indirect pathway to fiber toxicity. The order of induction of the expression of most oxidative stress related genes in AM, was amosite = rockwool > glasswool > woolastonite. Similarly, following direct treatment, mRNA levels for TNF-α, IL-1α and IL-6 were significantly enhanced in type II cells and AM with all fibers tested, in the order amosite > rockwool > glasswool > woolastonite, although the magnitute of induction was higher in AM. On the other hand, amosite treatment of type II cells resulted in a 5-fold increase in iNOS mRNA levels, while the increase induced by the other three fibers was only about 2-fold. COX-2 mRNA levels were increased to a similar degree by all fibers. Using the co-culture system, we were able to demonstrate the effects on type II cells as a result of fiber-mediated stimulation of AM: The expression of the cytokines TNF-α, IL-1α and IL-6 in type II cells was induced only after exposure of AM to amosite or rockwool, the magnitute of induction being similar for both fibers. Similarly, iNOS levels were highly induced by amosite and rockwool treatments of AM, but only moderately by glasswool and woolastonite. Finally, the induction of COX-2 followed the order amosite > rockwool > glasswool. Woolastonite gave no induction. The above results must be assessed in the context of the inflammatory and genotoxic effects of the same fibres in vivo. In this light, it is concluded that differences in the direct or indirect induction of iNOS and COX-2 expression in type II epithelial cells may play a significant role in the determination of the relative genotoxic potency of the different fibres in vivo. This work was funded by the European Union (project FIBRETOX, contract no. QLK4-CT-99-01629)

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Induction of inflammation, oxidative stress and genotoxicity in the respiratory system J.Topinka1, M.Hurbáková2, P.Loli3, P.Georgiadis3, Z. Kováčiková2, K.Volkovová2, E.Tatrai4, M.Dušinská2, T.Wolff1, and S.A. Kyrtopoulos3

1GSF-Research Center for Environment and Health, Institute of Toxicology, Neuherberg, Germany 2National Hellenic Research Foundation, Athens, Greece, 3Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic,4Fodor Jozsef National Center of Public Health, Budapest, Hungary. The potential of asbestos fibres (amosite) and two asbestos substitute mineral fibres (rock wool RW1 and glass wool MMVF10) to induce inflammation, oxidative stress and genotoxicity has been studied in rats. To analyze the induction of gene mutations by fibres in lung, male homozygous λlacI transgenic F344 rats were intratracheally instilled with single doses of 1 and 2 mg/animal of fibres or with multiple dose of 2 mg/animal administered weekly on 4 consecutive weeks. Subchronic exposure to amosite and rock wool RW1 for 16 weeks significantly increased mutation frequency (MF) in the lung in a dose-dependent manner, while glass wool MMVF10 did not exhibit any increase of MF at any dose. Rock wool fibres gave a significant increase of MF already at a dose of 1 mg, while amosite fibres induced an increase of MF at a dose of 2 mg indicating a stronger mutagenic effect of rock wool RW1. Four weeks after instillation, neither the single nor the multiple doses significantly increased MF for all 3 fibre types . To investigate mechanisms for induction of mutations, other genotoxicity markers and parameters of inflammatory and oxidative damage were determined in relation to MF. For this purpose wild type F344 rats were treated by the same regimen as transgenic rats with a single dose of 2 mg and a multiple dose of 4x2 mg/animal of fibres. A weak correlation of mutagenicity data with other genotoxicity parameters studied was observed: amosite fibres induced micronuclei in alveolar macrophages but not in lung epithelial cells, rock wool fibres did not induce micronuclei at all, while glass fibres, which were not mutagenic in the lung of transgenic rats, form micronuclei in both cell types studied. DNA strand breaks as measured by Comet assay were increased in alveolar macrophages and lung epithelial cells of amosite treated rats. Histological findings revealed that amosite treatment caused the highest extent of tissue damage. Amosite fibres also caused the most extensive lung inflammation as measured by release of neutrophiles into BAL (broncho- alveolar lavage). The effects were maximal 16 weeks post-exposure, indicating a progression of the pathogenic process during the exposure period. Minor differences in the extent of inflammatory processes were observed between the doses of 2 mg and 4x2 mg, suggesting that any threshold for inflammation lies below the dose of 2 mg. Rockwool RW1 gave rise to less intense inflammatory changes in the lung after the higher dose of 4x2 mg. Treatment with glass fibres caused little inflammatory changes. All 3 fibres studied exhibited a low extent of oxidative damage as measured by levels of malondialdehyde in BAL and lung tissue. The changes in antioxidant enzyme activities (SOD, GSHPx) were also weak. We conclude that a weak but chronic inflammation in the lung tissue of fibre treated rats characterized by moderate influx of inflammatory cells into BAL and by a weak oxidative damage is probably responsible for the observed mutagenic effect of amosite and rock wool RW1 fibres. Glass wool MMVF10 did not cause inflammation in the lung of rats and did not exhibit any mutagenic effect. Thus, moderate but chronic inflammation, more likely than acute inflammation or direct oxidative damage, may be the cause of fibre induced gene mutations in the lung. In an attempt to examine the interaction of various fibres with benzo[a]pyrene (B[a]P) as a model compound for exposure to cigarette smoke, λlacI transgenic rats were treated intratracheally by fibres as described above and simultaneously by B[a]P (10 mg/animal). Benzo[a]pyrene administered simultaneously with amosite or rock wool fibres exhibited strong synergistic effect on mutagenicity, the observed MF being much higher than the sum of the MF induced after separate administration of both agents. Our data suggest that DNA damage by B[a]P adducts together with cell proliferation induced by simultaneous B[a]P and fibre treatment leads to the strong increase of mutation frequencies. The work was supported by an E.U. grant, contract No. QLK4-CT-1999-01629. Induction of inflammation, oxidative stress and genotoxicity in mesothelial tissues K. Unfried, C. Schürkes and J. Abel Institut für umweltmedizinische Forschung (IUF) at the Heinrich-Heine-University Düsseldorf, Germany. Besides tumour promoting effects of fibres, genotoxicity and mutagenicity are proposed to be involved in fibre induced carcinogenesis. As possible molecular mechanisms of this mutagenesis, direct effects mediated by transition metals leaching from the fibres as well as clastogenic effects of fibres penetrating the nucleus are discussed. Indirect effects may be induced by the inflammatory response on fibre deposition in the target tissue. Inflammatory cells are able to induce reactive compounds which may result in DNA damage. In order to get indications for the relevance of these molecular mechanism in vivo we performed animal studies using the system of intra-peritoneal injection in rats, a model system for mesothelioma carcinogenesis in vivo. Treatment of animals with UICC crocidolite asbestos as well as with the positive control substance benzo[a]pyrene revealed significant increases in mutation frequencies in omenta of transgenic rats bearing bacterial reporter genes for mutagenesis testing. Molecular analyses of the mutations induced by crocidolite revealed a specific mutation spectrum different from spontaneous mutations. The most prominent mutation types were G to T transversions which are known to be induced by 8-hydroxydeoxyguanosine (8-OHdG) and small deletions which may result from double strand breaks induced by radicals. The induction of the pre-mutagenic DNA adduct 8-OHdG by fibres was measured in DNA of target tissues from animals treated with several different types of fibres. In crocidolite treated animals, significant increases of 8-OHdG compared to controls were observed. The number of macrophages in peritoneal lavages and the activation of these macrophages

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measured by TNF-α expression of these cells revealed comparable increases in signal strength. Animals treated with equal numbers of MMVF 11 fibres which are known to be much less carcinogenic, probably due to the low bio-durability, exhibited at these time points similar amounts of 8-OHdG and TNF-α as crocidolite treated animals. Crocidolite fibres which had been depleted of iron by pre-treatment with iron chelating agents as well as MMVF 11 fibres which contain very low amounts of iron did not induce inflammatory parameters or 8-OHdG levels significantly different from the levels induced by crocidolite. Therefore, iron as transition metal provided by the fibre seems to be of minor importance in this pathway of mutagenicity. From these data we conclude that crocidolite fibres are able to induce mutations via molecular mechanism significantly different from those inducing spontaneous mutations in vivo. Due to typical G to T transversions and increased 8-OHdG levels, this pre-mutagenic DNA adduct is proposed to be important for this mechanism of mutagenesis in vivo. The analyses of inflammatory parameters indicate that the induction of 8-OHdG is mainly linked to fibre induced inflammation. Mineral fibres and immunotoxicity. J. Tulinska1, M. Kuricova1, A. Liskova1, E. Jahnova1, M. Dusinska1, S. Ilavska1, M. Horvathova1, M. Hurbankova1, L. Wsolova1, S. A. Kyrtopoulos2 and L. Fuortes3 1 Institute of Preventive and Clinical Medicine, Bratislava, Slovakia, 2 Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece, 3 University of Iowa, College of Public Health, Iowa City, Iowa, USA Asbestos is a well-known cause of both benign and malignant lung disease (interstitial and pleural fibrosis, lung cancer and mesothelioma). Since a deranged immune system may play a role in cancer development, the general level of immunocompetence was studied in workers exposed to mineral fibres (amosite asbestos, rockwool and glass fibres). Our findings indicate that one of the immune targets of mineral fibre exposure seems to be specific cellular immunity. Using immune function assays, a significant effect of mineral fibres was found on the function of lymphocytes in all three worker populations examined. Workers from a former asbestos cement plant had significantly decreased proliferative capacity of lymphocytes upon stimulation by T-cell mitogen. Spearman correlation analysis revealed decreasing proliferative response of T and B-lymphocytes in the exposed population with age. While a suppressive effect on T-cell immunity was observed in the asbestos cement plan population, the proliferative activity of T-lymphocytes in people from rockwool and glass fibre factories was enhanced. Phagocytic activity plays an important role in the pathogenic processes underlying asbestos-induced lung damage. Surprisingly, in contrast to a marked suppressive effect of mineral fibres on the activity of phagocytes, observed in animal studies, no dramatic influence was found in worker populations. Only a statistically significant deterioration of phagocytic activity of monocytes was observed among smoking workers exposed to asbestos, in comparison with exposed non-smokers. Natural killer cells are a unique lymphocyte population, with the ability to rapidly lyse tumor cells independently of major histocompatibility complex gene products, and are thought to be the first line of defence against cancer cells. Phenotypic analysis of peripheral blood leukocytes showed significantly decreased expression of markers CD16+56 (natural killer cells) in people exposed to glass fibres in comparison with factory controls. Natural killer cell activity did not differ among exposed and controls even in the population exposed to asbestos (assay in glass fibre workers not done). Inflammatory cytokines are rapidly induced and early expressed in a disease or injury process. They mediate and modulate healing processes but, if overexpressed, may exacerbate the severity of a disease condition. People exposed to asbestos had significantly increased concentrations of IL-6. Similarly, significantly elevated concentrations of IL-8 were found in workers exposed to all three types of fibres. Pathologically relevant increases in the expression and function of adhesion molecules have been observed in humans with such pulmonary disease/conditions as bronchial hyperreactivity, allergic rhinitis, idiopathic pulmonary fibrosis or neoplasia. In workers from a former asbestos cement plant the expression of adhesion molecule CD62L (L-selectin) on monocytes and granulocytes was significantly increased. Increased levels of soluble adhesion molecules ICAM-1 were found in people who worked with asbestos and rockwool. Exposure to glass fibres enhanced the level of soluble E-selectin in workers’ sera. Increased levels of immunoglobulin E (asbestos and rockwool) and expression of marker CD66b on eosinophils in all three groups of workers indicates a status of hypersensitivity in the exposed populations. This work was supported by the European Union (project FIBRETOX, contract no. QLK4-1999-01629), NIEHS Grant # 510205240 00000 and Slovak Grant Agency for Science # 04.92.11.08.

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Biomarkers of effects of mineral fibres in exposed humans M. Dušinská, K. Volkovová, M. Staruchová, A. Horská, M. Barančoková, A Kažimírová, Z. Džupinková, B. Smolková, J.Tulinská, E.Jahnová, L. Wsólová, A. Kočan, J. Petrík, A. Collins2, V. Harrington2, B.Ratcliffe2, S. Kyrtopoulos3 1Institute of Preventive and Clinical Medicine, Bratislava, Slovakia, 2Robert Gordon University, Aberdeen, Scotland, 3National Hellenic Research Foundation, Athens, Greece. A molecular epidemiological study was conducted in 3 factories in Slovakia, producing asbestos, glass fibres and rockwool. Altogether 387 subjects (239 exposed, 148 controls) were investigated. Mineral fibre as well as PAH exposure was measured four times a year (including sampling time) in all three factories. Since the asbestos factory had stopped the production of asbestos, personal dosimetry was carried out in the time of sampling only in the rockwool and glass fibre factories. The levels of asbestos in the asbestos factory exceeded the Slovak occupational limit (0.001 fibre/cm3) by 3-5 times. Only low levels of basalt glass fibres were detected in the rockwool and glass fibre factories (10 to 1000 times below the Slovak limit). PAH contamination was also very low: individual PAH congener levels in the occupational atmosphere ranged from tenths to several hundred mg/m3. Biomarkers of exposure, effect and individual susceptibility were measured in lymphocytes: DNA damage (strand breaks [SBs], base oxidation and alkylation, using the comet assay); micronuclei and chromosome aberrations; genetic polymorphisms of xenobiotic-metabolising and DNA repair enzymes; and individual DNA repair capacity in lymphocyte extracts. Intrinsic antioxidants, antioxidant enzymes, humoral and cellular immune markers, growth factors and proinflammatory mediators were also measured. Exposed asbestos workers had significantly higher numbers of chromosomal aberrations (P=0.005) compared with controls. Associations between DNA damage, exposure, smoking and sex and interaction of exposure with smoking habit were found in all three factories. Asbestos-exposed non-smoking (NS) men had more DNA damage (SBs) compared with exposed NS women (P=0.02). Exposed men had higher level of oxidative DNA damage (net endonuclease III-sensitive sites) than controls (P=0.04). There was also a clear association between oxidative damage and length of exposure in this group (P=0.01). The rockwool-exposed workers had higher level of oxidative DNA damage (P=0.029) compared with controls. Exposed NS had higher SBs than control NS (P=0.004). Control smokers had higher DNA damage than NS (P=0.043). The level of oxidative DNA damage is dependent on age and duration of exposure (P<0.0001). The group exposed to glass fibres had elevated numbers of DNA breaks (P=0.014), and oxidative DNA damage (P<0.01). Associations between DNA damage, exposure, smoking, sex and genotype were found in all three populations. In the asbestos-exposed group, people with variant GSTP1 allele have higher levels of DNA damage (P=0.001). An association between GSTT1 and DNA repair was also found (P<0.05). In the rockwool factory, we saw associations of SBs with exposure, EPHX4 and smoking (P=0.035), and also between exposure and smoking (P=0.035). There was an interesting inverse correlation between DNA repair rate in oxidative damage and micronuclei especially in the rockwool factory. Our results partially support the hypothesis that oxidative damage is involved in the mechanisms of toxicity of mineral fibres. Funded by the EC (contract no.QLK4-CT-1999-01629) and by bilateral Slovak-Greek project. Man-made vitreous fibres: evaluation of cancer hazards Robert A Baan Unit of Carcinogen Identification and Evaluation WHO - International Agency for Research on Cancer, Lyon, France. Man-made vitreous fibres in the form of wools are widely used in thermal and acoustical insulation and in other manufactured products in Europe and North America. These products, including glass wool, rock (stone) wool, and slag wool, have been in use for decades and have been extensively studied to establish whether fibres that are released during manufacture, use, or removal of these products present a risk for cancer when inhaled. A Working Group invited by the International Agency for Research on Cancer has recently re-evaluated the cancer hazards of man-made vitreous fibres (IARC Monographs volume 81, 2002). Epidemiological studies published during the 15 years since the previous IARC Monographs review of these fibres (IARC Monographs volume 43, 1988) provide no evidence of increased risks for lung cancer or mesothelioma (cancer of the lining of the body cavities) from occupational exposures during manufacture of these materials. In addition, there is inadequate evidence overall of any cancer risk. Much industrial effort has gone into the development of newer materials that have insulation properties similar to those of the older products, but which disappear from body tissues much more rapidly; that is, they are less bio-persistent. Some of these newer materials have now been tested for carcinogenicity. Most are found to be non-carcinogenic, or to cause tumours in experimental animals only under very restricted conditions of exposure. The IARC Working Group made no overall evaluations of possible carcinogenic hazards of these products as there was no satisfactory general way to classify these materials by chemical composition. Furthermore, there were very few data on human exposure.

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The Working Group concluded that only the more bio-persistent materials remain classified as possibly carcinogenic to humans (Group 2B). These include refractory ceramic fibres, which are used industrially as insulation in high-temperature environments such as blast furnaces, and certain special-purpose glass wools not used as insulating materials. In contrast, the more commonly used vitreous fibre wools including insulation glass wool, rock (stone) wool and slag wool are now considered not classifiable as to carcinogenicity to humans (Group 3). Continuous glass filaments, which are used principally to reinforce plastics, are also considered not classifiable as to carcinogenicity to humans (Group 3). References IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 43, Man-made mineral fibres and radon, Lyon, IARCPress (1988) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 81, Man-made vitreous fibres, Lyon, IARCPress (2002) Research needs for the development of Occupational Exposure Limits for Man Made Mineral Fibres (MMMFs) Ziegler-Skylakakis K. European Commission, Unit D5 Health Safety and Hygiene at Work, DG Employment and Social Affairs, Luxembourg. Occupational Exposure Limits are an essential tool in the control of exposure to hazardous chemical agents. Thereby minimising the associated occupational diseases. The setting of OELs together with other associated measures form an essential part of the European Community's strategy on health and safety at work, upon which the legislative framework for the protection of workers from risks related to chemical agents is based. The European Commission is assisted by the Scientific Committee on Occupational Exposure Limits (SCOEL) in its work of setting OELs for hazardous chemical agents. The procedure for setting OELs requires sufficient information on the toxic mechanisms of an agent to differentiate between thresholded and non-thresholded mechanisms. In the first case a no-observed-effect level can be defined which can be the basis for a derivation of an OEL. In the latter case any exposure is correlated with a certain risk. If adequate scientific data are available, SCOEL determines the risk associated with a series of exposure levels. This can then be used for guidance. Man Made Mineral Fibres (MMMFs) are widely used at different worksites. MMMF products can release air borne respirable fibres during their production, use and removal. According to the classification of the EU system, all MMMF wools are considered to be irritants and are classified for carcinogenicity. EU legislation foresees the use of limit values as one of the provisions for the protection of workers from the risks related to exposure to carcinogens. For this reason SCOEL is currently working on setting OELS for MMMFs. In the following the research needs identified by SCOEL for the development of OELs for MMMFs will be presented.

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Molecular Epidemiology of Cancer (Joint Symposium with Molecular Epidemiology Group) Mutational spectra of individual human cancers M R Stratton1

The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, UK. A major application for the human genome sequence in elucidating oncogenesis will be as a template subserving genome-wide searches for somatic mutations in cancer cell genomes. A full description of changes at the DNA level in cancer cells will require information on all types of abnormality; copy number changes, rearrangements, point mutations and methylation. To begin the process of using a whole genome sequence, we have embarked upon systematic genome-wide searches for small intragenic mutations (base substitutions and small insertions / deletions) in cancer cell lines. We have currently screened for mutations over three megabases of genomic DNA in 20 cancer cell lines. To our knowledge, this is the largest amount of DNA screened in any cancer sample. This screen is beginning to reveal previously uncharacterised patterns of somatic mutations in individual cancers. These patterns may inform us about past exposures or defects in DNA repair. Genetic testing: epidemiological and ethical issues Paolo Vineis 1 1 University of Torino and ISI Foundation, Torino, Italy. The proportion of common cancers directly attributable to single low-penetrance genes is probably modest. Low-penetrance genes modulate the response of individuals to carcinogens, and for this reason they are extremely interesting from a scientific point of view, but are of little use for Public Health purposes, i.e. to reduce the burden of cancer in populations. First, if one examines the proportions of cancers or other diseases attributable to common risk factors, such as smoking, they are much greater than those estimated for low-penetrance genes. Therefore, decreasing environmental exposures, including smoking, is likely to be much more effective than selecting the high-risk individuals. A further complication arises from the fact that there are many genes that contribute to an increased risk of cancer, so that to find “normal” subjects can be rather difficult. For the workplace environment, investigators examined, by simulation analysis, how many workers for jobs with benzene exposure would have to be screened for CYP2E1 activity and NADPH-quinone oxidoreductase (NQO1) alleles, until 1000 "normals" (those without the known susceptible polymorphisms) could be hired. They found that 2500 workers would need to be screened to hire 1000 normal workers and thus prevent one case of benzene-induced cancer. However, this is likely to be an underestimate because the genes involved in the pathway from benzene exposure to leukemia or other diseases are only partially known. While genetic screening is clearly justified in the case of some highly-penetrant genes, particularly when effective preventive or therapeutic measures are available, screening for low-penentrant genes is currently not advisable and cannot be a substitute for primary prevention. Molecular epidemiology of oesophageal cancer 1Wild, C.P.1Olliver, J.R., 1Jolly J., 2Dexter, S., 2,3Chalmers, D., 4Sahay P., and 1Hardie L.J. 1Molecular Epidemiology Unit, and 2Academic Unit of Surgery, Medicine and Anaesthesia, School of Medicine, University of Leeds, 3Gastroenterology Unit, Leeds General Infirmary, Leeds LS2 9JT, 4Pontefract General Infirmary, Wakefield. Barrett’s oesophagus (BE) is a pre-malignant condition predisposing to oesophageal adenocarcinoma (EA). Gastro-oesophageal reflux disease is associated with an increased risk of both BE and EA. Reflux of acid and bile can cause mucosal injury and initiate inflammation possibly resulting in oxidative stress and DNA damage (see Wild and Hardie, Nature Reviews Cancer 2003). This process may contribute to the progression of the pre-malignant BE to dysplasia and carcinoma. In vitro experiments in oesophageal cell lines (Flo-1 and Het-1A) showed that both acidified media and some primary and secondary bile salts at neutral pH and physiological levels can induce DNA damage, measurable in the comet assay. The comet assay was also optimised for the detection of DNA damage (strand breaks and alkali-labile sites) in oesophageal biopsies with and without inclusion of the DNA repair enzyme, Fapy-DNA glycosylase (Fpg). Oesophageal biopsies were collected from BE patients and control subjects (undergoing endoscopy, but without BE). In the BE patients we observed an increased level of DNA damage in Barrett’s epithelium compared to matched squamous epithelium; Barrett’s epithelium (median % tail DNA [1st-3rd quartile]) (25.1% [21.7-29.6%]) compared to matched squamous epithelium (18.6% [16.9-21.4%]). Fpg sensitive sites were demonstrated in both tissue types, at similar levels. DNA damage levels in squamous epithelium were in both BE and non-BE groups. In some BE patients, methylene blue dye is used during endoscopy to improve the targeting of biopsies. It is known that methylene blue photosensitised by

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white light, as occurs during endoscopy, generates oxygen radicals that can induce DNA damage. In biopsies taken before and after chromoendoscopy with methylene blue from a series of 15 patients increases in DNA damage were observed in Barrett’s epithelium in every patient using the optimised comet assay (Olliver et al., The Lancet 2003). The raised levels of DNA damage in Barrett’s epithelium, either resulting from reflux or chromoendoscopy, are consistent with the generation of DNA lesions as a consequence of oxidative stress. This type of damage in the pre-neoplastic BE tissue may contribute to the accumulation of genetic alterations occurring in the progression to EC. Understanding these underlying carcinogenic mechanisms provides a basis for cancer prevention strategies as well as possible identification of biomarkers of disease progression for integration in to endoscopic surveillance programmes for BE patients. Data on altered expression of cell cycle regulatory genes (e.g. cyclin D1 and TP53) in BE are promising in identifying BE patients at highest risk of progression to malignancy (Bani-Hani et al., J. Natl., Cancer Inst., 2000). This work is supported by Yorkshire Cancer Research. Molecular epidemiology of liver cancer: aflatoxin, p53 and hepatitis viruses as a paradigm J. Groopman Johns Hopkins University, Baltimore, MD USA. Aflatoxin B1 has been suspected to contribute to liver cancer since the 1960s, when its potent activity as a carcinogen in many species of animals was first reported. On the basis of these animal studies, extensive efforts have been made to investigate the association between aflatoxin exposure and risk of liver cancer in humans. These studies were hindered by: 1) the lack of adequate data on aflatoxin intake, excretion, and metabolism in people; 2) underlying susceptibility factors such as diet and exposure to hepatitis viruses; and 3) incomplete cancer morbidity and mortality statistics worldwide. These deficiencies provided the impetus to develop biomarker technologies to assess exposure status. Subsequently these biomarkers were applied to human investigations to determine their relationships to risk, and findings revealed the strong interaction between hepatitis B virus exposure and aflatoxin for the development of liver cancer. Hepatitis B viral exposure increases the risk of developing liver cancer by 7 fold. Aflatoxin exposure increases the risk about 3.5 fold, but in an interesting example of the synergy of environmental exposures, when people are both positive for the hepatitis B virus and exposed to aflatoxin through their diets, their risk for developing liver cancer is 60 times that of unexposed people. The development of biomarkers for aflatoxin adducts and the genetic changes that might be induced by aflatoxin, such as codon 249 p53 mutations, have led to not only understanding the role of these agents in HCC etiology, but they have also been used to develop effective chemo-prevention and cancer delay strategies in experimental models that have applicability to human settings. We have hypothesized that reduction of biomarker levels by chemo-preventive agents would be mechanistically related to and therefore predictive of cancer preventive efficacy. While primary intervention strategies to reduce mycotoxin exposures at the post-harvest level may have a significant impact in high exposure populations, they are unlikely to eliminate exposure. In addition, these approaches cannot be targeted specifically to high-risk individuals, e.g., people with chronic HBV infection or other compromised health status. Thus, intervention strategies encompassing chemo-prevention, using compounds, which interfere with the absorption or metabolism of aflatoxins once ingested have been developed. Clinical trials have established “Proof of Principle” outcomes for two chemo-preventive approaches: (1) molecular complexing with chlorophyllin and aflatoxin to block carcinogen bio-availability and (2) induction of phase 2 genes such as glutathione S-transferases (GSTs) with oltipraz to enhance metabolic detoxification and elimination of aflatoxins. Molecular epidemiology of colorectal cancer G. Smith. Biomedical Research Centre, Ninewells Hospital & Medical School, Dundee, DD1 9SY. Colorectal cancer is one of the most common forms of cancer in the Western world, the incidence of which has been associated with a number of dietary and environmental risk factors, including dietary fat and red meat intake. We have investigated the influence of diet and genetic background on susceptibility to colorectal cancer. Our analysis of information obtained from detailed dietary questionnaires administered to more than 400 colorectal cancer patients and matched controls has demonstrated that red meat consumption is a significant risk factor for colorectal cancer (Barrett et al, Carcinogenesis, 24, 275-282, 2003). However, we found no evidence that dietary heterocyclic amine carcinogens, found at high levels in cooked red meat, contributed to increased cancer risk (Sachse et al, Carcinogenesis, 23, 1839-1849, 2002). Cancer risk was not modified by heterocyclic amine exposure, and we saw no differences in matched case-control analysis of polymorphisms in drug metabolising enzymes e.g. CYP1A2 and NAT2 responsible for the metabolism and disposition of dietary heterocyclic amines. In contrast, we found statistically significant differences

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between case and control allele frequencies for genes e.g. CYP1A1 and GSTM1 which contribute to the metabolism and disposition of polycyclic aromatic hydrocarbon carcinogens, found in vegetable oils and at high levels in cigarette smoke. As part of this work, we also performed a detailed characterisation of tumour mutation spectra in APC, Kirsten-ras and p53, the progressive accumulation of mutations in which had previously been implicated in the pathogenesis of colorectal cancer. In contrast to previous models, we demonstrated that only a minority of tumours had mutations in all three genes and that more than 30% of tumours had mutations in only one of APC, Kirsten-ras and p53. In addition, mutations in p53 and Kirsten-ras were rarely found together, suggesting that these mutations lie on alternate pathways of colorectal tumour development. Kirsten-ras mutations appear to be a significant risk factor for both progression and survival (Smith et al, PNAS, 99, 9433-9438, 2002). The heterogeneous pattern of tumour mutations in our patient cohort suggests that multiple alternative genetic pathways to colorectal cancer exist and that the widely accepted genetic model of cancer development is not representative of the majority of colorectal tumours. References Barrett JH, Smith G, Waxman R, Gooderham N, Lightfoot T, Garner RC, Augustsson K, Wolf CR, Bishop DT, Forman D and the Colorectal Cancer Study Group. Investigation of interaction between N-acetyltransferase 2 and heterocyclic amines as potential risk factors for colorectal cancer. Carcinogenesis, 24, 275-282, 2003. Sachse C, Smith G, Wilkie MJV, Barrett JH, Waxman R, Sullivan F, Forman D, Bishop DT, Wolf CR and the Colorectal Cancer Study Group. A pharmacogenetic study to investigate the role of dietary heterocyclic amine carcinogens in the etiology of colorectal cancer. Carcinogenesis, 23, 101-111, 2002 Smith G, Carey FA, Beattie J, Wilkie MJV, Lightfoot TJ, Coxhead J, Garner RC, Steele RJC, Wolf CR and the Colorectal Cancer Study Group. Mutations in APC, Kirsten-ras and p53 – alternative genetic pathways to colorectal cancer. Proceedings of the National Academies of Science (USA), 99, 9433-9438, 2002. Combinations of genetic variants in DNA repair genes enhance risk for non-small cell lung cancer O. Popanda1), CT. Phong1), T. Schattenberg1), D. Butkiewicz 2), A. Risch1), L. Edler3), K. Kayser4), H. Dienemann4), V. Schulz4), P. Drings4), H. Bartsch1) and P. Schmezer1) Departments of 1) Toxicology and Cancer Risk Factors, and 3) Biostatistics, German Cancer Research Center (DKFZ), Heidelberg, Germany; 3) Department of Tumor Biology, Center of Oncology - M. Sklodowska-Curie Memorial Institute, Gliwice, Poland, 4) Thoraxklinik Heidelberg-Rohrbach, Heidelberg, Germany. Lung cancer risk may, in part, be modulated by polymorphisms in DNA repair genes. Specific variants have been reported to be associated with such an increased risk including XPA (-4G/A), XPD (Lys751Gln and Asp312Asn), XRCC1 (Arg399Gln), APE1 (Asp148Glu), and XRCC3 (Thr241Met). These variants are localised in five genes involved in different DNA repair pathways. There is, however, little information on possible combinations of these variants. In an ongoing lung cancer case-control study, we are therefore investigating the effect of these variants alone and in combination with each other. 463 lung cancer cases (among them 204 adenocarcinoma and 212 squamous cell carcinoma) and 460 tumour-free hospital control subjects were investigated using PCR-based restriction fragment length polymorphism assays and melting point analysis of allele specific hybridisation probes (LightCycler, Roche). Odds ratios (OR) adjusted for age, gender, smoking and occupational exposure were calculated. Lung cancer risk was not affected by the XPD (Asp312Asn) or the XRCC1 (Arg399Gln) gene variants. For homozygous individuals carrying the Glu variant of the APE1 gene, a protective effect was found (OR=0.77, P=0.25). Individuals homozygous for the XPA-AA, XPD-751Gln or XRCC3-241Met variants, showed increased ORs (1.53, 1.39 and 1.29, respectively). However, only the risk increase correlated with the XPA variant reached a statistical significance of 0.05. When adjusted ORs were calculated for the adenocarcinoma cases only, enhanced ORs were found for individuals homozygous for XPA-AA (OR=1.62, P=0.05) and XRCC3-Met (OR=1.65; P=0.05). Furthermore, individuals carrying combinations of gene variants in DNA repair genes were investigated for cancer risk. 54 patients and controls were identified which were homozygous for two or three gene variants that exhibited an enhanced OR when analysed separately (i.e. XPA-AA, XPD-751Gln and the XRCC3-241Met). The XPA and the XPD gene are involved in nucleotide excision repair, the XRCC3 gene participates in double strand break repair. ORs were significantly increased when all patients (OR=2.37; CL=1.26-4.48), patients with squamous cell carcinoma (OR=2.83; CL=1.17-6.85) and with adenocarcinoma (OR=3.05; CL=1.49-6.23) were analysed. Combinations of polymorphisms in genes involved in the same repair pathway (e.g.:XPA+XPD or XRCC1+APE1) did not affect lung cancer risk. In conclusion, our results indicate that lung cancer risk is only slightly increased by single sequence variations in DNA repair genes. However, a specific combination of sequence variations markedly enhanced cancer risk. For a more comprehensive characterization of how gene variants affect lung cancer risk, analyses of additional DNA repair gene variants and of specific carcinogen exposures are warranted.

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Characterisation of BCL2-IGH translocation in peripheral blood lymphocytes of healthy individuals: Influence of occupational exposure to pesticides Sandrine Roulland1, Pierre Lebailly1,2, Jérôme Gallois1, Yannick Lecluse1, Didier Pottier1, Christian Bastard3, Michel Henry-Amar1,2 And Pascal Gauduchon1 1 Groupe Régional d’Etudes sur le Cancer (GRECAN) EA-1772, Université de Caen-Basse-Normandie 2 Registre Général des Tumeurs du Calvados, Centre François Baclesse, Caen. 3 Laboratoire de Génétique oncologique, Centre Henri Becquerel, Rouen. The incidence of non-Hodgkin's lymphoma (NHL) increased in most western countries in the last two decades. Farming and exposure to selected pesticides has been proposed as potential risk factors in many epidemiological studies. The translocation t(14;18) is a common rearrangement in NHL involving the BCL-2 oncogene and considered to be an early step in lymphomagenesis. It has also been detected in peripheral blood lymphocytes of healthy individuals. The current study, conducted in Normandy (France), was design to evaluate whether exposure to pesticides has an influence on the frequency and molecular characteristics of BCL2(MBR)-IGH translocation in peripheral blood lymphocytes of occupationally exposed individuals. In our area, pesticide use by agricultural workers follows a seasonal cycle with periods of intensive pesticide use and periods of little or no use. Peripheral blood lymphocytes were then collected from two groups: i) a non-exposed male population (n=33) with samples obtained at two time points: winter and spring ii) selected farmers occupationally exposed to pesticides (males and non-smokers), with an unique sample obtained at the height of pesticide use for 23 individuals or earlier before pesticide use (from 3 to 6 months) for 32 individuals. Exposure data were collected by face to face detailed questionnaire. BCL2(MBR)-IGH translocation was detected using nested PCR associated to a multi-tube approach. Prevalence of farmers exposed to pesticides bearing the BCL2-IGH translocation in peripheral blood lymphocytes, was 67% and 80% outside and during the period of intensive pesticide use, respectively. This was higher than the prevalence observed in the reference group for the same period (48 and 50%). A tendency to elevated frequency of BCL2-IGH translocation in the most exposed farmers (2.9x10-6 vs 6.9x10-6), defined on the period of pesticide use, was found, when no elevation was observed on the same interval of time in the reference group (3.9x10-6 vs 4x10-6). Presence of more than one BCL2-IGH positive clone was frequently observed, with almost the same pattern in both studied populations. No obvious difference was found in the distribution of BCL-2 breakpoint according to the period of pesticide use. Regarding these preliminary results, we could conclude that pesticide exposure has a significant effect on the prevalence of BCL2-IGH translocation. Confrontation between BCL-2/JH translocation characteristics and exposure data is currently ongoing for the farmer group. Cytogenetic biomarkers and metabolic polymorphisms in occupational exposure to diisocyanates Hannu Norppa1, Harriet Wikman1, Sabrina Bernardini1,2, Hilkka Järventaus1, Pekka Ylöstalo3, Katja Kääriä1, Harri Vainio4, Christina Rosenberg1, Ari Hirvonen1, and Claudia Bolognesi2

1 Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Helsinki, 2Istituto Nazionale per le Ricerca sul Cancro, Toxicological Evaluation Section, Genoa, Italy, 3 Department of Epidemiology and Biostatistics, Finnish Institute of Occupational Health, Helsinki, Finland, 4 International Agency for Research on Cancer, Lyon France. Cytogenetic biomarkers were studied in polyurethane foam workers exposed to toluene diisocyanate (TDI; n=17) and 4,4'-methylenediphenyl diisocyanate (MDI; n=56) and in unexposed age- and sex-matched referents (n=70). Exposure levels were higher for TDI than for MDI. The cytogenetic techniques applied included the analysis of chromosomal aberrations (CAs), sister chromatid exchanges (SCEs), and micronuclei (MN) in blood lymphocytes (17 TDI-, 41 MDI-exposed, 56 controls), and MN (studied by using pancentromeric fluorescence in situ hybridization) in buccal mucosa (17 TDI-exposed, 54 MDI-exposed, 64 controls). TDI exposure increased the frequency of lymphocytes with MN and chromatid-type CAs and centromere-negative (C-) MN in buccal cells - especially in non-smokers. MDI exposure was associated with a moderate increase in C- MN in buccal cells among non-smokers and in a slightly elevated SCE frequency: TDI-exposed workers with the GSTM1 null genotype showed an elevated frequency of lymphocytes with chromatid-type aberrations and MN. The NAT2 slow acetylator genotype was associated with increased baseline rates of lymphocytes with chromatid breaks and MN. Chromatid aberrations were also increased in TDI-exposed workers with the NAT1 tentative slower acetylator genotype. Subjects with the GSTT1 null genotype showed a higher baseline SCE frequency than GSTT1 positive subjects. Smoking increased the level of both CAs and SCEs. Women had more SCEs and MN in their lymphocytes than men, and lymphocyte MN frequency increased with age. Our results show that occupational exposure to TDI and MDI increases the level of cytogenetic biomarkers, suggesting that diisocyanates are a genotoxic hazard. In accordance with exposure levels, TDI exposure was associated with clearer cytogenetic effects than MDI. In buccal cells, the occupational exposure exclusively increased C- MN, in accordance with the suggested clastogenic effects. Polymorphisms of metabolic enzymes appeared to modulate the level of TDI-induced and baseline chromosome damage, indicating genotype differences in individual susceptibility. Our earlier studies have shown that the NAT1 tentative slow acetylator genotype (alone and in combination with the NAT2 slow acetylator genotype) and the GSTM1 null genotype (alone and in combination with the NAT1 tentative slow acetylator or the NAT2 slow acetylator

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genotype) are associated with an increased risk of isocyanate asthma. The present findings, with relatively few subjects, suggest that these genotypes are also associated with an increased cytogenetic response in exposure to TDI. DNA binding of tamoxifen in human colon after administration of a single 14C-labelled therapeutic dose Karen Brown, David Boocock, Elizabeth A. Martin1, Karen H. Dingley2, Esther Ubick2, Emma Parrott3, Peter B. Farmer, David Hemingway4, Ian N. H. White3. Cancer Biomarkers and Prevention Group, University of Leicester, LE1 7RH, U.K.; 1AstraZeneca, Genetic Toxicology Department, Cheshire, SK10 4TG, U.K.; 2Lawrence Livermore National Laboratory, Livermore, CA94550, U.S.; 3MRC Molecular Endocrinology Group, Dept. of Obstetrics & Gynaecology, Leicester Royal Infirmary, LE2 7LX, U.K.; 4Leicester Royal Infirmary, Leicester, LE1 5WW, U.K. Tamoxifen, which was recently approved for the prevention of breast cancer in healthy women, is known to increase the incidence of endometrial cancer and there has also been some concern that tamoxifen therapy may be associated with an elevated risk of colorectal malignancy. Tamoxifen induces hepatocellular carcinomas in rats through a genotoxic mechanism, involving the formation of multiple DNA adducts and whilst there is much debate over whether DNA adducts are a contributing factor in human endometrial carcinogenesis nothing is known about the ability of tamoxifen to form DNA adducts in human colon. Therefore, to investigate the DNA binding potential of tamoxifen in human colon we conducted a study using the sensitive technique of accelerator mass spectrometry to determine if 14C-tamoxifen DNA adducts are detectable in colon samples taken from ten women administered a single dose of 14C-radiolabelled drug (20mg, 1.85MBq) 18 hours prior to surgery. We have previously used this methodology in an analogous study to demonstrate the formation of low levels of tamoxifen adducts in human uterine tissue (Martin et al. Mut Res - Fundamental and Molecular Mechanisms of Mutagenesis, (2001) 483; P14-11). Quantification of 14C-radiolabel in colon tissue by liquid scintillation counting confirmed the presence of tamoxifen and its metabolites at a concentration of 653 ± 135 fmols tamoxifen equivalents/mg tissue (mean ± SEM, n=10), which is approximately 3 fold higher than the levels we reported in human uterus. Plasma levels of tamoxifen equivalents (15 ± 2 ng/ml) were however, comparable to that previously measured after a single dose. Preliminary results demonstrated DNA binding was detectable in all patients with average levels of 4356 ± 1050 adducts/1012 nucleotides. CYP3A4, the enzyme responsible for conversion of tamoxifen to its reactive α-hydroxylated metabolite, was detectable by Western blotting in extracts of colon tissue from all patients (1.01 ± 0.14 pmols/mg protein), although there was no correlation between CYP3A4 protein levels and DNA adduct numbers. It is possible that low levels of tamoxifen adduct formation in humans may be solely due to the intrinsic reactivity of α-hydroxylated metabolites towards DNA. The extent of adduction would therefore be dependent on the plasma concentration and distribution of α-hydroxylated derivatives in each tissue and also the amount of any locally generated α-hydroxylated metabolites in tissues expressing CYP3A4. The relatively high adduct levels detected in the colon compared to the uterus may therefore be attributed to higher tissue concentrations and the fact that CYP3A4 protein is present in the colon of all patients examined. Whilst we have previously shown than non-bound tamoxifen can be efficiently removed from uterine tissue during the DNA extraction process, the level of 14C-radiolabel was on average 3 fold higher in colon tissue, therefore the extraction methods also need to be assessed under these specific circumstances to determine whether the 14C-radiolabel detected in colon DNA by AMS is entirely due to adduct formation or to non-specific binding.

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Inducible Responses The use of DNA microarrays to characterize genotoxicity R.K. Newton Eli Lilly and Company, Genetic and Molecular Toxicology, Greenfield, IN. 46140 It has been proposed that patterns of induced gene expression changes may be characteristic of specific classes of genotoxic compounds and that distinctive fingerprints may be identified that help classify agents with different mechanisms of action. As part of the ILSI/HESI Genotoxicity Working Group’s project to assess DNA microarrays as a tool to characterize genotoxic responses, investigations with compounds from defined mechanistic classes were performed to test the above hypothesis. As part of this study, the group attempted to correlate gene expression changes with traditional genetic toxicology endpoints and examine the relative sensitivity of DNA microarray technologies. From our studies, the number of genes altered by a particular compound’s treatment was not as sensitive an endpoint as more traditional genetic toxicology endpoints such as DNA adduct induction or micronucleus formation. In addition, concentrations of compounds producing significant genotoxicity induced only modest changes in gene expression (< 3-fold). It is unclear whether this lack of sensitivity is due to the high noise-signal ratio of some of the microarray platforms or to the biology of the in vitro gene expression stress response. From our experience, it also became quickly apparent that the use of global arrays as a screening tool was impractical, since the data analysis portion of the experiment was quite time-consuming. A critical goal of the project was to determine first whether one could distinguish a genotoxicity profile from a cytotoxicity profile by comparing cisplatin (a genotoxin) with sodium chloride (a non-genotoxin) tested at equally toxic doses. Using principal component analysis, it was shown that the two agents induced responses that segregated nicely based on their gene expression profiles. In a further analysis using a supervised statistical approach, it was possible to identify subsets of genes from large global arrays and use these genes as a clustering tool to discriminate the mode of action of test compounds from other laboratories. This strategy of using directed arrays has been used previously to investigate gene changes in cell lines with different p53 status. Comparisons of gene expression changes integrated into targeted statistical test paradigms have enabled us to discriminate genotoxic agents that interact with DNA from those compounds that act via a non-DNA interactive mechanism. However, one of the more important sources of variability found in the technology was the method in which data was analyzed from the studies. An alternative analysis of Affymetrix data using Rosetta Resolver indicated that compounds cluster according to the laboratory performing the study, not by mode of action. Further refinements in data analysis tools in the future may help explain discordant results such as these. Overall, the group is encouraged that preliminary studies have indicated that toxicogenomic analysis may be a valuable tool for differentiating genotoxic mechanisms in vitro. The effect of altered p53 functions on mutagenesis and carcinogenesis in DNA repair-deficient Xpa mice Esther Hoogervorst, Conny Th. M. van Oostrom, Edwin Zwart, Rudolf B. Beems, Susan Wijnhoven, Tyler Jacks1, Annemieke de Vries and Harry van Steeg. National Institute of Public Health and the Environment (RIVM), Laboratory of Toxicology, Department of Carcinogenesis, Mutagenesis and Ageing, PO Box 1, 3720 BA Bilthoven, The Netherlands. 1Center for Cancer Research, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, USA. Xpa-deficient mice have a complete deficiency in nucleotide excision repair (NER pathway), and as such they display cancer predisposition after exposure to several (genotoxic) carcinogens. The p53 gene has been found to modulate the NER pathway. In order to further study the inter-relationship between NER, p53 and cancer, we exposed mice with defective Xpa and/or modified p53 functions to agents, which are substrate for NER, like UV-B, DMBA and 2-AAF. Upon 2-AAF exposure we found that the NER-deficiency had an effect on liver cancer, but not in the bladder. An opposite response was encountered in mice having heterozygously lost one p53 allele. In the liver gene mutation induction, monitored as lacZ reporter gene mutations, closely reflected the 2-AAF-induced tumor response, this was not found in the bladder. The most striking finding was that the high bladder tumor response in double mutant Xpa-/-/p53+/- was accompanied by a moderate lacZ gene mutation induction. Apparently, accumulation of gene mutations in general is not the mechanism underlying bladder cancer in mice. In order to get more knowledge on the process of bladder tumor formation we determined in mice, with different genetic defects, the effect of 2-AAF on both cell proliferation and apoptosis. Next, we also determined whether mutations in the remaining wild type allele of p53+/- are crucial to these processes. In contrast to gene mutation induction in general, it appeared that p53 is a primary mutational target in 2-AAF-driven bladder cancer. The observed changes in the p53 gene had early effects (within 1 week) on cell proliferation, but did not significantly affect apoptosis. Aberrant mutations, not reported earlier, in the p53 gene were found both in early non-neoplastic lesions as well as in the tumor endpoints. Implications of these findings will be discussed. In an attempt to further explore the role of the p53 gene in DNA repair and carcinogenesis in general, we created so-called “knock-in” mutants in the mouse. One of these mutants, i.e. p53.S389A, carries a mutation at codon 389 (392 in human), a serine was replaced by an alanine residue. Cells exposed to UV accumulate DNA damage (specifically substrate to NER) and also become phosphorylated at serine389 in the p53 protein. A phenomenon not seen in cells exposed to non-NER-related compounds like γ radiation. This indicates that serine389 might be crucial in directing NER

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and possibly in preventing cancer induced by NER-related compounds. Currently we are testing this hypothesis by exposing p53.S389A mice and cells derived from these to UV, 2-AAF and γ radiation. Thus far, our results, obtained both in vitro and in vivo, substantiate the view that p53 plays an active role in NER-related DNA repair and thereby by preventing cancer caused by NER compounds. This work is in part supported by the Dutch Cancer Society (grant RIVM2000-2352) and the NIH/NIEHS (Comparative Mouse Genomics Centers Consortium, grant 1 UO1 ES 11044). DNA damage induced GFP expression in yeast identifies most direct acting mutagens detected by the Ames test in addition to clastogens previously identified only in mammalian cell tests R.M.Walmsley. Biomolecular Sciences, UMIST Manchester, M60 1QD, UK A eukaryotic (yeast) genotoxicity assay has been developed which detects increases in the activity of the DNA repair system. It uses a reporter system consisting of the DNA damage inducible promoter of the RAD54 gene fused to a GFP gene. A large study has been carried out to assess the utility of the assay in routine genotoxicity assessment. A single 96 well microplate is used to assess 4 compounds at ten dilutions, and can be set up by a simple manual protocol for low throughput (<40 compounds/day) or with robotic systems for higher numbers. Test yeasts are added to compound dilutions, microplates are incubated overnight, then spectroscopic data is collected. Optical density is used to estimate cell proliferation (toxicity). Fluorescence, divided by absorbance, is used to estimate activity of the repair system. Data handling will be described, and sample data will be presented from tesalts with purified compounds as well as effluent samples. Less than 0.5ml of compound is required and over 250 toxic and non-toxic compounds have been tested, without S9 addition. The results demonstrate that this test provides useful additional information to that obtainable from screening versions of the Ames test. It is effective in identifying most of the direct-acting mutagens found by the Ames test in addition to many clastogens, normally only detected in mammalian cell assays. Several compounds that would require S9 in bacterial tests are also positive, however many aromatic amines and amides are not seen, showing that the metabolic potential of the current yeast strain falls short of that provided by S9 in bacterial tests. Interestingly several Ames-negative carcinogens are positive in this test, as well as some photomutagens (in normal laboratory lighting). It was also interesting to detect colchicine as positive, suggesting a clastogenic as well as aneugenic mechanism for this compound. Transgenic promoter/reporter mice and bioluminescent imaging – applications in genetic toxicology Anthony M Lynch Safety Assessment, GlaxoSmithKine, Park Road, Ware, Herts, SG12 0DP, UK. The metabolism of luciferin in cells or animals transgenic for luciferase (luc) produces light that may be detected in vitro (cell culture) or trans-vivo in living animals by an intensified CCD camera (biophotonics) and captured by image analysis (Zhang et al, 2001). Thus the generation of transgenic promoter-luciferase animals for genes regulated by specific toxic processes, coupled with real-time evaluation of site-specific gene expression may provide novel, non-invasive in vivo biomarkers which may be predictive of developing toxicity within a wide variety of tissues and organs. For example, studies in HO-1.luc mice, transgenic for the murine haem oxygenase-1 promoter show that the transgene is upregulated in liver, kidney and testis by treatment with cadmium chloride ie. known target organs for this toxin. Comparison of the HO-1.luc response with markers of toxicity measured ex vivo (RT-PCR differential gene expression, clinical chemistry and pathology) confirm that the expression of the transgene is correlated with the expression of the endogenous HO-1 gene and with more traditional endpoints of toxicology (Speight et al, 2002). We have previously shown that the expression of a number of genes associated with cellular responses to DNA damage are upregulated > 3 x fold in HepG2 cells following treatment with chemical clastogens, using RT-PCR (Smith et al, 2002). Using computational modelling, the promoter sequences for a number of these genes was determined (RAD50, RAD51, RAD52, RAD54b, REV3L and GADD45) and a couple of promoter-GFP reporter constructs were engineered (RAD50 and GADD45). Preliminary studies suggest that exposure to chemical clastogens results in the up-regulation of the GFP reporter in transfected cells. These promoter-reporter constructs may provide the basis for developing novel transgenic models which may be predictive of developing genetic toxicity using biophotonic image analysis to capture real-time, site-specific, gene expression changes associated with DNA damage in living animals.

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References: Zhang, W., Feng, J.Q., Harris, S.E., Contag, P.R., Stevenson, D.K. and Contag, C.H. (2001). Transgenic Research. 10, 423-434. Speight, B., Walker, S., Meakin, J., Driver, R., Schenck, E., Weir, L. and Lynch A.M. (2002). Mutagenesis 17, 569. Smith C.C., Gooderham, N.J. and Lynch A.M. (2002). Mutagenesis 16, 577.

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Industrial Genotoxicity and Regulatory Guidelines Validation of an in vitro model to demonstrate the interference of apoptosis in in vitro aberration assays D. Marzin, S. Meintiere Institut Pasteur de Lille, Laboratory of Toxicology - 1, rue du Professeur Calmette - BP 245 - 59019 LILLE, France. A model was developed to demonstrate the interference of apoptosis in in vitro chromosomal aberration assays (metaphase analysis and micronucleus tests). CTLL-2 cell line was transfected with Bcl2 gene in order to inhibit apoptosis induction in this transfected cell : when a compound induces a positive result in CTLL-2 cells and not in CTLL-2/Bcl2 cells, it is a proof of apoptosis interference (Meintières et al., 2001). This model was tested in our lab using a large number of clastogens, aneugens and pure apoptosis inducers (Meintières et al., 2003a ). This model was used to demonstrate that ghost cells were not induced by apoptosis and/or necrosis (Meintières et al., 2003b). A new step in progress is to validate this model system by a collaborative interlaboratory study. Five laboratories received coded compounds to be tested in the micronucleus test in the 2 cells lines using an agreed protocol. Non mutagens compounds inducing or not apoptosis, aneugens and clastogens compounds are tested using in vitro micronucleus in both cell strains. Each test compound is studied by at least 2 laboratories to study interlaboratory reproducibility. The first results of this interlaboratory study will be presented. They confirm the possibility to distinguish the role of apoptosis as an interference factor in the micronucleus test and the place of this test when interference of apoptosis is suspected as a confusing factor. MEINTIERES S., BIOLA A., PALLARDY M., MARZIN D. Apoptosis can be a confusing factor in the in vitro clastogenic assays Mutagenesis, 2001, 15(3), 243-250 MEINTIERES S., A. BIOLA, PALLARDY M., MARZIN D. Using CTLL-2 and CTLL-2 bcl2 cells to avoid interference by apoptosis in the in vitro micronucleus test Environmental and Molecular Mutagenesis, 2003(a), 41(1), 14-27 MEINTIERES S., F. NESSLANY, PALLARDY M., MARZIN D. The detection of ghost cells in the standard alkaline comet assay is not a good measure of apoptosis Environmental and Molecular Mutagenesis, 2003(b), 41(7) , 260-69 Current developments in regulatory control of genotoxic impurities in drug substances: An interim report P. Kasper Federal Institute for Drugs and Medical Devices, Bonn, Germany. The Safety Working Party of the CPMP is currently in a process of preparing a Position Paper which is intended to provide guidance on how to define acceptable limits of genotoxic impurities in new drug substances. According to the existing ICH Q3A Guideline, impurity acceptance criteria should be set no higher than the level that can be justified by safety data, and should be consistent with the level achievable by the manufacturing process and the analytical capability. How can these demands be applied to impurities with genotoxic properties? In current regulatory practice genotoxic compounds are usually considered to operate by a non-threshold mode of action and thus any level of exposure carries a risk. This conservative view implies that pharmaceutical (quality) measurements should be guided by the so called ALARA principle, i.e. where avoidance is not possible, genotoxic impurities must be kept As Low As Reasonably Achievable. From a toxicological point of view different approaches for assessing acceptable limits are suggested depending on the availability of data. (1) For known genotoxic carcinogens, where a full in vitro and in vivo genotoxicity and carcinogenicity set of data is available risk assessment can be carried out either by applying quantitative risk assessment based on mathematical modeling or using no effect levels of carcinogenic response modified by uncertainty factors. (2) For in vivo genotoxins of unknown carcinogenicity use of a No-effect-level-uncertainty-factor-approach is justified in those (probably rare) cases where adequate data for demonstrating a threshold for genotoxic effects are available. Whether such an approach is also applicable to in vivo genotoxins with no evidence of a threshold requires further discussion. (3) For most impurities, where there may only be limited in vitro data available a pragmatic approach is needed which recognises that the presence of very low levels of genotoxic impurities do not change the risk/benefit balance for patients in a meaningful way. The application of a target level for an acceptable daily intake of 1.5 µg/day based on the concept of Threshold of Toxicological Concern is currently under discussion for this purpose. From this threshold value a permitted level in the drug substance (ppm) can be calculated based on the expected dose to the patient. A higher threshold may be justified taking into consideration drug duration and frequency of treatment as well as indication and exposed populations.

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The revised EU Technical Guidance Document for mutagenicity testing and risk assessment of industrial chemicals and biocides Andrew Smith & Peter Howden Health & Safety Executive, Magdalen House, Stanley Precinct, Bootle, UK. Earlier this year, the European Commission’s Chemicals Bureau (ECB) published the 2nd edition of the Technical Guidance document (TGD) on Risk Assessment of Chemical Substances. The TGD can be found on the ECB website http://ecb.jrc.it/tgdoc. Although having no legal status, the TGD has been endorsed by all stakeholders, and it is expected to be adhered to in most cases. The section “Mutagenicity” was updated significantly in the revised TGD and its scope extended to include biocides as well as new and existing industrial chemicals. It includes some general advice about evaluating mutagenicity studies, but the main focus is on describing a new testing strategy to support the risk characerisation and classification and labelling of mutagens. The strategy offers more flexibility than the previous version and takes account of the pressures to minimise the use of animals in regulatory testing. There is an emphasis on applying expert knowledge to the testing requirements of individual chemicals. The base set consists of a bacterial test and an in vitro mammalian cell test, which should be one of: the chromosome aberration test, a mouse lymphoma gene mutation test or a micronucleus test. Decisions about the need for further testing of a substance are made on the basis of the results of these tests, its physico-chemical and toxicokinetic profile, its production level (new substances) and considerations of the scale and type of human exposure. A principal of the strategy is that in vivo tests should be avoided as far as possible. The bone marrow micronucleus test is no longer necessarily the automatic follow-up to a positive in vitro test result. There is a place in the strategy for new test methods such as the comet assay and gene mutation tests in transgenic animals. The strategy calls for tests in germ cells only when it is not possible to predict satisfactorily whether effects in somatic cells may cause heritable mutations. It is possible now to reflect on the early signs of changes the TGD is provoking on the regulatory testing of industrial chemicals and biocides in the EU. Additional technical guidance is in preparation for the risk characterisation (RC) of mutagenic chemicals. In most cases, it is assumed that there is no identifiable threshold for mutagencity, and that there is a risk, however small, at any level of exposure. However, it is accepted that certain modes of action (e.g. saturation of metabolic pathways, interaction with multiple non-DNA targets) can have a threshold that could be determined through appropriate experiments. When such a threshold can be demonstrated, the RC should be conducted using the conventional approach” that is taken for threshold endpoints. The burden of proof is on providing scientifically robust evidence to demonstrate a threshold: it is likely to be difficult to provide adequate experimental evidence to demonstrate such a clear cut threshold. Strategy for genotoxicity testing and classification of genotoxicity test results – report on initial activities of the IWGT expert group L. Müller Novartis Pharma AG, Basel, Switzerland

An expert group in the International Workshop on Genotoxicity Tests (IWGT) has started to work out a process to give guidance on a common stategy for genotoxicity testing and risk assessment. This IWGT group involves experts from government, industry and academia. All group members represent important national and international stakeholders and have actively contributed to the development of strategies and guidances for genotoxicity testing. Since the landmark paper of John Ashby in 1989 on a common genotoxicity strategy approach, which would be independent of the use of the test item, the field has moved forward and a number of national and international guidances have been developed. Despite these new and valuable guidance approaches such as the ICH guidances for pharmaceuticals or, more recently, the COM Guidances, the IWGT group noted that there is still a lack of common understanding of several issues that are considered important for the development of a modern general strategy for genotoxicity testing and the interpretation of test results for risk assessment.

The following areas were identified as important areas, for which a further exploration of the scientific basis, existing data and practices is needed, before a common strategy and risk assessment procedure can be delineated: 1) Examples of unique in vivo positives • Inappropriate metabolism • Low exposure to relevant metabolites (both in vitro and in vivo) • Which endpoints/tissues should be studied in vivo to confirm negative results in vitro? • What to do when structure activity data suggest conventional assays are irrelevant? 2) Examples of tumours at specific sites in carcinogenicity studies when chemical is clearly negative in vitro and in vivo in elemental data set.

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3) Examples or uniquely in vivo positives that would justify the use of animals in the base set of tests 4) Examples of major human metabolites not represented in vitro or in rodents in vivo • When to change exogenous metabolic system in vitro? • When to select different animal species in vivo? • When to synthesise/extract human metabolite and test separately? 5) Examples of threshold effects or non-relevant positive results • When do positive Ames results not indicate a human hazard? Does positive MLA confirm hazard? Can negative MLA overrule Ames positive? Examples of positives resulting from bacterial-specific metabolism?

• How to interpret positive results from in vitro MN? Aneugen? If clastogen, is there a threshold?

• Criteria for non-relevant/threshold results in chromosomal aberration or MLA tests in vitro. 6) Can any argument on potency be derived from in vitro test data? For the above tasks, work groups leaders have been identified and have started to establish sub-groups in order to accomplish the task. Several questionnaires have been formulated and sent out for input to various industrial and pharmaceutical companies. An update on the current status of these subgroups will be given. The ultimate goal is to formulate a common strategy position under the IAEMS umbrella by the next ICEM conference in 2005. A strategy for the testing of hair dye cosmetic ingredients for their potential mutagenicity/genotoxicity in the European Union Nicola Loprieno1 and Christian Laurent2 1University of Pisa,Italy,and 2University of Liège,Belgique.E.C. Scientific Committee for Cosmetic Products and Non-Food Products Intended for Consumers.Bruxelles, Belgique. An epidemiological study recently published by M.Gago-Dominguez et al. (Int.J.Cancer:91,575-579,2001) has shown an increased risk of bladder cancer among women who made regular use of permanent hair dyes over many years.The SCCNFP recommended the European Commission to take further steps to control the use of hair dye chemicals since the potential risks for the european women of using this category of substances give cause of concern. The European cosmetic industry uses a considerable number of permanent hair dyes and the safety of many of these has not yet been assessed by public authorities.The reasons for this include incomplete dossiers and non-conformation of experimental data to modern (current) methods of collection (SCCNFP/0553/02). Data on genotoxicity/mutagenicity are often not consistent with internationally accepted guidelines (OECD,EU) and/or with modern strategies.Of 25 hair dye cosmetic ingredients evaluated by the SCCNFP during the last three years,inadequate studies have been included (21% of in vitro studies,and 49% of in vivo studies). For 46 hair dye ingredients recently included in Annex III of Directive 76/768/EEC,the previously submitted data no longer comply to present standards.For all oxidative hair dye ingredients,information on their products of reaction between the bases and the couplers and the oxidants are lacking. In the contest of a most stringent and rigorous assessment strategy for hair dyes (SCCNFP/0553/02) a Proposal for “A Strategy for Testing Hair Dye Cosmetic Ingredients for Their Potential Genotoxicity/Mutagenicity” has been adopted by the SCCNFP (0566/02) and published on the Internet.Such proposal has received different comments;the Cosmetic Industry has presented and discussed it critically in a recent meeting with the SCCNFP (May 27th,2003). In this paper a definitive strategy recently adopted by the SCCNFP for testing hair dye ingredients and reaction products for their mutagenic /genotoxic/carcinogenic potential will be presented and illustrated for scientific comments (SSCNFP/720/03:June 24th,2003).

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Ecogenotoxicology Issues pertinent to research agenda in genetic ecotoxicology Lee R. Shugart1 1 LR Shugart and Associates, Inc. Oak Ridge, TN, USA. Although the importance of genetics to ecotoxicology has been noted and explored for many years, it was at the 1993 Conference on Genetic and Molecular Ecotoxicology (Environ. Health Perspec. 102,1994) that the field of Genetic Ecotoxicology was clearly defined. Furthermore, the scope, direction and goals of relevant scientific research that marries the two disciplines were also detailed. Genetic ecotoxicology is defined as “The study of chemical- or radiation-induced changes in the genetic material of natural biota. Changes may be direct alterations in genes and gene expression or selective effects of pollutants on gene frequencies”. The key objectives of research in genetic ecotoxicology are to explore the extent to which ecosystems are contaminated with genotoxicants and, more importantly, to identify adverse effects in populations, communities associated with genotoxicant exposure. This presentation will focus on three important issues pertinent to research in genetic ecotoxicology. First, current methods for measuring individual-level effects as well as those for detecting population-level effects will be discussed. Second, application of these methods for measuring genotoxicity and population genetics of field-exposed animals will be reviewed. And finally, implication of the Human Genome Project to genetic ecotoxicology with the potential for new research directions will be considered. Relative sensitivity of two bivalve mollusc species for the detection of geno- and cytotoxicity under laboratory and field conditions Victoria V. Cheung and Awadhesh N. Jha School of Biological Sciences, Plymouth Environmental Research Centre, University of Plymouth, Plymouth PL4 8AA, UK. In order to protect the environment it is important that sensitive species and their life stages are identified. Despite the fact that aquatic environment constitutes more than 70% of the planet and the invertebrates constitute more than 90% of the extant species, most of the currently available assays or biomarkers have been validated and applied to only a very limited number of species. It is frequently observed that at contaminated sites, either these species are not available or if they are, their responses might not be indicative of other species in the community. In this context, the marine bivalve Mytilus (mussels) species has been widely used as a sentinel species for environmental biomonitoring. This species requires rocky substrata for attachment, a prerequisite for its growth and proliferation. In contrast, the common cockle, Cerastoderma edule, is widely distributed in the fine sediment environment, which is the ultimate depository of many contaminants. In the present study, we have evaluated the relative sensitivity of these two species under laboratory and field conditions for biomarkers of genotoxicity (i.e. DNA strand break measurements using single cell gel electrophoresis or the comet assay) and cytotoxicity (i.e. lysosomal neutral red retention assay). For laboratory validation studies, responses in the two species were compared following exposure of the haemocytes to a range of concentrations of hydrogen peroxide under in vitro conditions. These experiments suggested the cockles to be more sensitive than the mussels. Following laboratory validation studies, the sensitivities of these two species were compared under field situations along the Tamar estuary, south-west Devon. Studies have indicated this region to have higher levels of a range of contamination. In these studies, attempts were made to find a correlation, if any, between the extent of damage (at 6 different sites including a reference site) and the presence of heavy metals in the sediment and in the body of the individual organisms (body burden). The response at individual sites was found to be variable. While the cytotoxicity (as measured by neutral red retention assay) did not show any statistically significant correlation, in general, the levels of DNA damage induced in the field samples correlated with heavy metal body burden, with greater variability for the ‘tail length’ and ‘tail moment’ observed in the mussels. Some of these observations could be explained by organism’s feeding behaviour (mussels being filter feeders while cockles directly feeding from sediments) and their ecological niche. The study emphasises the need for multiple species in biomonitoring programmes, which should include representative species from different groups including filter and detritus feeders.

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Stress responses in the haemocytes and gill cells of the deep-sea vent mussel Bathymodiolus azoricus David R. Dixon, Linda R.J. Dixon and Audrey M. Pruski. Southampton Oceanography Centre, Waterfront Campus, European Way, Southampton S014 3ZH, United Kingdom. In keeping with previous bivalve studies, apoptosis appears to play an important and sometimes confounding part in the genotoxicological and stress responses of the deep-sea vent mussel Bathymodiolus azoricus. A combination of Comet and gel electrophoresis analysis has shown that cage recovery was less stressful compared to deep-sea submersible or ROV recoveries. This finding was not surprising given the extended transit times that typically accompany some vehicular recoveries. However, regardless of the recovery method, all the animals showed signs of stress, in the form of increased levels of DNA damage/apoptosis, resulting seemingly from their decompression experience. Note all the animals used in this study were from a depth of 850m = 85 bar pressure, which is relative shallow in deep-sea vent terms. This initial pressure damage was reversible, with the majority of animals going on to survive in the laboratory for weeks or even longer at 1 atmosphere pressure. However, monitoring the baseline levels of DNA damage in blood cells and gill cells showed that while the damage levels fell after a few days post-collection, this DNA recovery was only short lived and by two weeks had given way to irreversible damage, regardless of the maintenance regime employed (i.e. particulate vs. chemosynthetic feeding). This means that there is only a restricted time window in which to perform laboratory exposure experiments on deep-sea vent animals and even then there is the risk of increased sensitivity caused by their stressed state. These findings raise serious concerns about the validity of in vivo experiments conducted on deep-sea organisms, and point to an urgent need to develop isobaric collection and experimentation systems. This work was funded by an EU RTD award: VENTOX EVK3-CT1999-00003 to DRD and by a Marie Curie Individual Fellowship to AMP. Time-course evaluation of DNA damage in mussels (Mytilus galloprovincialis) from an heavy polluted harbour environment Giada Frenzilli1, Francesco Regoli2, Marco Nigro1. 1Dipartimento di Morfologia Umana e Biologia Applicata, Università di Pisa, Italy 2Istituto di Biologia e Genetica, Università Politecnica delle Marche, Ancona, Italy. Harbours can be considered as model environments for developing and validating field monitoring procedures and to investigate mechanistic relationships between different biological responses. In this study several biomarkers were investigated in marine mussels caged for 4 weeks into an industrialised harbour of North West Italy. Organisms were collected at different time intervals (3, 7, 14, 24, 30 days) to better characterize the sensitivity, temporal variations and interactions of analysed responses. DNA damage was evaluated by Comet assay on mussel gill cells and data obtained were integrated with the antioxidant status. Besides single antioxidants (catalase, glutathione S-transferases, glutathione reductase, total glutathione), the Total Oxyradical Scavenging Capacity (TOSC) assay was used to analyse the capability of the whole antioxidant system to neutralize specific forms of radicals. Neutral Red Retention Time was carried out on circulating haemocytes to study lysosomal stability as other marker of cellular toxicity . Results showed that mussels sampled at the harbour site exhibited a lower degree of DNA integrity since the third day after the deployment and statistically higher levels of DNA damage were constantly found in harbour mussels in comparison with specimens sampled at the reference site during the entire experiment. A biphasic trend was observed for single antioxidants and TOSC, with an increase during the first 2 weeks of exposure to the polluted site followed by a progressive decrease up to a severe depletion in the final part of the experiment. These findings suggest an initial counteractive response of mussels toward the enhanced prooxidant challenge, while antioxidants appeared overwhelmed at longer exposure periods. The hypothesis of ROS-mediated toxicity is supported by the appearance of cell damages (DNA integrity and lysosome membrane stability), which exhibited a progressive enhancement during the course of the experiment with a maximum impairment after 30 days of exposure.

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Micronucleus test in marine animals, as a biomarker for genotoxic environmental contamination C. Bolognesi 1, M. Monteverde1, E. Perrone1 1 Environmental Carcinogenesis Unit, National Institute for Research on Cancer, Genoa, Italy. “Environmental effect monitoring” approaches provide signals about the frequency and amplitude of environmental changes that enable understanding the impact of these in advance. Genotoxic alterations represent the primary events for the environmental exposure to carcinogenic and mutagenic agents. Many genotoxicity assays have been applied in marine organisms, but only a limited number of biomarkers have been validated or are currently suitable for large scale field assessment. Micronucleus test, due to its simplicity and rapidity, seems to be one of the most promising techniques to identify genomic alterations in environmental biomonitoring studies. Micronuclei (MN) are small fragments of chromatin separated from the main cell nucleus which are index of chromosomal breaking or mitotic spindle dysfunctions. Micronuclei are produced after an irregular division process in which a chromosome fragment or a whole chromosome is not lost during the anaphase but is delayed with respect to the rest of chromosomes, constituting a small secondary nucleus. This test has the advantage that it can be applied in interphase to any proliferating cell population regardless of its karyotype. Micronucleus assay, originally developed with mammalian species and today widely applied to many proliferating cell populations, has been applied in fish and other aquatic organisms, including sea urchins, bivalve molluscs, crabs and worms. The micronuclei frequency provides an index of accumulated genetic damage during the lifespan of the cells and it is an index of the integrated response to the complex mixture of contaminants monitored or not that could contribute to the toxic load. The bioindicator organisms most commonly used in environmental biomonitoring studies are the bivalve molluscs in the mussel watch program, but teleost fish, due to their role in the biotic communities and in the food chain, are the most interesting sentinel organisms. The induction of micronuclei has been studied in the hemocytes of mussels as the main target of neoplastic diseases in these species, although the test has been now more efficiently applied in gill cells which are more directly exposed to environmental contaminants. Micronuclei can be analysed in different fish cell types such as gill cells, kidney and hepatic cells, but the use of erythrocytes is more diffused to avoid complex procedures of cell preparation and animal sacrifice. A high interindividual variability of the micronucleus frequency was also observed in native and transplanted mussel and in fish populations. Sources of variability that may influence the micronuclei frequency fall in two categories. The first one is inherent to the laboratory method and sample collection, that could easily be controlled by the standardization and intercalibration procedures. The other category includes biotic factors, such as sex, age, nutritional status, environmental and climate change processes. Multiple samplings during the year and the use of animals with the same size allow to overcome the seasonal effects. In addition in studies with transplanted animals the choice of an appropriate time of caging allows to improve the sensitivity of this test. Further factors modulating the susceptibility to genotoxic agents could not be controlled and they mainly reflect the development of resistant individuals or populations. One of the aims of our lab is the validation of the micronucleus test in bioindicator organisms in the field. The test was applied in native and transplanted mussels in selected contaminated and in reference areas along the Ligurian coast. Statistical significant increases of micronuclei frequency in polluted stations were detected with respect to reference areas. The frequency of micronucleated cells/1000 cells ranges from 1.78 ± 1.04 to 24.4 ± 12.9. Native animals accumulate much higher concentration of chemicals and facilitate quantifying both exposure and effects. Seasonal influence was also observed with highest values during the summer period. The kinetics of micronuclei and DNA damage was studied in mussels exposed in situ in a reference and in a polluted station for different caging periods (7, 15, 30 and 60 days). A time-dependent increase in MN frequency in gill cells was observed with highest values recorded after 30 days. Micronucleus was also applied in fish belonging to different species collected in reference and polluted area and during 5 cruises along the west Mediterranean coast. Nuclear lesions other than micronuclei such as lobed nuclei, vacuolated nuclei and nuclear evaginations (NEs) were also recorded. A parallel increase of MN frequency/1000 cells and NEs was observed in fish (Mullus barbatus) along a pollution gradient. Our experimental evidence suggests the importance for the recording of this anomaly in fish erythrocytes in order to improve the information obtained with this test. (Supported by UE contract No EUK-2000-00543-BEEP). Genome-wide identification of expressed sequence tags in Mytilus galloprovincialis P. Venier1, C. De Pittà1, F. Marsano2, A. Pallavicini3, A. Viarengo2 N. Vitulo1 and G. Lanfranchi1 1 Department of Biology, University of Padua, Italy 2Department of Science and Advanced Technology, University of Piemonte Orientale, Italy, 3 Department of Biology, University of Trieste, Italy. Marine bivalves of the genus Mytilus are model organisms which importance is established by their ecological role, marketable production as sea-food, and long-standing use in coastal pollution bio-monitoring. However, limited knowledge is available on the mussel genes and their expression in ordinary or stressing conditions. Providentially, the systematic production and sequencing of the 3’-end cDNA clones (Expressed Sequence Tags or ESTs) allows rapid and large-scale identification of protein coding genes also providing information on the relative abundance of the most common mRNAs. Therefore, we have started the construction of 3’-end cDNA libraries from normal and pollutant-treated mussels in order to investigate genes basically expressed and specifically modulated by the exposure to toxic and genotoxic pollutants. This experimental approach represents the starting point for the identification and functional characterization of specific genes and for genome-wide expression profiling in Mytilus galloprovincialis.

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RNA purification from multiple tissues of normal adult mussels and massive sequencing of the corresponding 3’-end ESTs allowed us to assemble 829 sequences in 524 clusters (98 virtual consensuses and 426 singletons). Similarity searches performed against the non-redundant NCBI database, identified a small number of mussel transcripts (e.g. actin, twitchin, COIII) and revealed significant similarities with a number of mitochondrial and nuclear housekeeping genes. Marked abundance of mitochondrial mussel transcripts was also evident and, gene functions potentially recruited in stress responses were putatively identified (e.g. myticin A, heat shock proteins, methallothionein). A large fraction (59%) of the consensus sequences obtained from the unstressed mussels showed poor or no similarity with any listed entry and it is indicative of unknown mussel genes. Work is in progress to enlarge the 3’-end EST collection from normal and pollutant-treated mussels and to produce a new tool for studying mussel transcription profiles. At the moment, 5664 ESTs from multiple tissues of normal mussels and 1731 independent sequence clusters have been produced. Biomarkers of carcinogenic exposure in estuarine fish species for the assessment of biological effects of contaminants B.P. Lyons1, G.D. Stentiford2, M. Longshaw2, S.W. Feist2. 1CEFAS Lowestoft Laboratory, Pakefield Road, Lowestoft, Suffolk NR33 0HT, United Kingdom and 2CEFAS Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB, United Kingdom. The estuarine environment is a major sink for many potentially hazardous chemical pollutants emitted from industrial and domestic sources. Inventory-based chemical monitoring programmes are restricted to the identification of a limited range of contaminants and provide no information on their biological significance. This has led to the utilization of biomarkers as evidence of the deleterious effects of such environmental contaminants. Here we report on combined biological endpoint data for biomarkers of carcinogenic exposure and histopathological alterations and in three species of estuarine fish captured from UK estuaries (the Tyne, Tees, Mersey and Alde) with different contaminant loads. The fish species used in this study, the flounder (Platichthys flesus), the sand goby (Pomatoschistus minutus) and the viviparous blenny (Zoarces viviparus) were chosen because of their close association with the estuarine environment. Fish captured from the three contaminated sites were found to show more pathological alterations to their tissues and organs than the same fish species captured at the reference site. The presence of pre-neoplastic and neoplastic toxicopathic lesions was highest in flounder captured from the Tyne, Tees and Mersey, when compared to fish from the Alde reference site. In particular, the prevalence of hepatic foci of cellular alteration (up to 43.3 %) and hepatocellular adenoma (up to 10 %) were highest in flounder captured from the Mersey estuary. The analysis of bile metabolites from flounder indicated that fish caught from the Tyne, Mersey and Tees were exposed to significant amounts of PAH. In addition, flounder and blenny captured from the Tyne estuary exhibited patterns of aromatic/hydrophobic DNA adducts in the liver typical of exposure to a complex mixture of genotoxic PAHs. Studies are on going, expanding number of estuaries examined and increasing the suite of biomarkers used to include a toxico-genomic approach. Fish Cells To Evaluate Genotoxicity In Environmental Risk Assessment Procedures Argelia Castaño. Animal Health Research Center. CISA- INIA, E-28130, Valdeolmos, Madrid, Spain. Evaluation of the genotoxic potential is an essential information for the hazard assessment of chemicals in the regulatory process, nonetheless, this requirement is not included in the ecotoxicological dossier of a chemical. From the ecotoxicological point of view, the evaluation of genotoxicity has a particular importance because of the delayed manifestation of the genotoxic effects, that may require years to manifest fully, and may be crucially important at the population level. Thus, predicting the effects of chemicals or complex mixtures upon populations, prior to their release, allows the establishment of safety levels in environmental risk assessment protocols. In vitro assays are a common tool in genotoxicity, making shorter, cheaper and easier the study of mechanistic effects of chemicals on the DNA. The assays currently used for regulatory purposes to measure genotoxic potencies of water samples or environmental chemicals, are mainly prokaryotic tests (Ames, Umu) and/or mammalian cell test. The use of fish cells provides a powerful alternative to the use of live fish for genotoxicity assays and offer several advantages over assays based on prokaryotic or mammalian cells. Fish cells appear to have a lower DNA repair activity compared to mammalian cells, and therefore, they may be more sensitive to genotoxins than mammalian cells. Comparative experiments using both piscine and rodent cell lines showed a higher sensitivity of the piscine RTG-2 cells than of the mammalian V79 cells to several chemical genotoxic carcinogens. In addition, significant species-specific differences in the metabolic conversion of xenobiotics are evident between mammals and fish as well as between various fish species, and, therefore, target organism-derived cells should be the preferred test system for genotoxicity assessment in fish toxicology.

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Fish cells have been applied with a large variety of genotoxicity endpoints. Since most fish karyotypes consist of large numbers of small, irregular chromosomes, chromosomal analysis are complicated and time consuming, therefore methods other than chromosomal analysis, is recommended to be chosen. In the last years we developed an semi-automatic micronuclei assay in fish cells using flow cytometry. We have applied this assay in both pure chemicals and complex mixtures and even in Toxicity Identification Evaluation in studies from urban sewage treatment plants. When testing pure chemicals, our results confirm higher sensitivity of fish cells when compared to results on mammalian cells. In a similar way than mammalian cells, differences depending on the endpoint used has been observed too, and may be up to one order of magnitude. Using the same cell line, RTG-2, but different endpoints, the genotoxic concentrations for B(a)P varies: 0.05 mg/L for altered DNA fingerprint, 0.1 mg/L for induction of micronuclei, and 0. 605 mg/L in the comet assay. Again, these concentrations are in the lower reported range of genotoxic concentrations when compared with those obtained in in vitro tests with mammalian cells (from 0.1 to 1670 mg/L) for the same chemical. In addition, the use of fish cells simplifies analysis of genotoxicity and could contribute to the study of genotoxic effects in fish populations. Genotoxic studies, based on alterations of DNA fingerprints are difficult because of a large genetic polymorphism in fish leading to variations in the genetic complement between individuals and populations. This fact makes genetic comparisons between control and exposed fish very difficult particularly in field situations. Fish cells have the advantage of being a population of homozygous individuals (clone). After exposure to genotoxic substances, detected alterations in the genomic fingerprint is the result of interaction of the agent with the DNA and not because of polymorphisms. Our results comparing rainbow trout individuals and RTG-2 cells, a rainbow trout cell line, demonstrated a good comparability between in vivo and in vitro systems with respect to their genomic pattern, with a inter-population similarity index in vivo/ in vitro of 0,931. On the basis that there is a good comparability between in vivo and in vitro systems with respect to genotoxic responses, genotoxicity detected in a fish cell line exposed to environmental genotoxicants will most likely also be manifested in vivo when fish are exposed to the same agent and with a high probability, the agent could also alter the genomic complement of a given population. Later studies on the same direction but using carps (Cyprinus carpius) and EPC cells, a carp cell line, demonstrate just he opposite: comparison between all carp individuals showed high analogy in the DNA bands patterns, in contrast low analogy is observed when comparing DNA fingerprints between carp and EPC cell line. A great genetic similarity of the in vivo and in vitro systems is an essential requisite in predictive genotoxicity assays, then before any predictive study, the similarity in the genetic pattern between in vivo populations and the candidate cell line should to be proved. Use of plant bioassays for the detection of genotoxins in soils and in the aquatic environment S. Knasmüller 1, B.J.Majer1 and T.Grummt 2

1 Institute of Cancer Research, University of Vienna, 2 UBA-Germany, Bad Elster. During the last decades several plant bioassays have been developed which enable the detection of genetic damage. To date the most widely used assays are micronucleus (MN) tests with Tradescantia (clone # 4430), Vicia faba and Allium cepa. Several comparisons indicate that the Trad-MN test, in which damage is recorded in meiotic chromosmomes, is more sensitive towards induction of damage than mitotic cells in root tips. We used these procedures to study the effects of contaminated ground and surface waters and of water disinfection procedures and compared the results with data from other tests, which are commonly used for water testing (Salmonella/microsome assays and tests with primary hepatocytes); furthermore correlation analyses were carried out with chemical data. For soil testing a new protocol was developed with Tradescantia (exposure of intact plants). We could demonstrate that the Trad-MN assay is a unique tool for the detection of genotoxic effects caused by heavy metals in soils. Metals which induce DNA damage and cancer in humans and rodents such as As, Cd, and Cr (VI) caused strong dose dependent effects in the plant assay. On the contrary, no effects were seen with Cr(III), Cu and Sb, which are not proven carcinogens in humans. Earlier investigations with individual compounds show that plant bioassays are also highly sensitive towards certain classes of environmental genotoxins (eg. certain pesticides, industrial waste chemicals, ionising radiation etc.) whereas weak or negative results are obtained with compounds which require metabolic activation (e.g. nitrosamines, PAHs, heterocyclic aromatic amines).

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Aneuploidy and Chromosome Stability Comparative genomic hybridisation: principles and application to analysis of brain tumours T.Warr Institute of Neurology, University College London, London, UK. The development and application of comparative genomic hybridisation (CGH) over the last decade has made a huge impact in the field of solid tumour genetics. Previously, one of the major limitations to cytogenetic analysis of solid tumours had been the difficulties in obtaining sufficient numbers of good quality metaphase chromosomes. CGH is a modified in situ hybridisation technique which allows a genome-wide analysis of DNA sequence copy number alterations in a single experiment without the need for prior cell culturing, The technique is based on competitive binding of differentially labelled tumour (usually green fluorescence) and normal (red fluorescence) DNA to normal metaphase spreads. Regions of increased or decreased copy number in the tumour DNA are mapped onto normal metaphase chromosomes as deviations in the green to red fluorescence ratio profile along the chromosomal axis using dedicated software. A major advantage of CGH compared to conventional karyotypic analysis is the ability to assign the chromosomal origins of double minutes and homogenous staining regions. CGH can also be applied to archival paraffin embedded tissues and universal in vitro amplification of genomic tumour DNA by degenerate oligonucleotide-primed PCR allows the use of very small amounts of starting material. The main limitations of CGH are that balanced chromosome rearrangements are not detectable, its sensitivity is limited to 10-20Mb, it does not provide quantitative information about gene dosage and imbalances are detectable only if they are present in a substantial proportion (at least 50%) of cells. Nevertheless, in a variety of tumour types CGH has identified novel regions of gain, loss and high copy number amplification which are involved in tumour development and progression. In brain tumours, CGH has been used to define subtypes of tumours based on their copy number profiles. The presence of particular copy number aberrations has also been correlated with clinical outcome (including tumour recurrence, malignant progression, response to therapy and length of survival) and with in vitro chemosensitivity. The frequencies of micronuclei induced by aneugens are dependent on caspase-3 activity Ilse Decordier, Enrico Cundari, Micheline Kirsch-Volders Vrije Universiteit Brussel, Laboratory for Cell Genetics, Pleinlaan 2, B-1050 Brussels, Belgium. The biological relevance of accepting thresholds in risk assessment for aneuploidy induced by non-DNA interacting mutagens is supported by data obtained in vitro in human lymphocytes exposed to inhibitors of tubulin polymerisation (1, 2, 3). The in vitro cytochalasin B micronucleus assay can be used to assess thresholds for aneuploidy. It was shown by us in vitro in human lymphocytes with the cytochalasin-B micronucleus assay that the threshold value for chromosome non-disjunction was in general lower than that observed for chromosome loss (2). The fact that apoptosis can modify the frequencies of micronucleated cells in the surviving cell fraction is of major concern for accurate assessment of hazard related to exposure to mutagens (clastogens and aneugens) which are also apoptogens, and for risk assessment of aneugens. We demonstrated in vitro in human lymphocytes that nocodazole and carbendazim are capable to induce apoptosis, with involvement of caspase-9, -8 and -3 and that micronucleated cells, and at a far less extent cells presenting aneuploid nuclei resulting from non-disjunction, are found in the apoptotic cell population (3). Here we compare the induction of micronuclei by nocodazole and MMS in two human paired MCF-7 cell lines, one expressing caspase-3 and one deficient for caspase -3. To confirm the results obtained in the caspase-3 deficient cell line, an inhibition experiment by addition of a caspase-3 inhibitor (Ac-DEVD-CHO) in the caspase-3 proficient cell line was performed in parallel. Moreover apoptosis was monitored in the different experimental conditions by annexin staining. The results show that : - deficiency in caspase-3 expression leads to a lower but not completely suppresssed nocodazole dependent apoptotic reponse at both concentrations tested and a different statistically significantly lower induction of micronuclei but at the highest concentration of nocodazole (0.1 µM) only. - addition of the caspase-3 inhibitor to the caspase-3 proficient cell line induced a dose-dependent reduction of the micronuclei frequencies found in the untreated cells and an almost complete suppression of the nocodazole dependent micronuclei in the treated cells. These data suggest that caspase-3 is directly involved in the induction of micronuclei by nocodazole. Since our well validated scoring criteria include a strict selection of micronuclei with normal non condensed chromatin, similar in staining to the macronucleus (5) one should consider either that all nocodazole induced micronuclei correspond to very early apoptotic bodies, what should be surprising in non-synchronised cells, or that caspase-3 plays besides its function as an effector caspase in the apoptotic pathway, a direct role in the formation of micronuclei, independently of apoptosis. References: 1. Elhajouji et al. (1995) Environ. Mol. Mutag. 26, 292-304. 2. Elhajouji et al. (1997) Mutagenesis 12, 133-140 3. Kirsch-Volders et al. (2003) Toxicology Letters, in press 4. Decordier et al. (2002) Mutagenesis 17, 337-344. 5. Kirsch-Volders and Fenech (2001) Mutagenesis 16, 51-58 Acknowledgements:

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This study was supported by the EU research programs ENV4-CT97-0471 and QLK4-CT-2000-00058.

Chromosome malsegregation: a link between Alzheimer disease and Downs syndrome? Lucia Migliore Dipartimento di Scienze dell’Uomo e dell’Ambiente, Università di Pisa, Italy. We are interested to a unified hypothesis trying to relate Down’s syndrome (DS), trisomy 21 and Alzheimer’s disease (AD). Aged DS patients have neuropathological features (high levels of Aβ deposition in the brain), similar to those seen in AD individuals, which are correlated with neuronal disfunction leading to dementia by the age of 40. In families in which the Alzheimer's disease is inherited as autosomal dominant trait an excess of DS children and haematological malignancies is registered. An increased frequency of AD (about fivefold) among young mothers of individuals with DS is reported (Schupf et al., Neurology, 57, 979-984, 2001).

Previous cytogenetic studies performed by us (Migliore et al. Cytogenet Cell Genet 87;41-46, 1999, Trippi et al. Mutagenesis,16,4, 323-327, 2001) showed an increased frequency of aneuploidy in peripheral lymphocytes and fibroblasts of AD patients and a preferential occurrence of chromosome 21 in malsegregation events was observed in lymphocytes of sporadic AD patients. The observation that both pathologies (AD and DS) are characterized by the presence of aneuploid cells, taken together with the findings concerning the location of the Aβ-precursor-protein gene (APP) on chromosome 21, supports the hypothesis that trisomy for chromosome 21 could lead to AD later in life. We have now investigated the cytogenetic characteristics of peripheral blood lymphocytes of mothers who has a DS child in young age (DSM) by means of cytokinesis-block micronucleus assay (MN). The assessment of the basal level of chromosome malsegregation in interphase cells (chromosome loss and gain) was performed by applying a dual-color fluorescence in situ hybridization (FISH) with a centromeric DNA probe for chromosomes 13 and 21 and a cosmidic probe for the region 21q22.2. The study was performed on a group of 36 women who gave birth to a DS child before the age of 35 years and on a group of 23 female controls, with comparable age, who had healthy children. Results of MN assay indicate significant (p<0.001) higher levels of micronucleated lymphocytes of DSM respect to the control group (13.3±5.8‰ vs.5.7 ±2.7‰). Preliminary data of FISH analysis show that lymphocytes of DSM would be particularly prone to chromosome malsegregation of both chromosomes assessed. Alterated inhibition of Diazepam-mediated centrosome splitting leads to mitotic checkpoint activation and apoptosis-like death in tumour cells I. Vitale 1, A. Antoccia1 , S. Leone1, P. Crateri2 , G. Arancia,2 and C.Tanzarella1 1Dipartimento di Biologia, “Università Roma Tre”, Roma, Italy ; 2Laboratorio Ultrastrutture ISS, Roma, Italy. . Recently, chromosomal instability (CIN) has been largely reported in many human solid tumours. It has been proposed that defects in the mitotic apparatus and in particular in centrosome functioning may play an important role in the process of cancerogenesis increasing CIN. Furthermore, a G2-to M transition checkpoint mediated by an impaired centrosome functioning has been also proposed. Human primary fibroblasts and tumour cells were treated for 12 hours with 80 µg/ml of Diazepam (DZ), a benzodiazepine known to affect centrosome separation, and different endpoints related to checkpoint alterations, morphology of mitotic spindle, and apoptosis induction were analysed. Cells were immunostained with γ- and α-tubulin, or analysed at the electron microscopy (EM) to monitor the presence of monopolar and bipolar mitotic spindles. Interestingly, tumour cells showed a high percentage of bipolar spindles with non-congressed chromosomes, suggestive of a failure of DZ to arrest cells at a prophase stage. Tumour cells that attempted to enter mitosis in the presence of the drug were not able to proceed toward anaphase, were arrested by a mitotic checkpoint and incurred in a sort of “mitotic catastrophe”. The EM analysis of treated samples allowed us to detect mitochondria with markedly disorganized cristae and apoptotic cells with a morphology described in the literature as apoptosis–like. Such observation was also confirmed by immunofluorescence and immunoblot analyses indicating a caspase-3 independent process. On the other hand, the observation that the apoptotic terminal substrate PARP was cleaved indicated that the apoptotic-like process involves proteases other than caspases. The basal level of proteins involved in the G2/M transition (cyclinB1 and p34CDC2) was also assessed by immunoblot to check whether a less stringent control of centrosome splitting might be related to over-expression of such proteins. HeLa cells transfected with an antisense cyclinB1 oligo showed a reduction in the percentage of mitotic cells and a lower rate of metaphases with non-congressed chromosomes. In conclusion our results indicate that normal cells greatly differ from the tumor ones in particular for a prophase to metaphase p53-independent checkpoint responsible for aberrant chromosome segregation accompained by the activation of an apoptosis-like pathway.

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Analysis of chromosome changes associated with the progression of Barrett’s oesophagus S.H.Doak, G.J.S.Jenkins, J.M.Parry and E.M.Parry. Centre for Molecular Genetics and Toxicology, School of Biological Sciences, University of Wales Swansea, Swansea, SA2 8PP, UK. Barrett’s oesophagus is a pre-malignant lesion which in 10% of patients progresses to adenocarcinoma. The initial changes leading to Barrett’s development are strongly associated with chronic gastro-oesophageal reflux. However, the factors which lead from metaplasia through low and high grade dysplasia to adenocarcinoma are poorly understood. As an aid to providing an understanding of the mechanisms of Barrett’s progression, we have undertaken cytogenetic studies to determine the nature of any chromosome change that may be associated with the various stages in biopsies taken from both histologically normal and abnormal tissue. In these studies we initially analysed samples for the presence changes in all chromosomes using comparative genomic hybridisation (CGH). Using the data obtained from CGH we then focused our studies upon the use of chromosome specific centromeric probes for chromosomes 4, 8, 9, 20 and Y and gene specific probes for p16, p53 and Rb. Our studies indicated that genomic instability leading to aneuploidy was an early event in progression. The most clearly defined chromosome changes observed were amplifications of chromosomes 4 and 8. Although changes in chromosome 8 have previously been observed in some studies, our observations of the amplification of chromosome 4 are unique and provide a new target(s) for investigation. Male-mediated development defects and resulting chromosomal abnormalities Diana Anderson Department of Biomedical Sciences, University of Bradford, Richmond Road, Bradford, BD7 1DP, West Yorkshire, UK. In recent years, the public has become more aware that exposure of males to certain agents can adversely affect their offspring. The hazards associated with exposure to ionising radiation have been recognised for nearly a century, but interest was aroused when a cluster of leukaemia cases was identified in young children living in Seascale, close to the nuclear processing plant at Sellafield in West Cumbria. There was a civil court case on behalf of two of the alleged victims of paternal irradiation at Seascale against British Nuclear Fuels. The case foundered on “the balance of probabilities”. Nevertheless, there was support for paternal exposure from Japanese experimental X-ray studies in mice. The tumours were clearly heritable as shown by F2 transmission. In addition in humans, smoking fathers appear to give rise to tumours in the F1 generation. Using rodent models, developmental abnormalities/congenital malformations and tumours can be studied after exposure of males in an extended dominant lethal assay and congenital malformations can be determined which have similar manifestations in humans. The foetuses can also be investigated for skeletal malformations and litters can be allowed to develop to adulthood when tumours, if present, can be observed. Karyotype analysis can be performed on foetuses and adult offspring to determine if induced genetic damage can be transmitted. Using this study design, cyclophosphamide, 1,3-butadiene and urethane have been examined and each compound produced positive responses: cyclophosphamide in all endpoints examined, 1,3-butadiene in some and urethane only produced liver tumours in F1 male offspring. This suggests the endpoints are determined by independent genetic events. The results from heritable studies with 1,3-butadiene have been used in the parallelogram approach to determine a risk assessment for the germ cells in man. Micronucleus induction in mouse peripheral reticulocytes by 5-nitrofurantoin A. Fucic1, D. Markovic2, Z. Ferencic2 1Institute for Medical Research and Occupational Health, Zagreb, Croatia,2 PLIVA Pharmacuetical Industry Inc. Dept. Medicinal Safety, Zagreb, Croatia. 5-nitrofurantoin (furagin) is used in therapy of urinary infections in adults and children. However, the genotoxicity potential of this compound is still under investigation. According to published data (Slapsyte et al, 2002) the genotoxicity effect in children is detectable after long term application and increased risk of transitional cell carcinoma was reported in patients with a history of treatment by this compound. The aim of the current study was to test 5-nitrofurantoin potency to induce micronuclei in mouse reticulocytes. The peripheral blood cells were collected from tail vein and in vivo micronucleus assay was carried out according to the method of Hayashi et al (1990). Animals were divided into three groups (each group was 3 males and 3 females). Concentrations of 10mg/kg and 50 mg/kg were applied to animals. Cyclophosphamide at concentration of 75mg/kg was used as positive control. Animals were sampled before injection and again 48 and 96 h after treatment. The mean value of micronucleus frequency of control samples was 0,1%. The mean value of positive control was 7,1 %.

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The incidence of micronucleus frequency at dose of 10 mg/kg was 0,43% after 48h and 0,2% after 96h. In animals treated with 50 mg/kg micronucleus frequency was after 48h 0,46% and after 96 h 0,38%. For both concentrations response peak which significantly deviates from control values was after 48h. At concentration of 50 mg/kg the micronucleus frequency remains significantly higher after 96h than at concentration of 10mg/kg suggesting slow rate of elimination of 5-nitrofurantoin and prolonged genotoxic activity. It could be concluded that 5-nitrofurantoin increase significantly micronuclei frequency in mice reticulocytes and that slow rate of elimination could be reason of 5-nitrofurantoin accumulation and risk of genome damage after long term application. Acording to the described difference in farmacokinetics of furagin in adults and children (Wierzba, 1982) further research should be focused on experiments in young animals.

Persistence of chromatid cohesion at anaphase results in aberrant chromosome segregation. F. Degrassi, D. Cimini, M. Mattiuzzo, L. Torosantucci, Institute of Molecular Biology and Pathology, CNR, University “La Sapienza” Rome, Italy The successful segregation of chromosomes at mitosis relies on the coordinated execution of cytoskeletal and chromosomal events. Although the importance of mitotic spindle disturbances in causing unbalanced chromosome segregation is widely acknowledged, the role of defects in the dynamics of the chromatin during mitosis in promoting genomic instability is still largely unexplored. In this work we investigated the mechanisms promoting abnormal chromosome segregation when histones are maintained hyperacetylated during mitosis by inhibiting histone deacetylation with the specific inhibitor trichostatin A (TSA). To selectively investigate the role of histone modifications prior to mitosis on the fidelity of the mitotic process the exposure regimen was built to influence histone deacetylation either during synthesis of late replicating heterochromatic regions or during prophase mitotic condensation in exponentially growing human primary fibroblasts. Inhibition of histone deacetylation prior to mitosis produced defective chromosome condensation and impaired mitotic progression in time-lapse experiments on living cells, suggesting that improper chromosome condensation may induce mitotic checkpoint activation and metaphase delay. Immunofluorescence detection of heterochromatin-associated protein 1 (HP1) on kinetochores showed that HP1 was less efficiently recruited to the centromeric region of TSA-treated PtK1 and human fibroblast cells, indicating that heterochromatin centromeric structure is modified when histones are hyperacetylated. The metaphase delay observed in TSA-treated cells suggests that the mitotic checkpoint can detect not only kinetochore microtubule attachment but also chromosome three-dimensional structure during mitosis. Conformational defects in the centromeric region associated to HP1 dispersal may be the architectural defect monitored. In situ hybridization analysis on anaphase cells using chromosome-specific centromeric probes has been used to demonstrate that chromatin bridges observed at anaphase were sister chromatids that had remained connected along their arms after centromere separation and chromosome migration. Thus, our results show that the presence of hyperacetylated chromatin during mitosis impairs proper chromosome condensation during the pre-anaphase stages, resulting in poor sister chromatid resolution. This will cause persistence of cohesion along sister chromatid arms after centromere separation and chromatin bridges at anaphase. Lagging chromosomes consisting of single or paired sisters were also induced by the presence of hyperacetylated histones, indicating that the less constrained centromeric organization associated with heterochromatin protein 1 depletion may promote the attachment of kinetochores to microtubules coming from both poles (merotelic orientation). In conclusion, both chromosome structural aberrations and defective chromosome segregation are produced when histones are not properly deaceylated prior to mitosis.

Combined effects of modeled microgravity and ionising radiation on human primary lymphocytes E. Bortoletto1, M. Mognato1, G.L. Buglia2, M. Ferraro2, R. Cherubini3, L. Celotti1 and A. Russo1 1Department of Biology, University of Padova, Italy, 2Department of Genetics and Molecular Biology, University "La Sapienza", Rome, Italy, 3 INFN, Laboratori Nazionali di Legnaro, Padova, Italy. Space missions expose humans to an exogenous environment not encountered within our biosphere: in particular, the contemporary presence of radiation and microgravity. It has been observed that altered gravitational conditions of space flights have adverse effects on the lymphoid and erythroid haematopoietic systems; studies on DNA repair suggest that a disturbance of cellular repair in the microgravity environment might not be a complete explanation for the reported synergism of radiation and microgravity (Horneck, Mutation Res., 430: 221-228, 1999; Pross et al., Radiat. Res., 153: 521-525, 2002).

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The aim of our study is to investigate the response of G0-phase primary human lymphocytes exposed to simulated microgravity (Modeled MicroGravity, MMG) after in vitro irradiation with gamma rays (in the dose range 0-3 Gy) or low-energy protons (28 keV/µm, 0.5 Gy). MMG was obtained by incubating cells in the bioreactor Rotatory Wall Vessel (RWV) in the 24 h immediately following radiation exposure. Primary lymphocytes were stimulated either after MMG or simultaneously to the onset of MMG condition. Different parameters including mitotic index, cell survival, mutant frequency at the HPRT locus, chromosome aberrations and sister-chromatid exchanges (SCE) have been determined. Moreover, in some cell populations quantitative fluorescence in situ hybridization (Q- FISH) was applied to evaluate telomere length variation. The results recorded in samples obtained from different donors suggest that microgravity does not reduce T-cell proliferation if mitogenic activation with PHA or IL-2 is carried out after MMG; interestingly however, we found that radiation exposure followed by 24 h MMG incubation of G0 lymphocytes affects, after stimulation and with respect to 1xg, the mitotic index and the clonogenic cell survival. A sharp reduction of the mitotic index was observed in addition when lymphocytes were stimulated in MMG, as expected from literature data. We also concluded that MMG decreases the proportion of cells responding to stimulation, but does not affect their proliferation rate if eventually starting to cycle: in fact, according to differential 5-bromodeoxyuridine (BrdU) labelling the proliferation rate of growing lymphocytes did not differ between MMG and 1xg. As far as the chromosome aberration frequency is concerned, no significant difference was observed by comparing G0-phase lymphocytes stimulated after 24 h of MMG incubation versus 1xg. The same parameter could not be assayed for lymphocytes growing in MMG, because the adequate sample size could not be reached due to the strong reduction of proliferation occurring under these conditions. The combined effects of ionising radiation and MMG produced an increased frequency of chromosome aberrations (P < 0.005) and HPRT mutants. This synergism has been also observed in experiments conducted on lymphoblastoid cells belonging to the TK6 cell line (paper in preparation). Furthermore, we observed a significant difference (P < 0.05) in SCE frequencies observed in lymphocytes stimulated after 24 h incubation in MMG with respect to those maintained at 1xg. The increase was found either in untreated cells or in cells exposed to 0.5 Gy proton irradiation. On the other hand, as expected SCE frequencies were not induced after irradiation, and no synergic effects were visible. Evaluation of telomere length variation is in course; preliminary results do not indicate an effect of MMG, alone or in combination with ionising radiation, on the telomere size.

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DNA repair and genome instability: from bacteria to man Stephen C. West. Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD, UK - stephen.west@cancer.org.uk In recent years, we have become aware of a relationship between genome instability and predisposition to cancer. The mechanisms by which cells repair potentially mutagenic lesions and DNA breaks are therefore critical to the faithful reproduction of the genome and tumour avoidance. Our interest lies in the mechanisms by which chromosomal DNA breaks are repaired. There are two primary mechanisms in somatic cells: (i) Homologous recombination using a sister chromatid as a coding template and (ii) non-homologous end joining. The mechanisms of homologous recombination are highly conserved from simple organisms, such as bacteria, to yeast, and to man. Many of the enzymes that promote this process in human cells have now been identified and isolated. Central to the process is RAD51, a homolog of the bacterial RecA protein, which promotes homologous pairing and DNA strand exchange leading to the formation of crossovers between interacting DNA molecules. These recombination events take place within a nucleoprotein filament that is easily visualized by electron microscopy. RAD51 activity appears to be controlled by the tumour suppressor BRCA2, a large 380 kDa protein that has a number of RAD51 binding sites. Interactions between BRCA2 and RAD51 maintain RAD51 in monomeric form, thereby blocking the formation of active nucleoprotein filaments. BRCA2 mutations are found in individuals predisposed to breast and ovarian cancer, and BRCA2-defective cell lines exhibit gross a chromosomal instability phenotype, presumably due to defects in RAD51-mediated recombinational repair processes.

Also important for efficient recombinational repair is RAD52, a protein that plays a dual role by stimulating RAD51-mediated reactions and by promoting RAD51-independent single-strand annealing (SSA). Recent structural studies indicate that RAD52 forms a heptameric ring structure in which the single-stranded DNA wraps around the outer surface of the ring. Other proteins necessary for repair include RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3. In new work, it was shown that these proteins are components of the ‘resolvasome’, a protein complex that is responsible for Holliday junction branch migration and resolution. Remarkably, the mechanisms of Holliday junction processing appear similar to those mediated by the E. coli RuvA, RuvB and RuvC proteins. What is good for bacteria also appears to be good enough for man.

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Posters Nutrition and Carcinogenesis P1 1

P2

Antigenotoxic effect of water extract of Terminalia chebula S. Kaur , S. Arora , and S. Kumar 1 1 2

Department of Botanical and Environmental Sciences, 2Department of Chemistry, Guru Nanak Dev University, Amritsar, India Nutrition is essential to support life, but at the same time may paradoxically be the main cause of cancer. Doll and Peto estimated that approximately 30% of deaths in US are due to cancer. It appears to be possible to increase or decrease our cancer risk by taking appropriate dietary measures. Recent research has highlighted the immense importance of secondary metabolites present in plants. The secondary metabolites i.e. phenols, polyphenols, flavonoids, alkaloids etc. are known for their anticancer/antimutagenic properties. Terminalia chebula is an important medicinal tree, the fruit of which contains a large amount of polyphenols and tannins. It is useful in treating cough, asthma, urinary disorders, constipation and heart diseases. It is one of the most active components of herbal drug “Triphala”. Water extract of T. chebula was examined for its antimutagenic properties by two modes of experimentation i.e. co-incubation and pre-incubation in Ames histidine reversion assay. The antimutagenic effects were observed against direct-acting mutagens [sodium azide and 4-nitro-o-phenylenediamine (NPD)] and S9-dependent mutagen [2-aminofluorene (2AF)] in strains TA98 and TA100 of Salmonella typhimurium. With direct acting mutagens, water extract showed maximum inhibition of 55.89% in pre-incubation against sodium azide. The effect was found to be maximum in S9 dependent mutagen 2AF in TA100 tester strain of Salmonella typhimurium. The maximum inhibition observed was 75.22% and the effect was dose-dependent. Pure polyphenolic compounds i.e. gallic and ellagic acids and a polyphenolic glycoside have been isolated from water extract and compared with standard using various spectroscopic techniques viz. IR, 1H-NMR and mass spectroscopy.

Antioxidants can prevent arsenate exacerbated ultraviolet and visible radiation-induced DNA damage N. Morley, A. Curnow, L. Salter, T. Clifford and D. Gould Cornwall Dermatology Research Project, Royal Cornwall Hospital, Truro, Cornwall, UK There is increasing evidence that exposure to arsenic results in an increased risk of skin cancer. Certain parts of the UK are heavily contaminated with arsenic, in particular the South West of England, which also has amongst the highest rates of skin cancer. While the mechanism of arsenate (As(V)) carcinogenesis is far from clear, there are reports that arsenate can be substituted for phosphate causing oxidative damage in cells possibly by uncoupling oxidative phosphorylation. Additionally, ultraviolet (UV) and visible radiation is known to induce DNA damage in cells directly or indirectly via reactive oxygen species (ROS) and previous work in our laboratory has shown that these genotoxic effects can be exacerbated in cultured human lung fibroblasts by arsenate exposure. In this study, single cell gel electrophoresis otherwise known as the comet assay was used to assess the DNA damage induced by sodium arsenate with or without UV and visible radiation in cultured human lung fibroblasts, skin fibroblasts and epidermal keratinocytes. It was found that arsenate could induce DNA damage in human lung fibroblasts without an irradiation insult being necessary and exacerbated the DNA damage induced by UVA and visible radiation (320 - 700 nm) but not UVB (280 - 320 nm). This supports the evidence that arsenate causes DNA damage via ROS. This hypothesis was further supported by the finding that a reduction in UVA and visible radiation-induced DNA damage could be produced by incubating the arsenate-exposed cells concurrently with antioxidants prior to irradiation. Four antioxidants were examined; the thiols N-acetylcysteine and ergothioneine and the polyphenolic compounds epigallocatechin gallate and quercetin. These antioxidants were found to protect against UVA and visible radiation-induced DNA damage in each cell type studied but the effect produced was dependent on concentration. The findings of this investigation therefore indicate that UVA and visible radiation-induced DNA damage is exacerbated by arsenate exposure probably via additional ROS production and that protection against these genotoxic effects can be afforded with antioxidants. This work was supported by The Duchy Health Charity Limited.

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P3

P4

Interference of Myristica fragrens on the genotoxicity of methylmethanesulfonate in somatic cells of Drosophila melanogaster N. Mezzoug1, M. Idaomar1, A Muñoz serrano2, and A. Alonso-Moraga2

1 Université Andelmalek Essaâdi, Unité de Biologie Cellulaire et Moléculaire (BCM), Tétouan Morocco, 2 Universidad de Córdoba, departamento de Genetica, Córdoba, Spain In the last several years, greater attention has been given to the identification of chemopreventive dietary agents. The chemoprevention presents an effective strategy for either modulation or inhibition of pathological processes that result from exposure to an increasing number of mutagenic and/or carcinogenic compounds present in the environment. Thus a large number of naturally occurring anticarcinogenic compounds present in the food have been reported recently. Of these anticarcinogens, many are also antimutagens and the most of the mutagenic inhibitors are basically of plant origin. Myristica fragrans (Nutmeg), traditionally used as food additive, is tested for its antigenotoxic activity on methyl methanesulfonate (MMS) that induced mutant eye spots in the somatic mutation and recombination test (SMART) of Drosophila melanogaster. Oregon K-yellow females were crossed to Oregon k-white males. The flies were permitted to lay eggs in bottles for 3 days on food supplemented with the aqueous extract of Myristica at 0.5%; 1% and 2% and/ or MMS dissolved in water before mixing it into the treatment medium. The MMS was used at 0.5 and 1mM. The genotoxic activity of the food additive was also evaluated. Our results do not show any significant effect of the Nutmeg alone but it reduces the mutagenic activity induced by another agent. The mutagen used in this study is an alkylating agent known for its ability to react directly with DNA through covalent modification. This suppressing effect on MMS suggests that the extract of Nutmeg interacted with intact mutagen agent or its active form, in a desmutagenic manner, and that they did not affect the repair of DNA damage induced by MMS in a bioantimutagenic manner.

Antigenotoxic properties of some strains of lactobacilli used in Armenia K. Nersesyan* Cancer Research Centre, Yerevan, Armenia In view of the high incidence of dietary-related tumors, an important research goal is to identify the nutritional factors that may decrease the activity of carcinogens. Many investigations have shown that certain strains of lactic acid bacteria (LAB) consumed with fermented milk products have antigenotoxic properties. The aim of the present work was to study possible antigenotoxic activity of some strains of lactobacilli used in Armenia (Lactobacillus acidophilus INMIA 9602 – biologically active food additive, produced by VITAMAX-E, Armenia; Lactobacillus acidophilus Er 317/402 - contained in fermented milk used for babies’ food, produced by Academy of Veterinary, Armenia; Bifidobacterinum bifidum N1, 791 – medicine Bifidumbacterinum, produced by BioMed, Russia). In in vivo experiments LAB were administered by stomach tube or injected into the abdominal cavity of rats, mice and gray Armenian hamsters at various doses. 6-24 h after cyclophosphamide or N-metyl-N-nitro-N-nitrosoguanidine (MNNG) [at doses that induced 15‰of micronuclei (MN) in bone marrow polychromatic erythrocytes (mice and hamsters) or 15% of metaphases with chromosomal aberrations (rats)] were injected into the abdominal cavity of rodents. 24 h after possible cytogenetic abnormalities were investigated in bone marrow. In in vitro experiments the isolated primary colon cells from rats were incubated with MNNG and LAB (30 min), then possible DNA damage was studied by means of the Comet assay. It has been shown that all LAB studied have antimutagenic (anticlastogenic) properties. All LAB decreased significantly clastogenic action of chemicals on bone marrow cells of all species of rodents. It is noteworthy that mutagenic action of indirect-acting chemical agent cyclophosphamide was decreased more substantially than that of MNNG – direct acting mutagen. It may be connected with the influence of LAB on the process of metabolic activation of chemicals in organism. The most potent antimutagenic property has Bifidumbacterinum. By means of the comet assay it has been shown that all LAB have antigenotoxic action, and prevent colon cells DNA damage induced by MNNG. As in in vivo studies, the most potent antigenotoxicant was Bifidumbacterinum. Lactobacillus acidophilus Er 317/402 showed high antimutagenic property in vivo, but was weakly active in the Comet assay. Further investigations of Bifidumbacterinum, which shows high antigenotoxic activity both in vivo and in vitro, on possible anticarcinogenic action of are certainly warranted. *This work was partly supported by UICC ICRETT award.

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Antimutagenic effect of essential oil of basil and linalool T. Beric-Bjedov, D. Mitic, B. Vukovic-Gacic, J. Knezevic-Vukcevic, D. Simic Chair of Microbiology, Faculty of Biology, University of Belgrade, Belgrade, Yugoslavia Essential oil of basil (Ocimum basilicum L.) and its major monoterpene-linalool show antimutagenic effect in microbial tests: E. coli, S. typhimurium and S. cerevisiae. Inhibition of spontaneous and UV-induced mutagenesis varies in the range of 49-77%, depending on screening test. Antimutagenic effect may be the concequence of multiple mechanisms involved in DNA replication and repair. The effect of essential oil of basil (EO) and linalool on DNA repair mechanisms was tested with E. coli assay system (Simic et al., 1997; 1998). In different genetic backgrounds, genetic endpoints such as spontaneous and induced reversions, excision repair, recombination, and induction of SOS repair were monitored. EO and linalool have no effect on spontaneous mutation frequency in both repair proficient and mismatch repair deficient strains. Antimutagenic potential of both EO and linalool against UV-induced mutations is diminished in excision repair deficient mutant. EO and linalool had no effect, either reduction or stimulation, on SOS induction, but sligthly stimulate intrachromosomal recombination in recombination proficient strain. Our data obtained with E. coli K12 assay-system indicate that EO as well as monoterpene linalool from basil inhibit mutagenesis by enchancement of error free DNA repair processes, and may be clasified as bioantimutagen.

Use of antioxidant vitamins C and E for the “protection” of human lymphocytes from the effects of external factors Κ. Maridaki 1 , G. Christopoulos , Ν. Messini-Nikolaki , S. Tsilimigaki 1 and S. M. Piperakis . 2, 2,1 2 1

1 DNA Repair Laboratory, Institute of Biology, NCSR “Democritos”, Athens, Greece, 2 Department of Cell Biology, University of Athens, Athens, Greece. Oxidative damage to DNA by reactive oxygen species may be important in mutagenic, carcinogenic, and aging processes. Although it is plausible that antioxidant vitamins may reduce oxidative DNA damage, evidence from human studies has been inconsistent. In the present work the antioxidant capacity of vitamins C and E to protect human cells from damage induced by UV, γ-radiation and H2O2 was investigated. Human lymphocytes pre-treated with various concentrations of vitamins C and E were exposed to these factors and the amount of DNA damage and repair was estimated with the comet assay. Our results indicate that the antioxidant vitamins C and E may offer some “protection” to the human lymphocytes when exposed to these mutagens.

Influence of folate status on genomic stability: in vitro effects on chromosome structure, chromosome segregation and mutagen sensitivity Paola Leopardi, Francesca Marcon, Andrea Zijno and Riccardo Crebelli Istituto Superiore di Sanita’, Rome, Italy Folic acid plays a key role in DNA metabolism, entering in the biosynthesis of DNA precursors and DNA methylation. Low folate status is associated with global DNA hypomethylation, depression of DNA synthesis, and genetic alterations such as expression of fragile sites and chromosome fragility, likely due to the impairment of DNA repair capacity. Such events are believed to be mechanistically involved in the clinical traits associated to low folate status, i.e. increased incidence of some cancer types and developmental defects. In addition to environmental (diet) and life style (smoking) factors, also genotype may contribute to unbalance folate metabolism. Polymorphic variants of the methylentetrahydrofolate reductase (MTHFR) gene have been identified in normal human populations. This gene codes for an enzyme which catalyses the key reaction of the folate cycle, i.e. the conversion of 5, 10-methylentetrahydrofolate into 5-methyltetrahydrofolate, the methyl donor for the methylation of homocysteine to methionine. The MTHFR variants, characterized by reduced enzymatic activity, in conditions of low folate status, have been associated to increased risk for tumors at various sites (colon, rectum, breast and ovary) and to an increased maternal risk of Down syndrome, highlighting the central role of gene-environment (diet) interactions in the phenotypic expression of unbalanced folate metabolism. The mechanism(s) by which low folate status and MTHFR deficiency interact raising the risk of tumors and misconception are not yet elucidated, though hyperhomocysteinemia, global DNA hypomethylation, and unbalance of nucleotidic pool have been proposed to play a causative role. In order to get some insights on the underlying mechanisms, in this study the effects of the MTHFR polymorphism on chromosome stability and segregation have been investigated in the presence of different levels of folic acid. To this aim, peripheral lymphocytes of MTHFR wild type and variant subjects (both homozygotes and heterozygotes) have been

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cultured in vitro in presence of high (120 nM), normal (60 nM) and low folate levels (24 and 12 nM) for eight days. Cytokinesis was blocked with 4.5 µg/ml cytochalasin B, cells were harvested 28 hrs later and analysed for the presence of micronuclei and other relevant nuclear abnormalities (buds and nucleoplasmic bridges). The results so far obtained show a significant increase of Mn in cultures grown in presence of low folic acid, irrespective of the MTHFR genotype: the incidence of cells with micronuclei was three to four fold higher at 12 nM than at 120 nM (48, 39, 46‰ at 12 nM vs 12.5, 14, 12.5‰ at 120 nM for MTHFR+/+, MTHFR+/- and MTHFR-/-, respectively). Mn were characterized by immunofluorescence using CREST antibodies, in order to distinguish chromosome fragments (Mn K-) from whole chromosomes (Mn K+): the results obtained show that the relative proportion of Mn K- and Mn K+ was not affected by the experimental conditions, i.e. that both chromosome breakage and chromosome loss were induced by folic acid deficiency in MTHFR wild type and variant genetic background. In addition to the effects on baseline levels of chromosome damage, the effect of folate level/MTHFR genotype on radiation sensitivity was also investigated. To this aim, lymphocytes were cultured eight days in presence of low concentrations of folic acid, and then challenged with 0.5 Gy 137Cs gamma-rays. Afterwards cells were cultured further 28 hours and the incidence of micronuclei evaluated by the cytokinesis-block method. Preliminary results indicate a higher sensitivity to ionising radiation for cells of both homozygous and heterozygous variant than wild type MTHFR subjects, when grown in conditions of partial folate deprivation: in irradiated cells cultured with 12 nM folate the incidences of micronuclei were 36, 71, and 88 ‰ for MTHFR+/+, MTHFR+/- and MTHFR-/-, respectively, while in the unirradiated cells the frequencies of Mn at 12 nM folic acid were 48, 39, 46 ‰ for MTHFR wild type, heterozygotes and homozygotes subjects, respectively. P8

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The role of dietary vitamins A, C and E in genotoxicity of N-nitrosomorpholine S. Robichová, D. Slameňová, and I. Chalupa Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia It is generally known that a high intake of fruits and vegetables can decrease the risk of cancer development. The dietary components including vitamins A, C and E, have the ability to quench the carcinogenic potential of reactive oxygen species and can modify the process of carcinogenesis. Hydrophilic vitamin C is considered to be a powerfull reducing agent in the venicity of DNA. Vitamins A and E, which are soluble in organic solvents and lipids, play a role in cellular membranes mostly. Protective effects of these vitamins against the genotoxicity of N-nitroso compound (NNC), N-nitrosomorpholine (NMOR), were studied. NMOR belongs to the group of N-nitrosamines which are indirect-acting, and need metabolic activation. By the comet assay we proved that NMOR caused a dose-dependent increase of DNA strand breaks in three cell lines with different activity of metabolizing enzymes (HepG2, V79 and VH10); however, the levels of DNA migration in electric field were unequal in individual cell lines. The number of chromosomal aberrations was significantly increased only in HepG2 and V79 cells, no changes were observed in VH10 cells. In further experiments HepG2 cells were pretreated with vitamins A, C and E. Each vitamin significantly decreased the percentage of tail DNA formed after NMOR treatment, but the reduction of the clastogenic effects of NMOR in HepG2 cells was observed only after pretreatment with vitamins A and E. In our experiments vitamin C did not alter the frequency of NMOR-induced chromosomal aberrations. On the basis of these results we suppose that NMOR induces DNA damage by two different ways; indirectly through N-hydroxylamines which are generated by drug-metabolizing enzymes and directly via formation of NO radical. In addition, pretreatment of HepG2 cells with vitamins A, C and E which can scavenge NO radical, decreased the genotoxic effects of NMOR.

Food-borne compounds and human colon sulphotransferases: bioactivation and chemoprevention W. Meinl, and H.R. Glatt German Institute of Human Nutrition, Potsdam-Rehbruecke, Germany The incidence of colon cancer in industrial nations is high with an increasing tendency. However, causing agents are largely unknown, although epidemiological observations suggest a prominent role of nutrition. Food is a complex chemical mixture containing nutrients, but also large quantities of non-nutritive components (xenobiotics) of natural origin or produced during food processing, for instance. Elimination of xenobiotics usually requires biotransformation conferred by a xenobiotic-metabolising enzyme system. Expression of this system is tissue-dependent. While various cytochromes P450, well known toxifying enzymes, are preferentially expressed in liver, some conjugating enzymes demonstrate different tissue distributions, often with high levels in the gut. Cytosolic sulphotransferases (SULT) play an important role in xenobiotic metabolism by transferring the negatively charged sulpho moiety of the co-factor 3'-phosphoadenosyl-5'-phospho-sulfate (PAPS) onto nucleophilic groups of their substrates, which often facilitates their excretion. However, the sulphate group, formed by O-sulphonation, is a good leaving-group in certain chemical linkages, e.g. sulphate-esters of benzylic or allylic alcohols or hydroxylamines, i.e. if the resulting carbenium or nitrenium ion is resonance-stabilised. Therefore, sulpho conjugation bears a relatively high risk of the formation of electrophilic metabolites that may react with DNA and proteins and eventually lead to the induction of mutations and neoplasias. Three SULT forms 1A1, 1A3, and

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1B1 were identified by immunoblotting in the human colon. Using Ames' Salmonella typhimurium TA1538 strains genetically engineered for the expression of these human SULTs, we show that human SULT1A1 is very efficient in the activation of N-OH-PhIP and of N-OH-MeAaC (metabolites of heterocyclic amines formed during cooking of meat or fish), It also activated 5-hydroxymethylfurfural (a major pyrolysis product of carbohydrates), nitrofen (a herbicide that has led to a wide contamination of food and feed in 2002 in Germany, although its use has been forbidden for a long time) and 1'-hydroxysafrole (a metabolite of a phytochemical present in various spice plants). Likewise, 4-oxocyclopenta[def]chrysene, an environmental pollutant from diesel exhaust, was reduced by human intestinal bacteria to the corresponding alcohol and then specifically activated by SULT1B1. We suspect that the major physiological function of human colon SULTs is the conjugation of aglycons released in the colon from secondary plant metabolites. We found that small amounts of fruit juices, dealcoholised wine, tea or coffee were able to prevent the activation of OH-PhIP in SULT1A1-expressing Salmonella strains. This effect was mimicked by various polyphenols. These phenols (caffeic acid, flavonoids and lignans) proved to be competing substrates and/or inhibitors of SULTs. For example, quercetin inhibited SULT1A1 with an IC50 of 30 nM. The results of these in vitro experiments suggest that human SULT may be mediators of colon carcinogenesis (via the activation of food-borne promutagens) and targets for chemoprevention (by administration of high-affinity ligands). P10 Immunotoxicity and genotoxicity of fumonisins: Implication for their carcinogenic effects E. Martinova1 and O. Ivanchenko2 1 State Institute of Nutrition RAMS, Moscow, Russian Federation 2 Kazan’ State Technological University, Kazan’, Russian Federation Fumonisins are the group of mycotoxins produced by fungi Fusarium proliferatum, F. moniliforme and allied species spread widely abroad. Fumonisins of group B (FB1, FB2, and FB3) were shown to be the human and animal carcinogens. Fumonisin B1 is predominated over the other members of its group although these compounds may contaminate the corn together. Besides the facts that fumonisins B disrupt the sphingolipid metabolism and possess the toxic effect on cells, the molecular basis for their carcinogenic capacities is still unclear. According our data, fumonisins B1 and B2 have the strong immuno-modulative and genotoxic capacities. Using C14Fumonisin B1, it was found that less then 0,01% of exposed FB1 was accumulated into immune organs but it was enough to disrupt the lymphocyte activation, differentiation, and immune response to T-dependent antigens. Fumonisins impair the CD3, CD4, CD8, CD25, CD69, CD71 receptor expression to decrease the number of antibody-forming cells. FB1 switches the T helper differentiation from Th1 to Th2 cells, regulates the cell memory formation and the secondary immune response. All fumonisins B modulate the lymphocyte receptor expression but exhibit the different dose- and time-dependent effect. FB1 and FB3 exposure in vivo to mice (BALB/c, CBA, ASnL, SGL, etc.) results in the similar effect on the lymphocyte surface receptor number and density whereas FB2 has the opposite action on the same targets. Being exposed together, three fumonisins may modulate the immune response (from strong inhibition to activation) dependent on this mixture composition. FB1 alters the adhesion molecule expression, cell adhesion to the different surface, cell morphology and cell-cell communication. In vivo, FB1 disrupt the lymphocyte differentiation in thymus to cause the autoimmune pathology. FB1 and FB3 block the [H3]thymidine incorporation into lymphocytes and prevent the immune cell proliferation in vivo and in vitro. FB2 action on cell proliferation is dependent on the dose and time of its exposure. At the same time, FB1 was shown to be mitogenes for many transformed cell lines. Taken together, these results may explain the secondary immune deficiency under fumonisins exposure that creates the basis for growth of transformed and malignant cells. Because about 90% of FB1 is egested from organism in the unmetabolized form through the gastro-intestinal tract, we were studied the FB1 effects on the colon ecosystem including the local immune response, probiotics, etc. It was clear shown that FB1 caused the DNA damage in the tester E.coli strains. FB1 prevents the growth of Lacto bacterium acidophilus, Bifidobacterium and E.coli. in the dose-dependent manner. In the normal lymphocytes obtained from thymus, spleen, peripheral lymph nodes, as well as in lymphocytes associated with intestine and colon, FB1 may initiate an apoptosis dependent on the phase of cell cycle. FB1 exposure after antigen recognition leads to the activation-induced apoptosis. Mechanism of FB1 effect on the immune cells is due to both, the sphingomyelin cycle initiation followed by ceramide production, and recognition of the FB1-specific receptors on the cell surface. Signaling pathways of FB1-initiated apoptosis includes, inter alia, mitochondrial alterations that allow inhibitors of the mitochiondrial respiration chains to regulate the FB1-dependent apoptosis. As the inhibitor of ceramide synthase, FB1 regulates the formation of pool of ceramide in the response of some receptor recognition. Because ceramide regulates the expression of antigen-specific receptor of T cells, TCR, as well as composition of the lipid microdomains included signaling molecules, FB1 may regulate many vital processes into cells. Wide spreading of fumonisins and contamination of most grass families by these mycotoxins predispose the human and animal organisms to the secondary immune deficiency, DNA damage, growth of the transformed cells, and, finally, neoplastic growth.

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Antimutagenic effect of resveratrol and diallyl sulphide and its effect on the immune response in mice Bárta, I., Šmerák, P., Šestáková, H.,1) Polívková, Z., Bártová, J., Turek, B.,1) Langová,M., Anděl, M. 3rd Faculty of Medicine, Charles University, Ruská 87, 100 00 Prague 10, Czech Republic National Institute of Public Health, Šrobárova 48, 100 00 Prague 10, Czech Republic Exposure of the population to carcinogenic substances represents an important factor in the incidence of tumor diseases. Aside of chemical carcinogens mostly anthropogenic in character, there exists a large group of natural substances that have genotoxic effects and are demonstrable mutagens and carcinogens. On the other hand there are various kinds of vegetables, fruit and other plants with anti-carcinogenic, anti-mutagenic, immunostimulatory or immunomodulatory effects. Resveratrol and diallyl sulfide were applied to mice for three days in doses of resveratrol 5mg/kg and diallyl sulfide 100mg/kg, the third day a carcinogen was also applied – either aflatoxin B1 (AFB1) 1mg/kg, 2-amino-3-methylimidazo[4,5-f]quinoline 20 mg/kg, or N-nitroso-N-methylurea (MNU) 20 mg/kg. Resveratrol and diallyl sulfide demonstrably inhibits the mutagenic activity of three reference mutagens: aminomethylimidazo-quinoline (IQ), aflatoxin B1 (AFB1), and N-nitroso-N-methylurea (MNU) in the mammalian model (micronucleus test) in the bone marrow of laboratory mice. In the prokaryotic model (Ames test) a strong inhibition of the mutagenic activity of IQ and AFB1 by resveratrol and diallyl sulfide has been demonstrated. The protective effect of resveratrol and diallyl sulfide was assessed by the chemiluminescence method detecting the production of oxygen radicals, namely the production of the complex of hydrogen peroxide-myeloperoxidase-halid cofactor during phagocytosis, and by the blastic transformation method testing the functional capacity of T-lymphocytes. The phagocytic function of murine peritoneal granulocytes was suppressed significantly by all three carcinogens tested just like blast formation from mitogen-stimulated murine splenocytes. There was no statistically significant difference between the groups of animals treated with resveratrol and diallyl sulfide alone and those not treated, in chemiluminescence follow-up and as regards blastic transformation. In all of the experiments resveratrol and diallyl sulfide to a significant degree repaired the immunosuppressive effects of all three carcinogens tested. Results of this study indicate that, resveratrol and diallyl sulfide may play an important role in the prevention of carcinogenesis by modulation of immunosuppression caused by carcinogens. Supported by project Grant No. NJ/7462-3/03 from IGA at the Ministry of Health of the Czech Republic and Research Plan No. MSM 111200001.

DNA repair gene expression analysis in primary human G0 lymphocytes cultured in simulated microgravity after in vitro irradiation with X-rays M.Mognato1 and L. Celotti1

1Dipartimento di Biologia, Università degli Studi di Padova, Italy Exposure to ionising radiation induces a large spectrum of DNA lesions which in healthy individuals are repaired by different pathways. We analysed by quantitative Real-Time PCR (RT-PCR) the transcriptional profiles of fourteen known DNA repair genes (DDB2, GADD45A, XPC, BRCA1, BRCA2, k70, k80, p21, XRCC1, RAD51, PCNA, XRCC4, DNA ligase I and DNA ligase IV) in primary human G0 lymphocytes irradiated in vitro with 2Gy of X-rays and immediately transferred to Modeled MicroGravity (MMG) for 24 hours. The aim of this work was to study if the reduced gravity simulated on Earth by the “Rotary Cell Culture System Bioreactor” (Synthecon, USA), could affect the expression of DNA repair genes. Two 50 ml vessels/experiment, filled with a 1x106/ml cell suspension, irradiated and non-irradiated respectively, were placed in the bioreactor inside the humidified incubator for microgravity simulation. In the rotating system, gravity is balanced by equal and opposite mechanical forces (centrifugal and Coriolis) and the gravitational vector acting on cells is reduced to about 10-2 g (Schwartz et al., 1992). Ground based (1xg) lymphocyte cultures, both irradiated and non-irradiated, were grown at the same cellular density in 75cm2 flasks during the 24 hours of simulated microgravity. 24h later, total RNA was isolated from samples and retrotranscribed. Quantification of each mRNA target was performed according to Pfaffl (2001) using SYBR Green kits as recommended by the manufacturer. Genes analysed in the present work are DDB2, GADD45A, XPC, XRCC1, p21 and PCNA involved in NER-BER repair pathway; k70, k80, XRCC4 and DNA ligase IV of the non homologues end joining pathway (NHEJ); BRCA1, BRCA2 and RAD51 playing an important roles in homologous recombination (HR); DNA ligase I involved in the repair of single strand breaks. MMG seems to affect gene expression with inter-individual difference among the nine donors analysed. The information obtained will be helpful to estimate risk exposure to cosmic radiation associated to the reduced gravity of space-shuttle crew and to clarify the molecular mechanisms of the early steps of carcinogenesis. References [1] Pfaffl M.W., A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research, 29, pp. 2003-2007 (2001). [2] Schwartz R.P., T.J. Goodwin, and D.A. Wolf, Cell culture for three-dimensional modelling in rotating-wall vessels: an application of simulated microgravity. J. Tiss. Cult. Meth. 14, pp. 51-58 (1992).

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P13 Contents of nuclear buds and micronuclei in folate-deprived human lymphocytes. Hanna K. Sandvik1, Xu Wang2,3, Hilkka Järventaus1, Ghita C.-M. Falck1, Hannu Norppa1, and Michael Fenech2 1 Laboratory of Molecular and Cellular Toxicology, Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Helsinki, 2 CSIRO Health Sciences and Nutrition, Adelaide BC, Australia, 3 School of Life Sciences, Yunnan Normal University, Kunming, Yunnan, People's Republic of China Micronuclei (MN) are formed from acentric chromosome or chromatid fragments and whole chromosomes that lag behind in anaphase and are left outside the daughter nuclei in the subsequent telophase. Nuclear buds (NB) resemble MN morphologically, with the exception that a thin nucleoplasmic connection joins them with the nucleus. The mechanisms of NB formation are presently unclear. An increase in both NB and MN has been observed in cultured human lymphocytes deprived of folate. Folate functions as a methyl donor in the synthesis of dTMP from dUMP, and its deficiency causes misincorporation of uracil into DNA, which can lead to chromosome damage. In tumour cells, NB have been proposed to be involved in the process of eliminating amplified genes from the nucleus during the S phase. Similarly, the NB observed in the folate-deprived cells might reflect extrusion of amplified DNA generated by the breakage-fusion-bridge cycle. Such NB should contain no centromeric or telomeric sequences. Alternatively, NB may be formed in cell division either from laggards left in the proximity of the nucleus or from remnants of broken anaphase bridges. In the former option, the contents of NB would be expected to resemble those of MN, while in the latter centromeric and telomeric sequences would probably be rare. The aim of the present study was to investigate if the con-tents of NB differ from those of MN, and to better understand how NB are formed in folate-deficient cultures. Lymphocytes from eight female donors were cultured for 9 days in the presence of phytohaemagglutinin, interleukin-2, and 12, 24, 60 or 120 nM folic acid. Cytochalasin-B was added on day 8 to inhibit cytokinesis. The frequencies of MN and NB were scored in 1000 binucleate cells per donor per experimental point, and FISH with directly labelled human pancentromeric (TRITC, red) and pantelomeric (FITC, green) probes was used to characterise the contents of a total of 738 MN and 541 NB. MN and NB containing only red (telomere) label were considered to harbour terminal acentric fragments. The presence of both green (centromere) and red label indicated entire chromosomes. If no signals were present, the contents were considered to represent interstitial fragments. NB containing only telomeric label and those without any signals formed the largest groups of NB. At 12 nM folic acid, NB containing only telomeric label constituted 51.4 % and those without signals 34.2 % of NB. Most MN harboured only telomeric signals. At 12 nM folic acid, 71.2 % of the MN showed only telomeric signals, while 11.3% were without signals. MN with telomeric label had more often two green spots (18.0%) than NB with telomeric label (3.4%), suggesting that chromosome-type acentric fragments were rarer in NB than MN. Whole chromosomes were more often found in MN than NB, being responsible for 16.6 % (12 nM) and 18.8 % (120 nM) of MN and for 10.9 % (12 nM) and 3.4 % (120 nM) of NB. Decreasing folic acid concentration from 120 nM to 12 nM had a dose-dependent increasing effect on the frequency (per 1000 cells) of NB with telomeric signals (2.1-fold) without signal (1.9-fold) and with both signals (10.4-fold), and on MN with telomeric signals (2.2-fold), without signals (1.8-fold) and with both signals (1.7-fold). In conclusion, the findings suggest that about half of all NB are formed from acentric terminal fragments, mainly of the chromatid-type, probably during cell division. About one third of all NB contain no telomeres or centromeres, and represent either amplified DNA that is being eliminated during the S phase or broken bridges formed during cell division. About 10% or less of the NB harbour whole chromosomes. In comparison with NB, MN contain more terminal acentric fragments (about 2/3 of all MN) and whole chromosomes (about 1/5 of all MN) but less interstitial DNA fragments (about 10% of all MN). Further studies are required to clarify if NB in non-tumour cells are really formed from amplified DNA. This mechanism would have important consequences for the interpretation of MN as well, since NB expelled from the nucleus during S-phase may transiently exist as MN in the cytoplasm before expulsion from the cell; i.e. MN originating from NB would not require the completion of nuclear division to be expressed. DNA Damage and Repair P14 The role of pol II, pol IV and pol V- SOS- induced DNA polymerases in spontaneous mutagenesis of E. coli AB1157 mutD5, proofreading defective strain E. Grzesiuk, C. Janion and A. Nowosielska Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland It has been observed (also in our lab, unpublished) that E. coli pol III bacterial mutants, defective in 3'-5'- proofreading exonuclease, chronically induce the SOS response. Fijalkowska and Schaaper (1996) suggested that this high frequency of induced mutations in proofreading-defective E. coli mutD5, results from too many mistakes made by pol III DNA polymerase in course of DNA replication that leads to inhibition of dam-dependent mismatch repair. Alternatively, enhanced mutation frequency in mutD5 strains might arise by the action of SOS-induced mutagenic DNA polymerases. It

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is possible that the misaligned pair of the bases at the primer terminus is poorly elongated by pol III and synthesis of DNA is much impaired. The role of SOS induced DNA polymerases might be to facilitate further elongation of the misaligned pairs, hence preserving mutations. Of the SOS-induced DNA polymerases only pol II possesses proofreading activity and hence it might excise the mismatched base and allow the right one to be incorporated. We constructed E. coli AB1157 mutD5 strains deleted for SOS DNA translesion polymerases in various combinations and examined frequencies and specificity of spontaneous mutations arising under growing and non-growing conditions. The results were unequivocal. While deletion of either umuDC (pol V) or dinB (pol IV) genes in AB1157 mutD5 causes a 2-3-fold reduction of the growth- dependent spontaneous mutations, deletion of the polB (pol II) gene increases it 1.6-fold. However, deletion of both dinB and umuDC (pol IV and pol V) or polB and umuDC (pol II and pol IV) leads to a decreased frequency of mutation by a factor of 2.8-fold and 5-fold, respectively. Deletion of polB and umuDC (pol II and pol IV), or deletion of all the SOS polymerases decreases growth-dependent spontaneous mutation only marginally. In a mutD5 strain incubated under non-growing conditions, deletion of SOS-dependent polymerase genes has a lesser effect on spontaneous mutation. Spontaneous mutagenesis in non-growing mutD5 dinB (pol IV), mutD5 umuDC (pol V) and mutD5 umuDC dinB (pol IV pol V) strains was about two- fold, or 1.5-fold higher than in a mutD5 strain. Note that in all these strains, DNA polymerase pol II was present. Moreover, the spontaneous mutations arising under growing and non-growing conditions also differed in mutational specificity. In strain AB1157 it is possible to differentiate between back mutations to wild type (at a UAA argEochre site) and suppressor mutations in tRNA genes. While spontaneous mutations in growing cells arise mainly by suppressor formation, in non-growing cells almost no suppressor mutations have been found. In summary, these studies show that mutagenic polymerases are involved in spontaneous mutations arising in growing and non-growing cells, and they are probably in competition with each other. In the case of non-growing cells (probably because there are less SOS-induced polymerases present) competition is much less, and pol II, as the only low level constitutively expressed SOS polymerase, increases slightly spontaneous mutability perhaps by reinitiating of the replication process.

P15 Measurement of 8-oxo-7, 8-dihydro-2’ deoxyguanosine and related nucleoside and nucleotide in human urine Badouard, J.-L. Ravanat, T. Douki, J. Cadet & A. Favier. Laboratoire des Lésions des Acides Nucléiques; DRFMC/SCIB & FRE 2600; C.E.A-Grenoble, 38054 Grenoble Cedex 9, France.

Oxidative stress exposes the cells to different radicals, such as the reactive hydroxyl radical (•OH) that, upon reaction with DNA generates, in addition to strand breaks, a wide range of pyrimidine and purine-modified DNA bases. 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is one of the most important lesions and has been extensively studied. There are two main pathways to repair DNA damage: base excision repair (BER) and nucleotide excision repair (NER). Oxidized DNA bases are expected to be mainly repaired by the BER mechanism and as a result, 8-oxo-7,8-dihydroguanine (8-oxoGua) is supposed to be excised and eliminated in urine. Therefore, the measurement of 8-oxoGua (and 8-oxodGuo that could be the product of the NER pathway) in human biological fluids has been proposed as a biomarker of in vivo oxidative stress (1). The purpose of the present work was to determine the relative amount of the different forms of 8-oxodGuo that could be detected in human urine. HPLC coupled to tandem mass spectrometry (HPLC-MS/MS) is used to determine the level of 8-oxoGua, 8-oxodGuo as well as the corresponding ribose derivative, namely 8-oxo-7,8-dihydroguanosine (8-oxoGuo) (2). In addition, attempts were made to determine if urine contains significant amounts of the corresponding nucleotide derivative (8-oxodGMP) as well as 8-oxodGuo containing oligonucleotides. Preliminary results and the different approaches that have been used to prepurified human urine, prior to HPLC-MS/MS measurement (subsequently to adequate enzymatic digestion) will be presented. The presence, in significant amounts of 8-oxodGuo containing oligonucleotides, could explain, at least partly, the discrepancies observed for the measurement of 8-oxodGuo in human urine using either HPLC-electrochemical detection or immunodetection using ELISA (3).

References: 1 Shigenaga, M. et al.. (1989) Proc. Natl. Acad. Sci. USA 86, 9697-9701. 2 Weimann, A. et al. (2001) Free. Rad. Biol. Med. 30, 757-764. 3 Shimoi, K. et al. (2002) Cancer Epidemiol Biomarkers Prev 11, 767-70.

P16 S phase checkpoint in human cells following treatment with topoisomerase inhibitors M.Fiore, S.Albini, R.Zanier, R.Ricordy and F. Degrassi Institute of Molecular Biology and Pathology, CNR, “La Sapienza” University, Rome, Italy The aim of this work is to study S phase checkpoint modulation in human cells after treatment with two topoisomerase inhibitors (camptothecin and etoposide). In order to elucidate a possible involvement of p53 and replication protein A

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(RPA) in the S phase checkpoint we have investigated Bromodeoxyuridine (BrdUrd) incorporation and RPA phosphorylation in three human cell lines that differ in p53 status. The results show that after topoisomerase inhibitors the DNA replication block is more efficient in p53 expressing cells and that in these cells RPA phosphorylation is a crucial event in S phase inhibition. With the intent to have a deeper understanding of p53 involvement in the control of DNA replication following topoisomerase inhibitors we used two murine isogenic cell lines: a wt-p53 line (32D puro) and a mutant-p53 line (32D p53Val 135). Samples of X-ray treated cells harvested after 24 h confirmed the reliability of the temperature-sensitive system of the mutant-p53: in p53-mutant irradiated cells G1/S transition was not impaired; in fact cells didn’t accumulate in G1 phase and S-phase fraction decreased, in contrast to what was observed in wt-p53 cells. Results after topoisomerase inhibitor treatment show that wt-p53 cells are proficient in blocking DNA synthesis; BrdUrd incorporation-dependent fluorescence, measured by flow cytometry, indicated that after 4h release, particularly from the end of CPT-treatment, most of S-phase cells were blocked in DNA replication. In contrast mutant-p53 cells partially failed the replication block, in fact after 4h release a significant fraction of cells still underwent DNA replication. An analysis of RPA phosphorylation was performed in AHH1 and K562 human cells in relation to intranuclear localisation of the protein, after CPT and etoposide treatment. The results suggest a relationship between RPA phosphorylation, recruitment of RPA to nuclear foci of repair and DNA synthesis arrest. Furthermore, using wortmannin and caffeine that inhibit differentially DNA-PK and ATR kinases, we observed that DNA-PK is the main kinase involved in RPA phosphorylation and that the ATR pathway is the major mechanism involved in the S-phase checkpoint component which is independent of RPA phosphorylation. P17 Influence of cytochrome P450 genetic polymorphisms on individual sensitivity to DNA damage induced by styrene measured by comet assay Pérez-Cadahía1,2, B. Laffon1,2, E. Pásaro2, J. Méndez1

1Dept. Cell and Molecular Biology, University of A Coruña, Campus A Zapateira s/n, 15071-A Coruña, Spain. 2 Health Sciences Institute, University of A Coruña, Spain. Styrene is a monomer used worldwide in many applications. The main exposure to this compound takes place by inhalation during the manufacure of polyester products reinforced with fiberglass, resins, protective coatings, etc. In humans, styrene is biotransformed by cytochrome P450 monooxygenases (CYP) to styrene-7,8-oxide [classified as probable human carcinogen (group 2A)], so that the main genotoxic effects of styrene exposure could be imputable to it. The objective of this study was to investigate if genetic polymorphisms of CYP1A1 (m1,m2 and m4) and CYP2E1 (Rsa I and Dra I) modulate the DNA damage induced by styrene in human leukocytes. The study was carried out in isolated peripheral blood leukocytes from 30 healthy donors treated with 5 and 10 mM styrene, using 1% acetone as solvent control. DNA damage was evaluated by means of the alkaline comet assay. The results of the present work showed higher values of comet tail length in heterozygote individuals for CYP1A1 m1 and m2 polymorphisms than in wild type homozygotes; nevertheless m4 heterozygote individuals showed significant decreases in this parameter. Regarding the CYP2E1, no statistically significant results were detected in the analysis of Rsa I polymorphism, due to the fact that only one heterozygote individual was present. Finally, for Dra I polymorphism higher levels of styrene-induced DNA damage were observed in heterozygote individuals in comparison with wild type homozygote individuals. In conclusion, the findings of this study suggest that CYP1A1 m1, m2 and m4 polymorphisms and CYP2E1 Dra I polymorphism may affect styrene induction of DNA damage in human leukocytes. P18 Ageing, DNA-damage, DNA-repair G. Christopoulos 1 , K. Maridaki , G. Karanastasi 1 , N. Anagnostakis 1 , N. Messini-Nikolaki2, 2,1 2, 2, 2, M. Kanioura , S. Doudounakis3 4, S. Tsilimigaki and S. M. Piperakis 1 1

1DNA Repair Laboratory, Institute of Biology, NCSR “Democritos”, Athens, Greece. 2Department of Cell Biology, University of Athens, Athens, Greece. 3Hemodynamics Laboratory, Evangelismos Hospital, Athens, Greece. 4Department of Cystic Fibrosis, Aghia Sophia Hospital, Athens, Greece.

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Evolutionary theory and empirical evidence from many lines of research suggest that ageing is a process of gradual accumulation of damage in cells and tissues of the body, leading eventually to frailty and increased risk from a spectrum of age-associated diseases. Therefore faithful maintenance of genomic information is crucial for the survival of a species. Consequently, DNA repair processes must have evolved early during evolution. DNA damage left unrepaired might cause mutations leading to cell death, increased cancer incidence and severe syndromes. In the present study lymphocytes from individuals of four different age groups (1-5, 20-25, 40-50 and 60+ years old) were exposed to increased concentrations of γ-irradiation and H2O2 and the amount of DNA damage and the repair efficiency were estimated. The comet assay, a rapid and very sensitive method for detecting DNA damage and repair in individual cells was the method used in this study. Our results indicate that cells from older aged-groups have increased levels of basal DNA damage. They have also been found to have a lower repair efficiency. P19 Radiation sensitivity and DNA repair genotype in healthy individuals Marcon F1., Zijno A.1, Verdina A2, Galati R.2, Rossi S.1, Andreoli C.1, Crebelli R.1. 1 Istituto Superiore di Sanità, 2 Istituto Regina Elena, Rome, Italy Inter-individual variation in response to carcinogen exposure has been ascribed to differences in carcinogens metabolism as well as to variability in DNA repair. In order to investigate the role of inherited factors on individual variation in DNA repair, a mutagen sensitivity assay was carried out on 31 healthy subjects genotyped for relevant genes involved in different DNA repair pathways: APE1 (Asp148Glu) XRCC1 (Arg399Gln) XRCC3 (Thr241Met) XPD (Lys751Gln). XRCC1 plays an important role in base excision repair (BER); after excision of a damaged base, it stimulates endonuclease action and acts as a scaffold in the subsequent restoration of the site. APE1 is a key enzyme in BER, and in concert with specific DNA glycosylases, DNA polymerase β, ligase III and XRCC1 removes apurinic sites generated from exogenous and endogenous factors. XRCC3 is a member of the Rad-51 DNA repair gene family; it is involved in homologous recombinational pathway for repairing double-strand breaks, and appears to be required for the maintenance of chromosome stability in mammalian cells. XPD participates in nucleotide-excision repair pathway, which recognizes and repairs a wide range of DNA lesions such as bulky adducts and UV-induced DNA damage. The XPD protein takes part in the unwinding of DNA and forms a complex with the basal transcription factor TFIIH during transcription-coupled repair. METHODS. Fresh blood samples were irradiated with γ rays (2 Gy) and the kinetic of DNA repair assessed by Comet assay 0, 15, and 30 minutes after irradiation. Whole blood cultures were set up to detect spontaneous and induced structural chromosome aberration in lymphocytes 48 hours after mitogen stimulation. RESULTS. APE1: Radiation-induced DNA damage, detected by comet assay immediately after irradiation, was not significantly higher in wild type homozygotes (Asp/Asp) (N=22) than in carriers of variant genotypes (Asp/Glu and Glu/Glu) (N=9). Also, residual damage at 15 minutes was 75% and 56% and at 30 minutes 51% and 40% in wild type homozygous and in carriers of variant genotype, respectively. Number of aberrant cells, deletions, exchanges and total breaks were all slightly higher in Asp/Asp individuals than in combined Asp/Glu and Glu/Glu. XRCC1: wild type Arg/Arg (N=9) subjects displayed slight, not significantly, higher damage immediately after irradiation than combined Arg/Gln and Gln/Gln subjects (N=22). Residual damage at 15 and 30 minutes and chromosome aberrations did not show relevant differences among genotypes. XRCC3: No relevant differences was observed between Thr/Thr subjects (wild type, N=14) and Thr/Met heterozygotes (N=16). No Met/Met homozygote was found in this group. XPD: Similar levels of damage were measured immediatly after irradiation irrespective of the XPD genotypes. However, residual damage after 30 minutes and chromosome aberrations were slightly higher in Lys/Lys homozygotes (wild types, N=13) than in combined Lys/Gln and Gln/Gln subjects (N=18). When carriers of variant alleles at three or four loci (N=9) were compared to subjects with variant alleles at two or less loci (N=21), significantly less (p < 0.05, Mann-Whitney test) aberrant cells (23.1 and 27.7, respectively) and exchanges/cell (8.9 % vs 12.2 %, respectively) were observed in the former group. Previous results indicated that smoking habit and GSTM1 genotype may have a role in modulating the individual response to radiation-induced DNA damage. In this work no interaction has been observed between repair genotypes and smoking habits or GSTM1 genotyepe. CONCLUSIONS: The results obtained indicate that each of the genetic polymorphisms analysed has by itself a negligible effect on radiation-induced DNA damage, at least following treatment with a relatively large dose of radiation (2Gy γ rays). On the other hand, the presence of multiple, low penetrance alleles sems to hamper significantly DNA repair efficiency and influence the final processing of primary DNA damage in complex structural chromosomal aberrations. P20 Assessment of DNA damage in leukocytes of CBA mice induced by epirubicin V. Garaj-Vrhovac1, A. Horvat-Knežević2, N. Kopjar1, and I. Bašić2

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1Institute for Medical Research and Occupational Health, Laboratory of Mutagenesis, Zagreb, Croatia, 2Faculty of Science, Department of Animal Physiology, Zagreb, Croatia The alkaline comet assay was employed to assess the primary DNA damage induced by anthracycline drug epirubicin in leukocytes of CBA mice. Inbred mouse strains have been used for many years as preferable animal models due to their cytogenetic, molecular and histopathological characteristics that are comparable to those seen in human. Epirubicin has been shown to have mutagenic and carcinogenic properties. The precise mechanism of action of epirubicin is unknown, but is primarily related to intercalation of the planar ring with DNA and subsequent inhibition of DNA and RNA synthesis. Genotoxic effects of epirubicin were evaluated on CBA mice with mammary carcinoma (MCa) and animals without tumor. On third and seventh day of study mice were treated intravenously with epirubicin in concentration of 10 mg/kg. Corresponding negative controls were also selected. After 20 days of study blood sampling was done and alkaline comet assay was performed. To assess the levels of primary DNA damage in mice leukocytes comet tail length and tail moment were evaluated. In both epirubicin-treated groups higher levels of DNA damage compared to both negative controls were observed. Statistical analysis showed no significant difference between DNA damage levels of both negative controls (with or without mammary tumor). However, the levels of DNA damage recorded in epirubicin-treated mice were significantly increased compared to both negative controls. In summary, our data indicate that epirubicin induces harmful genotoxic effects in leukocytes of CBA mice. They also confirmed that the alkaline comet assay is a very sensitive technique for detection of primary DNA damage in single cells which has a widespread use in research of genotoxic effects of different chemicals on animal models. P21 Application of the comet assay in the evaluation of DNA lesions induced in vivo by melphalan F. Pacchierotti (2), E. Cordelli (2), S. Cinelli (1), A. Lascialfari (1), R. Ranaldi (1,3) RTC, Research Toxicology Centre S.p.A – Pomezia, Rome (Italy). (2)Section of Toxicology and Biomedical Science, ENEA, CR Casaccia, Rome (Italy). (3) Dipartimento di Agrobiologia e Agrochimica, Università degli Studi della Tuscia, Viterbo (Italy). Melphalan is an alkylating substance used as a therapeutic agent; its mutagenicity is related to its ability to produce monoadducts and to form DNA intra- and inter-strand cross-links. The alkaline comet assay is a useful test for the detection of DNA lesions. However, cross-links are not easily detected under standard conditions. Recently, modifications to the test have been introduced to measure cross-links by evaluating the reduction of induced DNA migration. In this work the standard comet assay and an assay modified by prolonging the electrophoresis time to 45 min have been applied to evaluate DNA lesions induced by 4 or 26 weekly oral administrations of melphalan to p53+/- knockout and to isotype parental mice. Melphalan doses tested were 0, 0.5 and 2 mg/kg; mice were sacrificed 3 hours after the last treatment. Cells were analysed from the liver, bone marrow, peripheral blood and the distal intestine. Moreover, a further protocol in which DNA migration was induced by X-ray treatment was applied to show DNA cross-links in bone marrow cells and the sensitivity of the different methods was compared. Results obtained by the standard comet assay did not show a consistent pattern; for example, liver cells of p53 +/- mice treated with the low dose showed a statistically significant increase in tail moment, while, after exposure to the high dose, cells of the same organ and genotype showed a statistically significant decrease in this parameter. Comparing the standard and the prolonged migration time, the majority of groups examined by the standard protocol showed no difference compared to controls, while 2/3 of the treated groups analysed by prolonged electrophoresis showed a significant decrease in tail moment values compared to matched controls. Only the protocol based on X-ray in vitro irradiation showed the presence of melphalan-induced cross-links in bone marrow cells exposed to 2 mg/kg, demonstrating that this was the most sensitive approach for detecting this type of lesion. DNA lesions were evident in all the organs analysed. However, according to the performance of the standard and modified comet methods with respect to each other, the contribution of cross-links and repair associated breaks in each organ might have been variable. In bone marrow, for instance, DNA damage was more evident as repair associated breaks rather than cross-links compared to the other organs. The pattern of response observed after 4 and 26 weekly administrations appears slightly different, suggesting that processes of DNA damage induction and repair might change during the course of chronic treatment. After comparison between genotype-matched treated and control groups, a significant effect was shown more frequently in p53 +/- than in wild-type groups. P22 In vivo, in vitro and acellular bioavailability of polycyclic aromatic hydrocarbons from commercial carbon blacks assessed by postlabelling of DNA adducts

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P. Borm1, G. Cakmak2, E. Jermann3, R. Duffin1, F.J. Van Schooten3, and R. Schins1 1 Institut für Umweltmedizinische Forschung (IUF) and 3 Medizinisches Institut für Umwelhygiene (MIU) at the Heinrich Heine University Düsseldorf, Germany, 2 Gazi University, Ankara, Turkey, 3 Maastricht University, The Netherlands Although there is general consensus that lung tumours induced by diesel exhaust particles (DEP) and carbon black (CB) are the result of persistent inflammation causing secondary genotoxicity, the possible role of polycyclic aromatic hydrocarbons (PAH) on the particle surface is still unclear. Experiments were performed with four commercial CB (Printex 90, Sterling V, N330, Lampblack 101), all with different size, surface and PAH content. Leaching of PAH was examined in acellular systems in the presence of dipalmitoyl phosphatidyl choline (DPPC), using HPLC. In all cases, PAH content in the fluid containing DPPC was below the detection limit. Subsequent in vitro studies in A549 human alveolar epithelial cells using 32P-postlabeling with Nuclease P1 or butanol extraction were performed with the same CB types as well as their organic extracts, and the extracted CB particles. Ultrafine TiO2 particles coated with BaP, BaP (0.03 µM) and PAH-mixture (EPA) were used as controls. Organic extracts of Sterling V showed PAH-DNA adducts with a clear dose-response between virtual BaP concentrations of 0.006 and 0.84 µM. No adducts were seen after incubation with extracted CB particles up to 100 µg/cm2. Upon incubation with CB particles at concentrations between 30 and 300 µg/cm2 in A549 cells, Sterling V showed a spot which co-located with BPDE-DNA adduct. However, the spot was not dose-dependent and not due to the major PAH in Sterling V, i.e. benzo(ghi)perylene. None of the original CB showed detectable DNA adducts at equivalent mass amounts of CB extracted. Interestingly, also TiO2 coated with BaP showed no detectable adducts up to 300 µg/cm2, while its extract showed a significant BPDE-spot. The in vivo bioavailability of PAH from two different CB (Printex 90, Sterling V) after 3 months inhalation of 50 mg/m3 was studied in lung homogenates immediately after exposure. The data showed no extra or increased spots related to PAH-DNA adduct formation compared to sham exposed rats. The results suggest that PAH are very tightly bound to CB particles since bioavailability of PAH was not detected in the surfactant leaching studies as well as in the in vivo experiments. In addition, in vitro bioavailability based on PAH-DNA adducts in target cells were not detected in three CB types examined. In one CB (Sterling V), it is not possible to interpret the data due to difficulty in the identification of the adduct and more importantly, the fact that there was no dose-response relationship. This study was funded by a grant from the International Carbon Black Association. Mutagenicity of plasmid treated with α-acetoxytamoxifen replicated in GM00637 normal cells or in GM04429 XPA cells K I E McLuckie1, K Brown1, E A Martin2, M N Routledge3 and P B Farmer1. 1 Cancer Biomarkers and Prevention Group, The Biocentre, University Road, Leicester, LE1 7RH. 2 Genetic Toxicology, AstraZeneca, Alderley Park, Macclesfield, SK10 4TG. 3 Molecular Epidemiology Unit, University of Leeds, Leeds, LS2 9JT. Tamoxifen, a breast cancer drug, is also used as a chemopreventative agent for this disease. However, tamoxifen causes hepatic carcinomas in rats by a genotoxic mechanism and increases the risk of endometrial tumours in women, through an as yet undetermined mechanism. In this study the mutagenicity of DNA adducts caused by α-acetoxytamoxifen, a model of a reactive tamoxifen metabolite, was investigated. When reacted with DNA, this compound produces the same major DNA adducts formed in the livers of tamoxifen treated rats. pSP189 plasmid DNA containing the supF gene was treated with increasing doses of α-acetoxytamoxifen and adduct levels determined by 32P-postlabelling. Adduct numbers increased with dose giving approximately 0.5, 1 or 2.5 adducts per plasmid for doses of 10, 25 and 50µM respectively. Plasmids were transfected into human fibroblasts, either DNA repair proficient (GM00637 cells) or DNA repair deficient from patients with Xeroderma pigmentosum complementation group A (GM04429 cells). Following replication, plasmids were recovered and screened in indicator bacteria. Whilst mutation frequency increased with increasing adduct levels there was no difference between the two cell lines, which suggests nucleotide excision repair does not play a major role in the removal of these particular tamoxifen DNA adducts in human cells. As expected, since the major site of tamoxifen adduct formation is the N2 position of deoxyguanosine, the majority of mutations (84-100%) occurred at GC base pairs. The predominant mutation induced in repair competent cells was the GC→AT transition, whilst in the repair deficient cells GC→TA transversions were the most frequent base changes. Cellular background has been shown to influence mutagenic spectra and the variations observed in this case may be due to differences in cell type. The inefficient repair of mutagenic tamoxifen DNA adducts in human cells suggests that if they are not excised, they may potentially contribute to the initiation of endometrial carcinogenesis.

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P23 Investigation of the mutagenicity of ethylene oxide induced DNA damage in human Ad293 cells using the supF forward mutation assay Jatinderpal K. Sandhu, Keith I. E. McLuckie, Margaret Gaskell, Elizabeth K. Wright, Peter B. Farmer, Karen Brown. Cancer Biomarkers and Prevention Group, University of Leicester, LE1 7RH, U.K. Ethylene oxide is widely used as an intermediate in the chemical industry, for example in the production of ethylene glycol, polyester fibres, and as a fumigant and sterilant of food and medical products. It is classed as a human carcinogen but there is conflicting evidence regarding its ability to induce cancers in humans, therefore there is a need to assess the consequences of exposure to ethylene oxide. Ethylene oxide is known to damage DNA primarily by reacting with the N7 position of guanine forming N7-(2-hydroxyethyl)-2’-deoxyguanosine. Other adducts include O6–(2-hydroxyethyl)-2’-deoxyguanosine and N3-(2-hydroxyethyl)-2’-deoxyadenosine which are formed in smaller amounts. The aim of this study is to investigate the mutagenicity of ethylene oxide treated plasmid in human cells, using the supF forward mutation assay. Aliquots of pSP189 plasmid (100µg in water) were incubated with increasing doses of ethylene oxide (0, 10, 25, 50, 100, 200 and 500µM) for 4h at room temperature and then purified for subsequent analysis. Initially a 32P-postlabelling method coupled with HPLC separation and incorporating a nuclease P1 enhancement step, has been developed to enable quantification and characterization of ethylene oxide derived adducts formed at each dose. Using an isocratic mobile phase of 1% methanol in 2M ammonium formate at least 8 32P-postlabelled adduct peaks can be detected in digests of ethylene oxide exposed calf thymus DNA, whilst analysis of treated pSP189 plasmid showed one major peak which is possibly the N7 adduct. To determine the mutagenicity of ethylene oxide, control and exposed plasmid was transfected into human Ad293 kidney cells. Following replication the plasmid was recovered and used to transform indicator bacteria to screen for the presence of mutations in the supF gene. Preliminary results indicate that at the low doses used there is a dose dependent increase in the number of mutants, with concentrations of 10, 25 and 100µM resulting in mutation frequencies of, 0.01, 0.02 and 0.03 respectively. This increased mutation frequency was accompanied by a decrease in plasmid survival as shown by a reduction in the number of transformants. At higher doses the number of transformants was greatly reduced, with none present at the highest dose of 500µM ethylene oxide. This suggests that the amount of damage induced at ethylene oxide doses greater than 100µM is extensive and may inhibit plasmid replication in the human cells. Results to date therefore demonstrate that ethylene oxide is mutagenic in human cells and based on these findings further studies will now be conducted using lower doses of ethylene oxide in order to determine the mutation spectrum of this compound in human cells. P24 Frequency of homologous recombination in two cell lines differing in DSB repair ability M. Wojewódzka, T. Bartłomiejczyk, M. Kruszewski Institute of Nuclear Chemistry and Technology, 03-195 Warszawa, Poland In mammalian cells DSB are repaired by nonhomologous end-joining (NHEJ) with DNA-dependent protein kinase (DNA-PK) being the key enzyme and by homologous recombination (HR), where the participating proteins are, among others, MRE11, BRCA1 and RAD group. We examined frequency of spontaneous and X-rays induced HR in Chinese hamster cell line CHO-K1 and radiation sensitive, DSB repair defective mutant line, xrs-6. The latter is defective in DNA-PK –mediated DSB repair pathway due to the deficiency in Ku80 protein. To assess the recombination frequency in studied cells we used pLrec plasmid. pLrec plasmid carries two nonfunctional copies of the β-galactosidase (lacZ) gene in a tandem array and neo gene conferring resistance to Geneticin (G418). In result of HR the copies of LacZ gene give rise to a functional copy of β-galactosidase gene. Activity of the β-galactosidase protein can be assayed either colorimetrically, fluorimetrically or histochemically. In this set of study, the expression of the lacZ gene was monitored in cell extracts by fluorescence-based assay that employs fluorogenic substrate - 4-methyl-umbelliferyl-β-D-galactopyranoside. A stable, single copy transfectants with pLrec incorporated into the genome were used in this study. This experimental design is eminently suitable to study HR because the plasmid is integrated into chromosome and, in contrast to episomal plasmids, is subjected to the same processes as chromatin. Therefore, the measured recombination frequency better corresponds with the actual processes that take place in the cell, than that examined with the use of episomal plasmids. Once isolated on a medium containing G418, transfected cells were cultured on medium containing G418 or allowed to growth for 5-6 days without G418. This treatment allows to distinguish between reciprocal gene exchange/HR (lost of neo gene) and gene conversion (neo gene is still present and cells are resistant to G418). Recombination frequency was measured either in non-treated cells (spontaneous recombination) or in cell irradiated with 2 Gy of X-rays (induced recombination). Our preliminary data indicate that the level of β-galactosidase activity (reflecting the frequency of spontaneous HR) in CHO-K1 cells was lower (2.9 U/g protein) than in xrs-6 cells (7.7 U/g protein). However, significant differences between individual clones have been observed. Irradiation of the cells with 2 Gy of X-rays significantly elevated the level of enzyme in both cell lines. As expected, in non-irradiated cells, release of the selective pressure (5-6 days without G418) significantly elevated level of β-galactosidase activity as compared with in cells cultured with G418. Our results suggest that the defect in NHEJ-mediated DSB repair pathway result in elevated frequency of HR.

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P25 Role of labile iron pool in nitric oxide-induced genotoxicity M. Kruszewski1, H. Lewandowska1, R.R. Starzyński2, T. Bartłomiejczyk1, T. Iwaneńko1 and P. Lipiński2 1Institute of Nuclear Chemistry and Technology, Poland, 2Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Poland Nitric oxide (NO) is widely recognised as a molecule strongly altering intracellular iron metabolism. Numerous and complex interventions of NO in iron metabolism include regulation of expression of genes responsible for maintaining iron balance as well as inhibition of iron-containing enzymes participating in mitochondrial respiration, heme and DNA synthesis. All these changes lead to a massive loss of intracellular iron, which is a common feature observed in cells exposed to NO. On the other hand, NO is a potent inducer of cell death through the apoptosis pathway. Here, we elucidated the role of iron in the NO-induced genotoxicity focusing on the relationship between NO and labile iron pool (LIP), a cytosolic fraction of metabolically active and potentially toxic iron composed of low molecular-weight iron complexes. A pair of mouse lymphoma cell lines L5178Y-R (LY-R) and L5178Y-S (LY-S) differing in LIP level was used as an experimental model. SpermineNONOate (SperNO) was used as a NO donor. In both cell lines NO induced DNA damage in a dose-dependent manner, as measured with comet assay. However, in LIP-reach LY-R cells the yield of DNA damage was higher as compared with LIP-depleted LY-S cells. Induction of DNA damage corresponded to the rapid increase of LIP level in both cell lines, as measured with a fluorescent probe, calcein. In parallel, as expected, we detected a rapid increase of electron paramagnetic resonance (EPR) signal with characteristic peaks at g=2.041 and g=2.015 corresponding to the paramagnetic complex [(NO)2Fe(SR)2]. Interestingly, we also observed decrease of the activity of cytosolic aconitase, a protein containing an 4Fe-4S cluster at the active site. Pretreatment of cells with salicylaldehyde hydrazone (SIH), a highly permeant iron chelator and subsequent exposure to SperNO resulted in a decrease of DNA damage and EPR signal. This demonstrates that SIH-chelatable iron may be involved in the generation of NO-induced DNA damage. A plausible source of iron released by NO to the LIP can be iron-containing proteins, such as Fe-S cluster proteins, that are known to loose Fe-S cluster upon the action of NO. P26 Light-dependent generation of reactive oxygen species from benzo[a]pyrene is mediated via its DNA adduct(s) M. Yamada1, S.-R. Kim1, K. Kokubo1, 2, K. Matsui1, N. Yamada3, Y. Kanke2, M. Fukuoka3,T. Nohmi1 1 National Institute of Health Sciences, Tokyo, JAPAN, 2 Otsuma Women’s University, Tokyo, Japan, 3Showa Pharmaceutical University Background: Benzo[a]pyrene (BP) is a potent environmental mutagen and carcinogen. The best characterized pathway of mutagenicity and carcinogenicity of BP is initiated by the metabolic activation to electrophilic metabolites capable of covalent binding to macromolecules such as DNA. The metabolic activation involves oxidation by cytochrome P450 to form the 7, 8-epoxide, hydrolysis by epoxide hydrase to the trans-7, 8-diol and oxidation to the 9,10-epoxide by cytochrome P450 to give a diol epoxide (BP-7,8-dihydrodiol-9,10-epoxide; BPDE). BPDE forms a major (~90%) covalent reaction product with DNA at the N2 of guanine, i.e., (+)-trans-anti-BP-N2-dG adduct, which is shown to be tumorigenic in experimental animals. Interestingly, BP generates reactive oxygen species (ROS), i.e., singlet oxygen, superoxide and hydrogen peroxide, even without metabolism if visible light is present. ROS interacts with DNA, thereby inducing a variety of mutagenic lesions such as 8-oxo-guanine (8-oxo-G) in DNA. This “photosensitization” pathway of BP to induce mutations could be important and interesting because it may represent the mechanisms of environmental photosensitizers, such as fluoroquinolone antibacterial agents or riboflavine, which induce a variety of photoproducts in DNA in the presence of visible light. Here, we examined the light-dependent mutagenicity of BP in Salmonella typhimurium strain TA1975 and its derivatives deficient in the capacity to repair DNA damage. The results suggest that DNA adducts of BP, rather than free BP itself, mediate the generation of oxidative damage in DNA in the presence of visible light. Results and Discussion: Treatments of S. typhimurium strains with BP and white light (370-750 nm, 750 lux) for 2 days enhanced the number of His+ revertants per plate without exogenous metabolic activation. No increase in the number of revertants was observed without visible light even in the presence of BP. The order of the sensitivity of tester strains (induced His+ revertants per plate per �g BP) was dependent upon DNA repair capacity of the strains: YG3001 (mutMST

- uvrB-) > TA1535 (uvrB- ) > YG3002 (mutMST) > TA1975. Relative sensitivities of YG3001, TA1535 and YG3002 to TA1975 are 134, 33, and 3, respectively. These strains are all isogenic except for the repair function. The mutMST gene encodes 8-oxo-G DNA glycosylase that removes 8-oxo-G in DNA and the uvrBST gene is involved in the excision repair of bulky DNA adducts such as (+)-trans-anti-BP-N2-dG adduct. DNA sequence analyses of the hisG allele of the revertants of YG3001 indicated that G:C to T:A transversions, expected from the presence of 8-oxodG in DNA, occurred. Introduction of the plasmid carrying the hOGG1 gene that is functionally equivalent to mutMST in humans substantially

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reduced the light-induced mutagenicity of BP in the strain YG3001. Since deficiencies in both mutMST and uvrBST are required for the maximum mutagenicity of BP in the presence of visible light, we suggest that the formation of DNA adducts of BP is needed to efficiently induce oxidative damage in DNA in the presence of visible light. If Uvr functions effectively remove BP adducts in DNA, the mutagenicity was much less pronounced (as strain YG3002). This suggests that free BP molecules do not efficiently induce oxidative damage in DNA even in the presence of visible light. We also suggest the DNA adducts of BP that induce oxidative damage in DNA in the presence of visible light are not (+)-trans-anti-BP-N2-dG adduct because bacteria does not possess epoxide hydrase. They might be adducts of BP 4,5-oxide or 7,8-oxide, which are the initial products of oxidization of BP by ROS generated by photoactivation of BP. Conclusion: BP is mutagenic without metabolic activation if visible light is present. We propose that DNA adducts of BP, rather than free BP in the cell, generate 8-oxo-G in the surrounding DNA when the visible light is present, thereby inducing G:C to T:A oxidative mutagenesis. P27 Misreading of 1,N6-ethenoadenine rearrangement derivatives by selected DNA polymerases E. Speina, and B. Tudek Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland 1,N6-ethenoadenine (εA) is an exocyclic DNA adduct introduced to DNA by vinyl chloride and related compounds as well as in the consequence of oxidative stress and lipid peroxidation (LPO). This highly genotoxic DNA damage is chemically unstable and either depurinates or converts into pyrimidine ring-opened secondary lesions. We have previously studied the structures of derivatives formed during εA chemical rearrangement and identified enzymes repairing one of the rearrangement products. Rearrangement involves a water molecule addition to the C(2)-N(3) bond of εA, resulting in formation of pyrimidine ring-closed B1 product, which is in equilibrium with pyrimidine ring-opened B2 compound. B2 further deformylates to yield compound C. [Speina et al., J.Biol.Chem. 257 (2001) 21821-21827]. In the present study we elucidated the fate of ethenoadenine rearrangement products during in vitro translesion DNA synthesis (TLS) by the followin DNA polymerases: phage T7 exo-, Thermophylus aquaticus (Tag), Thermococcus litoralis (Tli), E. coli pol I, Klenow fragment of pol I, exo+ and exo-, and calf thymus pol β. During TLS, C is much easier bypassed by DNA polymerases, than compound B, and also than the parental εA as well as than the AP-site. DNA polymerases T7 exo-, Tag, pol I, and pol β were used to in vitro replicate oligonucleotides containing either �A or its rearrangement products in the presence of only one of the four dNTPs in order to investigate the possible misinsertions opposite these modified bases. Under our experimental conditions almost no incorporation was observed opposite product B with all DNA polymerases tested. Bypass beyond C proceeded mainly by misinsertion of adenine and guanine, or by insertion of thymine, the latter restoring the parental A:T pair. Alternatively, looping out of adducted nucleotide alone or with the adjacent one generated one- or two-nucleotide deletions. This may explain the previously reported 20-fold higher mutagenic potency of product C in comparison to �A in Escherichia coli [Biochemistry 32 (1993) 12793–12801]. P28 Detection of the fate of DNA damage induced by the comet assay in HepG2 cells Yu F, Sasaki Hachinohe National College of Technology, Hachinohe, Japan It includes a main purpose of a genotoxicity test to detect a mutagen and to predict a risk in humans. The prediction of carcinogenicity from mutagenicity is done broadly, which plays an important role in the development of new medicines. For this purpose, the comet assay is recently used widely. Although this assay is a sensitive method to detect DNA lesions, no information on the fate of damaged cells is obtained. It is important to the fate of organs targeted by chemical carcinogens whether the DNA damage is repaired or persists. In general, DNA damage is fixed as chromosome aberrations after S phase and they can be detected as micronuclei. So, to know whether DNA damage detected by the comet assay is repaired or fixed, we compared the results of the comet and micronucleus assays in HepG2 cells. HepG2 cells were established from human hepatoma in 1979 by Aden et al. It is for some metabolic activation enzymes to have appeared as for the maximal characteristic of HepG2 cells. HepG2 cells were exposed to 40 pro-mutagens for 4 h in the absence of S9 mix. Immediately after the exposure, slides for the comet assay were prepared. For the micronucleus (MN) test, cells were cultured for 24 h with CytB. Binucleate cells (BNC) with MN and frequency of BNC were scored. From the data, chemicals were divided into five. For pro-mutagen in first group, both comet and MN assays are positive at the same concentration. Aniline was in this group. Comet assay-detectable DNA damages by the pro-mutagens in this group were considered to be fixed as chromosome aberrations. Second, the MN test was negative at the concentration at which comet assay was positive. B[a]A was in this group. The pro-mutagens in this group induced comet detectable DNA damages that were not fixed as chromosome aberrations. Third, cell death was observed at the concentration at which the comet assay was positive. Trp-P-1 and some azo dyes were in this group. Chemicals in this group induced comet detectable DNA damages that led to cell death during the culture period with CytB. Fourth, MN-

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induction was observed at the lower concentration at which comet was negative. DEN was in this group. Their induced DNA damages that were lower level than the sensitivity of the comet assay seemed to become chromosome aberrations. Fifth, neither comet nor MN was negative. Based on our results, parallel discussion of initial and fixed DNA damages would give more useful information to assess risk of mutagenic chemicals to human. P29 Induction of DNA damage in A549 cells by a binary mixture of mutagens Csilla Mišľanová1, Kay L.M. White, Kate Healey, Elizabeth C. Smith, Laura J. Hardie, Christopher P. Wild, Mariá Dusinská1, and Michael N. Routledge Molecular Epidemiology Unit, Academic Unit of Epidemiology & HSR, School of Medicine, University of Leeds, LS2 9JT, UK 1Department of Experimental and Applied Genetics, Institute of Preventive and Clinical Medicine, Bratislava, Slovakia Many human exposures to mutagens occur as mixtures. We have previously investigated the genotoxicity of a binary mixture of mutagens, benzo[a]pyrene diol epoxide (BPDE) and UV irradiation. We observed an enhancement of mutagenicity in the supF assay when BPDE adducted plasmid was UVC irradiated in comparison to the individual BPDE and UVC treatments (Routledge et al, 2001, Carcinogenesis, 22, 1231). One possible cause of the enhanced mutagenicity was an increase in oxidative DNA damage. To investigate this possibility in cells we exposed a human lung epithelial cell line, A549 cells, to benzo[a]pyrene (BP) and UVC, either as individual treatments or as a combination. The comet assay was used to analyse induced DNA damage and DNA was extracted from the cells for the analysis of 8-oxo-dG by HPLC-ECD. In the comet assay BP + UVC induced 68% comets compared to 18% for BP and 24% for UVC alone at the doses used in the combination experiment (5 µM BP and 17 J/m2 UVC). In cells from the same cultures, there was no enhanced induction of 8-oxo-dG. Levels of 8-oxo-dG were 1.9 (+/-0.18), 5.8 (+/-0.46), 7.1 (+/-0.34), and 12.6 (+/-0.8) in 106 dG, for control, BP, UVC and BP+UVC, respectively. The level of 8-oxo-dG in cells exposed to the combination treatment was, therefore, no more than the additive value for the individual BP and UVC exposures. Hence, UVC irradiation of BP exposed cells led to enhanced DNA damage measured by the comet assay, but no enhancement of 8-oxo-dG. P30 Arsenite and its biomethylated metabolites induce oxidative DNA damage and interfere with DNA repair in cultured human cells and subcellular systems T. Schwerdtle, I. Walter, and A. Hartwig Institute of Food Chemistry and Toxicology, University of Karlsruhe, Germany Inorganic arsenic is a naturally ubiquitous occurring environmental pollutant of mayor concern, since chronic exposure to low concentrations via drinking water and the air has been associated with increased risk of skin and inner organ cancer. However, the underlying mechanisms of arsenic carcinogenicity are only poorly understood and also the role of biomethylation, which has long been considered to be one major detoxification process, is still a matter of debate. In the present study we compared the induction of oxidative DNA damage by arsenite and its trivalent and pentavalent methylated metabolites in cultured human cells and isolated DNA and the impact of the arsenicals on DNA repair in cultured human cells as well as their effects on two isolated DNA repair enzymes. As measured by alkaline unwinding all arsenic compounds investigated induced dose-dependently oxidative DNA damage at non-cytotoxic concentrations in HeLa S3 cells. Surprisingly, 3 h incubation with doses as low as 10 nM arsenite, a concentration comparable to blood concentrations of the general population, induced a high amount of oxidative DNA base modifications and some DNA strand breaks. Whereas arsenite-induced damage was rather low after 18 h incubation, trivalent (≥ 100 nM) and pentavalent (≥ 10 µM) methylated metabolites showed a pronounced dose-dependent induction of Fpg-sensitive sites after 3 and 18 h incubation. Additionally DMA(V) and MMA(III) generated dose-dependently DNA strand breaks. In isolated PM2 DNA only DMA(III) (≥ 10 µM) induced DNA strand breaks, whereas all other arsenic compounds showed no effects up to 10 mM. Besides the induction of oxidative DNA damage the interference with DNA repair processes is discussed to contribute to arsenic-induced carcinogenicity. As a model repair system we examined the removal of stable BPDE-DNA adducts in A549 lung cells by a highly sensitive HPLC/fluorescence assay. Arsenite and its methylated metabolites increased formation and diminished DNA repair of BPDE-DNA adducts at non-cytotoxic concentrations. Thus MMA(III) (≥ 2.5 µM) and arsenite (≥ 25 µM) increased DNA-adduct formation up to 40 and 60 %, respectively. In addition arsenite, its trivalent and pentavalent methylated metabolites inhibited the repair of the induced adducts, starting at 5, 2.5 and 250 µM, respectively, yielding 75, 90 and 30% repair inhibition, respectively. Potential molecular targets may be zinc finger enzymes, a family of proteins where zinc is complexed through four invariant cystein and/or histidine residues. Thus, in a previous study we demonstrated a suppression of poly(ADP-ribosyl)ation, a process which is predominantly mediated by the zinc finger enzyme PARP-I, at low nanomolar concentrations in HeLa S3 cells (Hartwig et al., 2003, Int. J. Cancer). Within this study we examined the effect of the arsenicals on two isolated zinc finger DNA repair enzymes, the mammalian xeroderma pigmentosum group A protein (XPA), essential for the assembly of the DNA

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damage recognition/incision complex during nucleotide excision repair, and the E.coli formamidopyrimidine-DNA-glycosylase (Fpg), involved in the repair of certain types of oxidative DNA damage. Our data demonstrate that all trivalent arsenicals can release zinc from the examined XPA peptide in the low micromolar concentration range, with the trivalent methylated metabolites being more active as compared with arsenite; the pentavalent metabolites MMA(V) and DMA(V) showed no effect. With respect to Fpg, only the trivalent methylated metabolites MMA(III) (≥ 1 mM) and DMA(III) (≥ 100 µM) were able to inhibit Fpg activity towards oxidatively damaged DNA, whereas arsenite and the pentavalent metabolites showed no such effects up to 10 mM. Taken together, our results indicate that both the induction of oxidative DNA damage and the interference with nucleotide excision repair may contribute to arsenic-induced carcinogenicity. As one potential target for repair inhibition we identified zinc finger repair enzymes. Furthermore, the demonstrated genotoxic potential especially by the trivalent methylated metabolites provide further evidence that arsenic biomethylation may contribute to arsenic-induced genotoxicity/carcinogenicity. P31 Substrate specificity of E.coli AlkB and human DNA repair dioxygenases P. Koivisto, T. Duncan, S.C. Trewick, T. Lindahl and B. Sedgwick Cancer Research UK, London Research Institute, Clare Hall laboratories, Potters Bar EN6 3LD , UK We have characterised the novel DNA repair activity of E.coli AlkB and two human homologs, ABH2 and ABH3. These proteins directly demethylate 1-methyladenine and 3-methylcytosine in DNA by an oxidative mechanism releasing formaldehyde. AlkB also repairs 1-ethyladenine generating acetaldehyde, however this lesion is repaired inefficiently by the two human homologs. Release of acetaldehyde from 1-ethyladenine indicates that AlkB oxidises the carbon atom of the alkyl group that is adjacent to the base nitrogen. By examining survival of bacteriophage exposed to simple epoxides, hydroxyalkyl lesions were also identified as AlkB substrates. Hydroxyalkylated adducts were generally less efficiently repaired than similar alkyl adducts. We have also determined the kinetic parameters for AlkB oxidation of 1-methyladenine in poly(dA) and in short oligodeoxyribonucleotides. Adenine-N1 and cytosine-N3 in single stranded oligonucleotides are susceptible to methylation by SN2 alkylating agents, while thymidine residues are rarely alkylated. Short oligonucleotides containing a single 1-methyl adenine residue and a varying number of thymidines were prepared by long exposures to dimethylsulphate and purified by HPLC. Methylated poly(dA) was the best AlkB substrate tested. However, AlkB repair of short oligonucleotides was efficient if the alkylated base was 5’ phosphorylated. Repair of these short oligonucleotides by the human ABH3 protein was inefficient and not detectable for ABH2. P32 Oxygen-induced DNA damage in freshly isolated brain cells compared with cultured astrocytes in the Comet assay E. Cemeli1, I.F. Smith2, C. Peers2, J. Urenjak3, O.V. Godukhin3, T.P. Obrenovitch3 and D. Anderson1 1Department of Biomedical Sciences, University of Bradford, Bradford, UK, 2Institute of Cardiovascular Research, Leeds University, Leeds, UK, 3School of Pharmacy, University of Bradford, Bradford, UK Brain cells are continuously exposed to reactive oxygen species generated by oxidative metabolism, and in certain pathological conditions defence mechanisms against oxygen radicals may be weakened and/or overwhelmed. DNA is a potential target for oxidative damage, and genomic damage can contribute to neuropathogenesis. It is important, therefore, to identify tools for the quantitative analysis of DNA damage in models of neurological disorders. The aim of this study was to compare the susceptibility of DNA to oxidative stress in cells freshly dissociated from the mouse brain, to that in cultured brain cells. Both primary cultures and a continuous cell line of astrocytes were considered. All cells were treated with xanthine/xanthine oxidase (i.e. a superoxide generator) or hydrogen peroxide, applied alone or in the presence of the oxygen radical scavengers superoxide dismutase, catalase or ascorbic acid. DNA damage was quantified with the Comet assay. DNA damage was consistent in all the different cell preparations exposed to oxidative stress, and was attenuated in similar ways by superoxide dismutase and catalase (i.e. scavengers of the superoxide anion and hydrogen peroxide, respectively). The results with ascorbic acid were more variable, presumably because this compound may switch from anti- to pro-oxidant depending on its concentration and other experimental conditions. Overall, similar responses were found in freshly dissociated and cultured brain cells. These results suggests that the Comet assay can be directly applied to cells freshly dissociated from the brain of mouse models of neurological disorders, such as stroke models and animals genetically engineered to reproduce human diseases.

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P33 The influence of aluminium ions on DNA damage and repair in human peripherial blood lymphocytes in vitro Banasik, A.1, Lankoff, A.1, Lisowska H. 1, Kuszewski T.2, Góźdź, S.2, Wójcik, A. 1,3 1Department of Radiobiology and Immunology, Institute of Biology, Świętokrzyska Akademy, Kielce, Poland 2Świętokrzyskie Cancer Center, Kielce, Poland 3Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, Warsaw, Poland The progressive increase in the use of aluminium compounds has enhanced the possibility of human exposure. The accumulation of Al in tissues may lead to several disturbances of function of organism. Aluminium combines with many compounds, important with respect to their function, e.g. proteins, ATP, DNA, RNA, phosphatases, fluorides, affecting their biological functions. Aluminium has proven to be carcinogenic to humans, but its mechanism of action is not yet well known. Taking it into consideration we have decided to determine the influence of aluminium ions on DNA damage and repair in human peripherial blood lymphocytes in vitro. The comet assay was used to analyze DNA lesions and DNA repair kinetics in single cells after treatment with 1, 2, 5, 10 and 25 µg of aluminium chloride. Human peripherial blood lymphocytes were treated in G1, S/G2 and the whole cell cycle. In order to determine the influence of Al on the repair of radiation-induced damage, lymphocytes were irradiated with a dose of 2 Gy (60Co). Our results indicate that after treatment with AlCl3 during S/G2 phase, the level of DNA damage in lymphocytes changed in a dose and time – dependent manner. We also observed an influence of AlCl3 on the radiation-induced DNA damage repair. Work supported by the KBN (Poland) grant number 3P05D 038 22. P34 DNA damage and repair in human peripheral blood lymphocytes following treatment with cyanobacterial toxins and ionizing radiation Anna Lankoff 1, Anna Banasik 1, Halina Lisowska 1, Łukasz Krzowski 1, Tomasz Kuszewski 2,Stanisław Góźdź 2, Andrzej Wójcik 1, 3

1 Department of Radiobiology and Immunology, Institute of Biology, Swiętokrzyska Academy, Kielce, Poland 2 Świętokrzyskie Oncology Center, Kielce, Poland 3 Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, Warsaw, Poland Cyanobacterial toxins present acute and chronic hazards to human health. These toxins possess potent tumor-promoting activity mediated by inhibition of protein phosphatases PP1 and PP2A. Phosphatases PP1 and PP2A are involved in many cellular processes such as cell signalling and DNA repair. Since damage at the DNA level and disturbances in DNA repair are included in the process of cancerogenesis we decided to determine the effect of cyanobacterial toxin – microcystin-LR on the cellular response to ionizing radiation. The comet assay was used to analyse DNA lesions and DNA repair kinetics in single cells. To determine the level of DNA damage, lymphocytes were treated with 1, 10 and 25 µg/ml of microcystin-LR for 6, 12, 18 and 24 hours.To assess the kinetics of DNA repair, lymphocytes were pretreated with 1 µg/ml of microcystin-LR for 4 hours and then irradiated with 2 Gy 60 Co-rays. Our results indicate that microcystin-LR induces DNA damage in a dose- and time-dependent manner. Moreover, longer treatment with microcystin-LR caused an increase in the number of apoptotic comets. Lymphocytes irradiated with 2 Gy 60 Co-rays showed an increased level of DNA damage. The repair of DNA after irradiation was comparable to the results available in the literature. The level of DNA repair in lymphocytes treated with microcystin-LR and simultaneously irradiated with 2 Gy 60

was decreased.These results suggest that microcysystin-LR may inhibit the cellular DNA repair capacity. This work was supported by the Polish Committee for Scientific Research No6PO5D01320. P35 Potent radioprotective effect of therapeutic doses of ranitidine and famotidine against gamma-rays as assayed by the single cell gel electrophoresis and micronucleus test M. Shahidi1 and H. Mozdarani1 1Department of Radiology, School of Medical Sciences, Tarbiat Modarres University, Iran As our previous investigations have revealed, cimetidine -a histamine H2-receptor antagonist- has proved to show radioprotective effects against gamma- and neutron-induced micronuclei in bone marrow erythrocytes. In this study, the anticlastogenic effects of famotidine and ranitidine, which act similar to cimetidine as histamine H2-receptor antagonists, were investigated in male Balb/c mice lymphocytes and bone marrow cells by means of the comet assay and micronucleus test respectively. Various doses of these two drugs were injected i.p. to the mice two hours prior to 1 or 2 Gy gamma irradiation. Results indicate that gamma irradiation alone can cause DNA damage and a high frequency of

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micronuclei formation and decrease cell proliferation ratio. Pre-irradiation injection of famotidine and ranitidine, of various doses, effectively reduced the migration of DNA and the number of micronucleated polychromatic erythrocytes (MnPCEs), yet has no effect on cell proliferation ratio (PCEs/PCEs+NCEs). In fact, these two drugs reduce the clastogenic effects of gamma rays, while they are ineffective against the cytotoxic properties of gamma rays. The dose reduction factor (DRF) calculated, show a DRF=2 for famotidine and a DRF=1.8 for ranitidine which is indicative of a high radioprotective property of these drugs. The mechanism in which these drugs reduce clastogenic effect of gamma radiation is not fully understood. It might be due to their antioxidant, and free radical-scavenging properties. P36 Amifostine can differentially modulate DNA double-strand breaks and apoptosis induced by idarubicin in normal and cancer cells. Gloc, K. Wozniak, and J. Blasiak Department of Molecular Genetics, University of Lodz, Lodz, Poland Amifostine [S-2(3-aminopropylamino-) ethyl phosphosulphate acid, WR-2721] is a pro-drug used in the protection of normal cells against chemotherapeutic drugs and radiation in a variety of cancers. We have recently shown that amifostine may differentially modulate the DNA-damaging effect of idarubicin in normal and cancer cells. To investigate further this effect we examined the influence of amifostine on double-strand breaks (DSBs) and apoptosis induced by idarubicin in normal human lymphocytes and p53-deprived chronic myelogenous leukaemia K562 cells expressing BCR/ABL, an oncogenic fusion tyrosine kinase, which, as we previously showed, may stimulate DNA repair. We used the neutral comet assay and pulsed field gel electrophoresis for detection of DNA double strand breaks. The measurement of caspase 3 activity, examination of morphological changes in the chromatin as well as DNA fragmentation assessed by the alkaline comet assay were employed to evaluate the process of apoptosis resulted from the action of the drugs. Amifostine at 14 mM decreased the extent of idarubicin-induced DSBs and apoptosis in normal cells, but it increased these effects in leukemic cells. Our results suggest that normal and cancer cells may diversely response to combination of idarubicin and amifostine resulting in different level of DSBs and apoptosis and that p53 and/or BCR/ABL tyrosine kinase may be involved in this diversity. This work was supported by the grant 505/431 from the University of Lodz. P37 Vanadyl sulfate can differentially damage DNA in normal and cancer cells K. Wozniak, and J. Blasiak Department of Molecular Genetics, University of Lodz, Lodz, Poland Using the comet assay, we showed that vanadyl sulfate induced DNA damage in human normal lymphocytes and HeLa cells. Vanadyl at 0.5 and 1 mM produced DNA single- and double-strand breaks (SSBs and DSBs) in lymphocytes, whereas in HeLa cells we observed only SSBs. Post-treatment of vanadyl-damaged DNA from lymphocytes by formamidopyrimidine-DNA glycosylase (Fpg), an enzyme recognizing oxidized purines, gave rise to a significant increase in the extent of DNA damage. Similar effect was observed in HeLa cells, but, using endonuclease III, we detected also oxidized pyrimidines in DNA of these cells. There were no differences in the extent of DNA damage in lymphocytes and HeLa cells in the alkaline and pH 12.1 version of the comet assay, that indicates that strand breaks and not alkali-labile sites (ALSs) contributed to the measured DNA damage. Study of DNA repair of vanadyl-damaged DNA revealed that HeLa cells retained the ability to repair of their DNA at a concentration ten times higher than lymphocytes. Incubation of the cells with spin traps: DMPO, POBN and PBN decreased the extent of DNA damage, which might follow from the production of free radicals by vanadyl sulfate. The presence of vitamin A, C and E caused an increase of DNA damage in HeLa cells, whereas in lymphocytes such an increase was observed only for vitamin C. Our data indicate that vanadyl sulfate can be genotoxic for normal and cancer cells. Vitamins A, C and E can increase its genotoxic potential. This work was supported by the grant 505/431 from the University of Lodz. P38 Diabetogenic streptozotocin can induce DNA damage in normal and cancer cells Sikora1, K. Wozniak1, J. Drzewoski2, and J. Blasiak1 1 Department of Molecular Genetics, University of Lodz, Lodz, Poland, 2 Department of Clinical Pharmacology, Medical University of Lodz, Lodz, Poland

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Streptozotocin is an antibiotic, which can be used to induce diabetes in experimental animals and this can be exploited in research on pathogenesis of diabetes. To use streptozotocin as a diabetogenic substance, its molecular mode of action should be understood. Using the alkaline comet assay we showed that streptozotocin at concentrations from the range 0.01-100 µM induced DNA damage in normal human lymphocytes and HeLa cells in a dose-dependent manner. Treated cells were able to recover within a 120-min incubation. Vitamins C and E at 10 and 50 µM diminished the extent of DNA damage induced by 50 µM streptozotocin. Pretreatment of the lymphocytes with the nitrone spin trap, �-(4-pyridil-1-oxide)-N-tert-butylnitrone (POBN) or ebselen, which mimics glutathione peroxidase, or pyrrolidine dithiocarbamate (PDTC) reduced the extent of DNA damage evoked by streptozotocin. The cells exposed to alloxan and treated with endonuclease III (Endo III), formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (AlkA), the enzymes recognizing oxidized and alkylated bases, displayed greater extent of DNA damage than those not treated with these enzymes. These results suggest that free radicals may be involved in the formation of DNA lesions induced by streptozotocin. The drug can also methylate DNA bases. This broad range of DNA damage induced by streptozotocin indicates that the drug may seriously affect genomic stability in normal and pathological cells. This work was supported by the grant 505/431 from the University of Lodz. P39 Interchain cross-linking formed by halomalonaldehyde derivative Koissi Niangoran, Neuvonen Kari, Mikkola Satu, and Lönnberg Harri Department of Chemistry, University of Turku, FIN-20014 Turku, Finland Structural characterization of products formed by DNA alkylation provides the molecular basis for subsequent drug design and genotoxic evaluation. Formation of interstrand cross-links in DNA creates a severe challenge to a cell. Cross-link is per se an absolute block to replication for being present in both strands. Therefore, many efforts have been devoted to this type of structures. Previous studies from our group and others have revealed that BMA reacts with nucleic acid bases to generate cyclic adducts. Nevertheless, no cross-link was observed. A cross-linked dinucleoside, di(N6-Guanosyl)ethylene and other products have been isolated from the reaction of guanosine with bromomalonaldehyde. Adducts suggested a decomposition of BMA into glyoxal. Few such adducts have been isolated and characterized up to date. That has been aided by the fact that the cross-linking reactions of alkylating agents are irreversible. We will present series of results on the reaction of guanosine and BMA. Adducts were characterized by 1H and 13C NMR, UV, and mass spectra. Nevertheless, consistent structures and mechanism have been proposed. 1 Mikkola S, Koissi N, Ketömäki K, Rauvala S, Neuvonen K, Lönnberg H. Eur. J. Org. Chem. 2000, 2315 1 BMA: Bromomalonaldehyde Molecular Epidemiology of Cancer P40 DNA repair genes XRCC1, XRCC3, XPD polymorphisms and risk of multiple head and neck cancers. M. Rydzanicz1, M. Gajecka1, M. Wierzbicka2, M. Kujawski1, K. Szyfter1,2 1 Institute of Human Genetics, Polish Academy of Sciences, Poznań, Poland. 2 Department of Otolaryngology, K. Marcinkowski University of Medical Sciences, Poznań, Poland. DNA repair plays a critical role in maintaining the genome stability. Polymorphisms in DNA repair genes may be associated with differences in ability to repair DNA damage induced by carcinogens, and may influence an individual risk of head and neck cancer. We examined polymorphisms of three DNA repair genes: XRCC1 (G28152A and C26304T), XRCC3 (C18067T) and XPD (A35931C and C22541A). The choice of genes was connected with their involvement in three different DNA repair pathways. Genotyping was done in material derived from 167 patients with a single primary tumour, 63 subjects with multiple head and neck squamous cell carcinoma (HNSCC), and 118 healthy controls (matched by age, gender and tobacco use). Genotypes were identified using PCR-RFLP technique. The statistic analysis was performed to calculated odds ratio (ORs) and 95% confidence intervals (CIs). The XPD 35931AC and 22541CA genotype variants were more frequent in multiple HNSSC (53,2% and 59,7%, respectively) than in patients with a single cancer (41,9% and 45,8%, respectively) and controls (48,3% and 54,2%, respectively), but did not reach a level of statistic significance. Though

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22541CA genotype was much more frequently found in subjects with multiple cancer (59,7%,) than with single primary cancer (45,8%,) and it was associated with a borderline increased risk (OR = 1,84; 95% CI = 0,96-3,53; p = 0,064) of multiple HNSSC. The pattern of XRCC1 and XRCC3 genes distribution were similar in all subgroups and no statistically significant differences between controls, single primary tumor and multiple cancer patients were found. The finding concerning an association between XPD polymorphism and a risk of second (multiple) primary tumors is deduced from relative small number of the studied subjects and it needs to be verified by further investigations. Having larger groups, gene-gene interaction should be also taken into account. P41 Simultaneous evaluation of mitomycin C induced DNA damage and repair in the overall genome and in specific DNA sequences using a modified Comet-FISH assay. M. Gallus1, D. McKenna1, S. McKeown2, S. Downes1, V. McKelvey-Martin1

1 Cancer and Ageing Research Group, Faculty of Life and Health Sciences, University of Ulster, Coleraine, Northern Ireland, UK; 2 School of Applied Medical Science and Sports Studies, University of Ulster, Jordanstown, Northern Ireland, UK. The alkaline Comet assay (ACA) is a sensitive and rapid method to detect and quantify DNA strand breaks in individual cells. We have developed two modifications of standard protocols to investigate more thoroughly the effect of the DNA cross-linking agent mitomycin C (MMC). The first enables the quantification of DNA cross-links by inducing strand breaks with γ radiation and measuring the reduction of DNA migration caused by prior treatment with MMC. Using this modification a clear dose response was demonstrated in RT4 bladder cancer cells treated with 0, 5, 50 and 200µg/ml mitomycin C (MMC). When cells were treated with 50µg/ml MMC and allowed to repair, the MMC-induced cross-links require at least 24 hours to be completely repaired. In a second application the ACA has been combined with fluorescent in situ hybridisation (FISH) to study the localisation and sensitivity of specific gene domains within cells. Using the Comet-FISH assay the number and location of hybridisation spots of specific genes can be recorded for each cell. Using a probe for TP53 we showed the number of spots per cell, and per comet tail, decreased as the dose of MMC increased. In repair experiments, the number of spots, especially in the comet tail, increased as repair time increased. In particular, our results suggest that following MMC treatment the TP53 gene region repairs within 4 hours which is considerably faster than the average global DNA repair which takes ~24 hours. In conclusion, the Comet-FISH assay provides a novel method to study DNA damage and repair of selected gene regions in response to crosslinking agents. P42 Cytogenetic monitoring of occupational exposure to aromatic hydrocarbons: characterization of GSTM1 genotypes F.Festa1, R.Cozzi1, M.Giachelia2, R.Ranaldi2, D.Tirindelli2, A. De Marco3, M.Owczarek3 and A.Testa2 1 Dipartimento di Biologia, Università degli Studi “Roma Tre” Italy 2 Sezione Tossicologia e Scienze Biomediche, ENEA Casaccia, Roma, Italy 3 Centro Genetica Evoluzionistica, CNR, Roma, Italy A cytogenetic investigation (chromosomal aberrations (CA), sister chromatid exchanges (SCE), micronuclei (MN) and DNA primary damage (Comet assay) was carried out on a group of 25 workers (car painters) (12 smokers, 13 non-smokers) exposed principally to aromatic hydrocarbons and on 37 subjects (14 smokers, 23 nonsmokers) of a control group (from healthy blood donors) matched for gender and age. All subjects (mean age = 46 years) were asked to fill in the personal healthy questionnaire proposed by the International Commission Protection against Environmental Mutagens and Carcinogens. Workers were also selected using a questionnaire concerning the individual occupational exposure to chemical substances. Ambient air samples at working places were regularly analysed and high values of benzene, toluene, xylene and ethyl-benzene were always found. Furthermore, in the attempt to elucidate the interaction between genome and environment, we analysed in the same samples the GSTM1 polymorphism. Based on results of the cytogenetic assays, the exposed group shows an higher frequency of genetic damage compared to the controls if we consider non-smokers subjects. As far as smoker subjects are concerned, a slight statistical difference between exposed and controls was found in the frequency of total chromosomal aberrations. Comet parameters were lower in exposed smokers than in control ones. This work was partially supported by a grant from Ministero del Lavoro (contract n.1072) and from MIUR on “BIOLOGICAL BASIS ON HUMAN SUSCEPTIBILITY”

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P43 Screening of TP53 mutations in tumours and measurement of oxidative DNA damage in lymphocytes taken from a case-control study related to oesophageal cancers in Lower-Normandy (France), a European high incidence area J. Breton1, M. Lechevrel1, V. Prevost1, J. Marnay2, D. Arsène3 and F. Sichel1

Groupe Régional d'Etudes sur le Cancer (GRECAN), EA 1772 - Université de Caen / Basse-Normandie, CRLCC François Baclesse, Route de Lion / Mer, 14 076 Caen cedex 05 - France. 1 UFR des Sciences Pharmaceutiques de Caen and Laboratoire de Cancérologie Expérimentale, CRLCC François Baclesse, 2 Laboratoire d'anatomie pathologique, CRLCC François Baclesse, 3 Département d'Hépato-Gastroentérologie, CHU Côte de Nacre, 14 033 Caen cedex. In Europe, the major area for the incidence of oesophageal cancer is located in North-Western France. We recruited patients suffering from cancer of the oesophagus and controls in Lower-Normandy. With the aim to more efficiently characterise etiological factors in this area, various biomarkers were studied among which TP53 mutations and the oxidative DNA adduct 8-OHdG (8-hydroxy-2'-deoxyguanosine). TP53 mutations can be employed as a molecular signature of carcinogen exposure (e.g. aflatoxin B1 and G to T transversions at codon 249 in liver cancers). In order to establish such relationships in oesophageal cancers, we first screened TP53 alterations in 53 tumours with DGGE (Denaturing Gradient Gel Electrophoresis) followed by sequencing. We used the results to validate a more recent and automatic screening tool: DHPLC (Denaturing High Performance Liquid Chromatography). With ninety-seven % (33/34) of squamous cell carcinomas presenting at least one mutation or polymorphism, we corroborated data obtained in three former studies and showing mutation rates exceeding 80% in Normandy and Brittany. G to A and G to T substitutions were the most frequent. These mutations could be linked to nitrosamines, acetaldehyde, benzo[a]pyrene or nitrogen and oxygen reactive species. In adenocarcinomas, the alteration frequency was 69% (11/16) with a majority of G to A transitions at CpG dinucleotides. These mutations could be related to endogenous and inflammatory process located in lower oesophagus and cardia (e.g. Barrett's oesophagus and gastro-oesophageal reflux disease). These latter pathologies generate oxidative compounds. Oxidative stress in oesophageal tissues could also be generated by xenobiotic metabolism, hot alcoholic beverages or dietary deficiencies. We therefore decided to study 8-OHdG, a commonly used biomarker of oxidative DNA damage. The determination of this adduct remains quite delicate owing to the low limit of detection required and artifactual oxidations during sample treatments. This report presents validation stages of our HPLC-ECD (ElectroChemical Detection) method and the first measurements in lymphocytes of controls and patients. Levels are ranged between 2 and 10 8-OHdG for one million 2'-deoxyguanosines and seem to be in agreement with latest estimates of the ESCODD (European Standards Committee on Oxidative DNA Damage). In the near future, our aim will be to link these biomarkers with data regarding etiological factors (tobacco, alcohol, diet) and collected from patients and controls. Moreover, we will attempt to validate relationships between carcinogens, TP53 mutations and 8-OHdG with in vitro and in vivo testing. P44 Screening of TP53 mutations by DHPLC and sequencing in central nervous system cancers with an occupational exposure to pesticide or solvents V. Loyant1, D. Provost2, J. Breton1, I. Baldi2, S. Dutoit1, A. Vital2, H. Loiseau3, P. Lebailly1, P. Gauduchon1 1 GRECAN and EA1772, Université de Caen, France, 2 Laboratoire Santé Travail Environnement, Institut de Santé Publique, d’Epidémiologie et de développement, Université Victor Segalen, Bordeaux, France, 3 Centre Hospitalier Régional Universitaire, Bordeaux, France Several epidemiological studies described an association between pesticide exposure and brain cancers. But at present we cannot consider this exposure as a causal factor for this cancer. TP53 mutations could reflect exposure in certain cancers. In gliomas, one of the most frequent brain cancers, TP53 mutations occur in about 40% of cases. We explored the hypothesis that pesticide exposure could directly or indirectly increase the frequency of TP53 mutations in the central nervous system, thus contributing to the brain carcinogenesis. A population based case-control study on occupational and environmental factors of central nervous system cancers was carried out in south-western France between 1999 and 2001 and found a significant increase in risk for the individuals the most occupationally exposed to pesticides (OR=2.44, CI=1.24-4.78). We investigated TP53 mutations in exons 2 to 9 by DHPLC and sequencing and p53 accumulation by immunohistochemistry in cerebral tissue of 34 cases from this study. These patients were all cases with occupational exposure to pesticides or solvents from whom tumoral tissue was available. 17 were exposed to pesticides, 12 to solvents, and 5 both to solvents and pesticides. Histology of tumors was as follows: 14 gliomas, 7 meningiomas, 8 schwannomas, 2 central nervous system lymphomas and 3 other histologic types. At present we detected 3 mutations in a glioblastoma and in the two central nervous system lymphomas, two mutations leading to an amino acid exchange and the other to a stop codon. Mutations were found at codon 280 (exon 8), 271 (exon 8) and 132 (exon 5). Two mutations are transitions and one is a transversion. To our knowledge on mutations in the different histologic types of brain cancers, we expected 4 mutations in our sample. Moreover 2 patients with TP53 mutation were in the lowest pesticide exposed group. These preliminary results are not in favour of an association between pesticide exposure and TP53 mutations.

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P45 Frequency of micronuclei in human population occupationally exposed to radionuclides Jovičić D,Novakovic M,Rajačić I, Milačić S, Kovačevoć R CCS, Institute of Occupational Medicine and Radiological Protection, Belgrade, Yugoslavia, Medical Faculty, University of Banjaluka, Bosnia and Herzegovina This study presents possibilities for evaluation of radiation lesions induced by exposure to radionuclides.Occupational exposure is particularly delicate because of chronic exposure to low doses of ionizing radiation where, due to cumulative effect it is important to get insight into biological response to the body to the given exposure conditions. Genetic monitoring was conducted by cytochalasin-block (CB) micronucleus test in subjects working with open source of radiation (nuclear medicine).The study comprised two groups of subjects: 23 individuals that worked in once nuclear medicine center (the first group) and 11 individuals that worked in the other nuclear medicine center (the second group).Analysis of controls were those free from exposure to mutagenic agents (23 individuals). The average incidence of number of micronuclei (MN) per 1000 CB cells was 13.7 ±1.5 in the first group of subjects and 8,27±3,35 in the second, while the respective value was 6.0±0.6 in controls. Comparison of the exposed and control groups revealed significant difference (p<0,05) between them. The results have also shown that the MN count in the exposed subjects correlated with duration of exposure (R=0,290) positively. The changes in the genetic material of occupationally exposed subjects can be correlated with exposure to ionizing radiation, inadequate occupational protection, poor education and individual sensitivity to radiation. P46 Seasonal variation of DNA adduct patterns in farmers and non-exposed population J. Le Goff, V. Andre, P. Lebailly, D. Pottier, M. Henry-Amar and P. Gauduchon GRECAN (UPRES-EA 1772, Université de Caen), Centre François Baclesse, route de Lion / mer, 14076 CAEN cedex 05, France Since 1995, our group has lead an epidemiological study in order to estimate the risk of cancer in relation with farming and more specifically with occupational exposure to pesticides. Standard approach in epidemiology, based on the carrying out of cohorts and cancer incidence follow-up, is associated with the use of biomarkers of genotoxicity in blood and urinary samples. In this way, DNA adducts were evaluated by 32P-post-labelling method in a pesticide exposed group (crop farmers, n=26) and in a non-exposed group (n=29). For both groups, two blood samples were collected per individual: the first in January (S0) corresponding to a period of non exposure to pesticides for farmers, and the second in May-June (S4) during intensive spraying operations. Seasonal variations on leukocyte DNA-adduct patterns were analysed. Overall, DNA adduct levels appear relatively low compared to literature. However, for farmers, the mean DNA-adduct level increases significantly from S0 to S4 (RAL = 3.8 ± 3.2 x10-10 Vs 13.0 ± 15.6 x10-10, p=0.004) while no variations are observed in referents (RAL = 4.1 ± 3,8 x10-10 Vs 3.5 ± 2.2 x10-10, p=0.4). Therefore, open field crop farming, including repeated use of pesticides, seems to be linked to the global increase of DNA adduct levels during the spraying season. Qualitative analysis of DNA-adduct patterns tends to indicate an increase in the number of individuals with more than two distinct spots, only for S4 samples in the farmer group. However, the spot diversity remains comparable for the two groups, independent of the sampling period. In all cases, the most frequent spots were localised in an almost central area on chromatograms. Thus, the DNA-adduct increase observed in the farmer group seems to be the result of the reinforcement of pre-existing spots and/or the formation of new ones under occupational exposure conditions. P47 N7-Methyldeoxyguanosine levels in the DNA from patients with and without colorectal adenomas K.L. Harrison1, N.P. Lees2, C.N. Hall2, G.P. Margison3, A.C. Povey1. 1Biomarkers laboratory, Centre for Occupational and Environmental Health, School of Health Sciences and Epidemiology, University of Manchester, 2Wythenshawe Hospital, Manchester, 3Cancer Research UK Carcinogenesis Group, Paterson Institute for Cancer Research, Manchester, UK. Diet-derived and endogenously formed alkylating agents may be important in human colorectal tumourigenesis. We have previously developed an assay to quantify the predominant adduct produced by methylating agents, N7-methyldeoxyguanosine (N7-MedG) in DNA (Harrison et al, Chem. Res. Toxicol. 2001, 14, 295). We have now used this assay in a case control study to determine whether N7-MedG levels in colorectal DNA differ in patients with and without

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adenomas and in patients with and without a K-ras mutation in their adenoma. Biopsy samples were obtained at colonoscopy, DNA was extracted and N7-MedG levels were determined in DNA of normal mucosa from 73 cases and from 73 age, sex and site matched controls. Adenoma DNA was extracted from paraffin-embedded tissue blocks and examined for K-Ras mutations using polymerase chain reaction techniques. DNA from the majority of cases (89%) and controls (93%) had detectable levels of N7-MedG (>0.08µmol N7-MedG/mol dG) with mean levels of 0.91 ± 1.19 (range 0.08-7.73) and 0.94 ± 0.97 (range 0.10- 4.07) µmol N7-MedG/mol dG respectively. There was no significant difference in N7-MedG levels between normal mucosa of all adenoma cases and matched controls or after subdivision into small (< 10mm; n =19) and large (> 10mm; n =54) adenoma groups or with regard to patient age and gender. There was no difference in N7-MedG levels between the colon and rectum except that levels were lower in the rectum of small adenoma formers (0.37 ± 0.47; n =7) than in both the rectum of matched controls (1.26 ± 1.24; n =7; p < 0.05) and the colon of adenoma formers (1.48 ± 2.07; n = 2; p < 0.05). No overall correlation between N7-MedG level and histological grade and stage of adenoma was detected. But higher N7-MedG levels were associated with increased grade of dysplasia for both tubular and tubulovillous adenomas respectively in large adenomas. For tubulovillous adenomas the difference in N7-MedG levels between moderately dysplastic adenomas (median 0.204 µmol N7-MedG/mol dG; n= 12) and severely dysplastic adenomas (median 0.573 µmol N7-MedG/mol dG; n= 15), was of borderline statistical significance (p = 0.059). K-Ras mutations in codons 12 and 13 were found in 44% of the adenomas cases (n = 63) with the GC→AT transition mutations, that are associated with alkylating agent exposure accounting for 68% of all the mutations detected. N7-MedG levels were not significantly different between normal tissues of the K-Ras GC→AT mutated adenomas than in their matched controls. Likewise, there was no significant difference in N7-MedG levels between the normal tissue of the K-Ras non-mutated adenomas and the mutated adenomas. In summary, alkylation damage in colorectal DNA is ubiquitous with N7-MedG levels varying approximately 100-fold. Factors associated with this high variability in methylating agent damage are unknown but are likely to include both exposure and DNA repair. Whilst N7-MedG levels are not associated with the presence of an adenoma such alkylation damage may influence the histological progression of large adenomas. On the other hand, levels of alkylation damage in normal tissue are not directly associated with mutation in the adenoma itself in this study. This would suggest that factors, other than exposure to genotoxic agents, are important in influencing mutational risk in adenomas. P48 The effect of ethyl methane sulfonate (EMS) and hydrogen peroxide (H2O2) on DNA damage and repair in control individuals, p53+ cells and p53- cells from non-Hodgkin’s lymphoma (NHL) patients in the comet assay Behzad Foroutan1, Afruj Ali Ruf 2 and Diana Anderson3 1Department of Biomedical Sciences and Pharmacy, University of Bradford, UK, BD7 1DP 2Department of Haematology, Airedale General Hospital, Steeton, Bradford, UK 3Department of Biomedical Sciences, University of Bradford, UK, BD7 1DP One of the most important problems of cancer treatment is the development of drug resistance in cancer cells. Although the first course of chemotherapy may destroy most of the cancer cells some resistant cells may survive and proliferate. In some patients, the resistant cells may grow again and ultimately kill the patient. Though the problem of drug resistance in clinical setting is complex, it is necessary to continue research for a better understanding of the problem. The present study has investigated DNA damage and repair in peripheral lymphocytes from control individuals, p53+ and p53- NHL patients. The p53+ patients have shown a poor response to chemotherapy (CHOP regime). This is not the case for p53- patients. Cells have been treated with the model alkylating agent, EMS and model free radical agent, H2O2. Our results have shown that for DNA damage with H2O2, cells from p53+ patients had the greatest damage and control cells the lowest, while p53- cells showed intermediate damage. For repair with H2O2, p53+ cells have shown no repair capacity, whilst control cells repaired totally and p53- cells showed same intermediate repair. In contrast, with EMS for DNA damage, the control cells showed the highest damage, then p53- cells an intermediate level and p53+cells the lowest damage. For repair with EMS the control cells showed the highest repair capacity, p53+ cells little repair capacity and p53- cells on intermediate repair capacity. For kinetics of repair with EMS, p53+ cells did not show good repair kinetics however, control cells did and p53- cells showed an intermediate response. Cytotoxicity was determined with trypan blue exclusion for all samples during the experiments. Control cells were 99% viable and the clinical samples were more than 80% viable. The cell density of the control cells were 1x106, in p53- cells were 0.28x 106 and p53+ cells were 0.21x106. Mutations in genes such as the p53 suppressor oncogene may lead to drug resistance by allowing cells to undergo transformation and inactivating apoptosis so causing continuing proliferation and drug resistance. Ineffective repair can increase such mutation. Regardless of the mode of action of the chemical treatment in the present study the relationship between p53+ and p53- cells and repair remained constant and the poor repair response, particularly in p53+ might help explain their drug resistance. Reference: Hall and Johnson. The role of DNA repair in the prevention of cancer

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P49 A study on the DNA damage and repair efficiency of lymphocytes from breast cancer patients N. Anagnostakis1,2, N. Messini-Nikolaki2, G. Karanastasi1,2, G. Christopoulos1,2, K. Maridaki1,2, S. Tsilimigaki1, M. Kanioura3 and S.M. Piperakis1 1DNA Repair Laboratory, Institute of Biology, NCSR Democritos. Athens, Greece. 2Department of Cell Biology, School of Biology, University of Athens, Athens, Greece. 3Hemodynamics Laboratory, Evangelismos Hospital, Athens, Greece. Women with breast cancer and a family history of breast cancer and some with sporadic breast cancer seem to be deficient in the repair of radiation-induced DNA damage compared with normal donors with no family history of breast cancer. In the present study the effects of H2O2 and γ-irradiation on lymphocytes from breast cancer patients was investigated. Using the comet assay technique we estimated the DNA damage and the repair capacity on the above population in comparison to corresponding controls. Our results indicate a decreased DNA repair capacity in lymphocytes from breast cancer patients after the effects of the above mutagens. P50 Lung cancer patients’ lymphocytes DNA damage and repair efficiency Karanastasi1,2, N. Messini-Nikolaki2, N. Anagnostakis1,2, K. Maridaki1,2, G. Christopoulos1,2, K. Gourgoulianis3, K. Christou3, S. Tsilimigaki1, M. Kanioura4 and S.M. Piperakis1 1DNA Repair Laboratory, Institute of Biology, NCSR Democritos, Athens, Greece. 2Department of Cell Biology, School of Biology, University of Athens, Athens, Greece. 3Pulmonary Department, University of Thessaly, Larisa, Greece. 4Hemodynamics Laboratory, Evangelismos Hospital, Athens, Greece. Although lung cancer is the paradigm of tobacco-induced malignancy, host-specific factors modulate susceptibility to tobacco mutagenesis. Variations in DNA repair may influence the rate of removal of DNA damage and of fixation of mutations. To test the hypothesis that genetically determined DNA repair capacity modulates lung cancer susceptibility we conducted a study of patients with newly diagnosed previously untreated lung cancer and controls matched to the cases. In particular the effects of γ-irradiation and H2O2 in peripheral blood lymphocytes of lung cancer patients compared to healthy individuals was investigated. Using the comet assay we estimated the DNA damage and the repair capacity of the above populations. Our results indicate that lung cancer patients have a reduced DNA repair capacity. P51 Effect of GSTM1 and ERCC2/XDP gene polymorphisms on anti-BPDE-DNA adduct formation in mononuclear white blood cells of coke oven workers. S. Pavanello1, G. Coppola1, A. Pulliero1, E. Siwinska2, D. Mielzynska 2 and E. Clonfero1 1Departement of Environmental and Public Health- Occupational Health Section, University of Padova, (Italy).2Institute of Occupational Health, Sosnowiec (Poland). We investigated the influence of genetic deletion polymorphism of GSTM1 (*0/*0) and polymorphisms of XPD gene (codon Lys751Gln and Asp312Asn) on levels of anti (±)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE)-DNA adduct in the peripheral blood lymphocyte plus monocyte fraction (LMF) of highly BaP-exposed coke oven workers. A total of 95 male Polish coke oven workers (60% current smokers) comprised the sample population. PAH exposure was assessed by means of the urinary excretion of 1-pyrenol (mean + SD: 6.93+ 7.20 µmol/mol creatinine; frequency of subjects exceeding the proposed BEI (2.3 µmol/mol creatinine) were 70%). Anti-BPDE-DNA adduct levels were detected by HPLC/fluorescence analysis of the anti-BPDE tetrol I-1 released after acid hydrolysis of DNA samples. Genotypes were determined by PCR on genomic DNA of each subject. Mutated allele frequencies XPD 312 Asn (0.37) and 751 Gln (0.45) were those found in a Polish population. GSTM1 null frequency (0.33; 95% CI: 0.23 -0.43) was fairly lower than that found in a Caucasian population of 10451 subjects (0.53; 95% CI 0.42–0.60) and that found in a Polish population of 145 ones (0.49; 95% CI 0.41-0.57).Coke oven workers without GSTM1 (GSTM1 *0/*0, 33%) had significantly higher adduct levels

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than those with GSTM1 active (GSTM1*1/*1) (5.90+5.59 vs 3.25+2.01 adducts / 108 bases , Mann-Whitney U-test , z=2.53, p=0.011) being rather equal PAH-exposure in the two subgroups (7.06±6.83 vs 6.67±8.00 1-pyrenol µmol/mol creatinine). The highest presence of GSTM1 null subjects belonged to the quartile with the highest adduct levels (i.e; >4.67 adducts/ 108 nucleotides). That is coke oven workers with GSTM1 *0/*0 genotype had a significantly higher risk of having high adduct levels than individual with GSTM1 *1/*1 genotype (Fisher exact Odd Ratio (OR) = 4.145, 95% CI 1.000-18.761; p=0.0355). XPD genotypes did not affect alone the levels of BPDE-DNA adducts, while the more susceptible combination XPD Asn312Asn+Gln751Gln and GSTM1 null (n=5) gives the highest adducts levels (7.62±11.08 adducts/ 108 nucleotides). A progressive decrease of DNA adduct levels was found in workers with less-risk genotype combinations: XPD Asp312Asp+Lys751Lys / GSTM1 null (n=9) > XPD Asp312Asp+ Lys751Lys /GSTM1 active (n=17) > XPD Asn312Asn+Gln751Gln/ GSTM1 active (n=8) (5.64±3.36 > 2.91±1.14 >2.47±1.46 adducts/ 108 nucleotides). In the whole multiple linear regression analysis showed that the increase in anti-BPDE-DNA adduct levels in LMF was significantly related to the, high and long-term, occupational exposure to PAHs (BaP) of coke oven workers (t=3.206, p=0.0019) and to the lack of GSTM1 activity (t=3.518, p<0.001), but no statistically significant effect of XPD 312 and 751 polymorphisms was detected. Our results raise the possibility that the XPD Asn312Asn +Gln721Gln genotype may increase BPDE-DNA damage; this effect is evident in individuals who accumulate DNA damage because they are highly PAH-exposed and lack of some detoxification capacity. In this study the influence of GSTM1 detoxifying genotype on anti-BPDE-DNA adduct formation in LMF of coke oven workers was clearly demonstrated. This genetic factor appears to have an importance in the same size order of the occupational exposure to PAHs. This fact could be possibly at the basis of a higher lung cancer risk in coke oven workers by GSTM1 null genotype. P52 Influence of cytochrome P450 1A1 (CYP1A1) and myeloperoxidase (MPO) genotypes on cigarette smoking-associated urinary mutagenicity S. Pavanello1, G. Coppola1, S. Lupi2, P. Simioli2, and Erminio Clonfero1 1 Section of Occupational Health, Department of Environmental Medicine and Public Health, University of Padova, Italy 2Section of Hygiene and Occupational Medicine, Department of Clinic and Experimental Medicine, University of Ferrara, Italy. The polymorphic enzymes cytochrome P450 1A1 (CYP1A1), and myeloperoxidase (MPO) activate, the latter by radical oxygen generation, carcinogens in tobacco smoking including BaP and aromatic amines. An increased CYP1A1 activity was experimentally found in the CYP1A1 * 2 allele, while a reduced MPO activity was found in carriers the mutated alleles MPO A463A or A463G. CYP1A1 *2 has been related to lung cancer risk, conversely the mutat MPO 463 A allele seems to be protective against lung cancer. We investigated the CYP1A1 and MPO genotypes in relation to cigarette smoking-associated urinary mutagenicity detected on YG1024 Salmonella typhimurium strain with S9 mix in 96 smokers. In each subject, cigarette smoke intake was checked by analysis of internal tobacco smoke exposure (i.e., urinary 1-pyrenol, nicotine and its metabolites and trans, trans-muconic acid), CYP1A1 (*1 and *2 alleles) and MPO (G463A) genotypes were determined by RFLP. Frequencies of subjects carrying CYP1A1 *2 (0.29) and MPO 463A (0.42) alleles were those found in other Caucasian populations. The lowest mutagenic activity was seen in subjects poor activators, CYP1A1 *1/ *1 and MPO A/G or A/A (n=26), compared to the smokers with higher CYP1A1 (*1/ *2 and *2/ *2, n=28) and MPO (G/G, n=56) activities (1464±948 vs 1864±1489 and vs 1789±1516 revertants/mmol creatinine, respectively. Mann-Whitney U test, not significant). On the other hand the combination CYP1A1 *1/ *2 and *2/ *2 (high activity)/ MPO A/G and A/A (slow activity,) (n=14) gives the highest levels of urinary mutagens (1464±948 vs 2193±1658 revertants/mmol creatinine, Mann-Whitney U test, z=1.57, p=0.29) suggesting a slight predominant role of CYP1A1 activity in the increase of mutagenic activity. In multiple regression analysis, among the indicators of internal exposure the influence of urinary 1-pyrenol on urinary mutagenicity was predominant, followed by those of urinary trans,trans- muconic acid and nicotine plus metabolites (t= 5.09, 2.56 and 1.92, p<0.001, p=0.012 and p= 0.057, respectively). No statistically significant effect of CYP1A1 and MPO genotypes in raising (activating) urinary mutagens was found. In conclusion our results show that smoking exposure is the major determinant of mutagenic urine among smokers and the best correlation with urinary 1-pyrenol, designates the combustion processes of tobacco as the determining step for the formation of urinary mutagens. The lowest mutagenic acivity we found in urine of smokers with the genotype combination CYP1A1 *1/ *1 and MPO A463G or A463A can be slightly related to their poor activating capacity, preventing them from exposure to lung cancer-inducing, cigarette smoke-associated, carcinogens.

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P53 Application of the alkaline comet assay and sister chromatid exchange analysis for monitoring DNA damage in white blood cells of breast cancer patients under cytostatic therapy N. Kopjar1, V. Garaj-Vrhovac1, and I. Milas2

1 Institute for Medical Research and Occupational Health, Laboratory of Mutagenesis, Zagreb, Croatia, 2 The Universitiy Hospital for Tumors, Zagreb, Croatia The alkaline comet assay and sister chromatid exchange analysis were employed to evaluate the genotoxic effects of cytostatic therapy in 20 female patients with breast cancer. During study, all patients were given adjuvant chemotherapy according to CMF (cyclophosphamide, methotrexate, 5-fluorouracyl), FAC (5-fluorouracyl, adriamycin, cyclophosphamide) and AC (adriamycin, cyclophosphamide) protocols. To quantify the levels of primary DNA damage in white blood cells before and after chemotherapy two comet parameters were studied: tail length and tail moment. Pre- and post-therapy frequencies of sister chromatid exchanges and mitotic activity have been evaluated in PHA-stimulated lymphocytes. The results obtained using both methods indicate considerable interindividual variations between the levels of DNA damage in white blood cells of breast cancer patients before chemotherapy. After intravenous administration of antineoplastic drugs in all patients significantly increased levels of DNA damage compared to their pre-treatment values were observed. Following chemotherapy the lymphocyte mitotic activity in vitro was also significantly disturbed, with marked delays and retardations in cell cycle progression. With regard to the results obtained it can be concluded that the patients with breast cancer show increased levels of DNA damage and chromosomal instability which is strongly modulated by chemotherapy protocols employed. Alkaline comet assay and sister chromatid exchange analysis were powerful biomarkers that enable sensitive detection of critical DNA lesions produced in white blood cells of breast cancer patients after administration of standard chemotherapy protocols. Besides both tests could be used in biomonitoring of cancer patients in remission, they might be also helpful in pre-clinical evaluation of particular chemotherapy protocols before starting with cancer therapy. P54 Correlation between DNA adduct levels, determined by 32P-postlabelling and chemiluminescence immunoassay, in tumourous and normal peripheral lung tissues from lung cancer patients Schoket1, E. Győrffy1, L. Anna1, Z. Győri2, J. Segesdi2, J. Minárovits2, I. Soltész3, S. Kostič3, A. Csekeő3, and M.C. Poirier4 1 József Fodor National Center for Public Health, Budapest, Hungary, 2 Béla Johan National Center for Epidemiology, Budapest, Hungary, 3 Korányi National Institute of Pulmonology, Budapest, Hungary, 4 National Cancer Institute, Bethesda, MD, USA A comparative study of smoking-related carcinogen-DNA adducts by the 32P-postlabelling method and the recently developed benzo[a]pyrene -7,8-diol 9,10-epoxide-DNA chemiluminescence immunoassay (BPDE-DNA CIA) has been performed to explore primary DNA damaging processes induced by cigarette smoke in human lung. Within the framework of an ongoing molecular epidemiological study in a Hungarian lung cancer population, pairs of tumour and normal peripheral lung tissue samples were obtained from 63 patients, who underwent lung resection. Information on smoking history was obtained by self-reporting. Levels of aromatic DNA adducts were determined by the 32P-postlabelling method with nuclease P1 enrichment. Polycyclic aromatic hydrocarbon (PAH)-DNA adduct levels were determined by the competitive BPDE-DNA CIA. Statistical analyses included the Wilcoxon matched pair test and the Spearman correlation test. DNA adduct levels were in the same range, between 0.3 and 30 adducts in 108 nucleotides, using both methods. Undetectable PAH-DNA adduct levels were observed in 6% of samples by BPDE-DNA CIA. A smoking-related increase of adduct levels was significant in both normal and tumour tissues, as determined by 32P-postlabelling, and non-significant, as determined by CIA. Both methods clearly indicated lower DNA adduct levels in the lung tumours compared to the normal tissues, with approximately two-fold higher DNA adduct levels in the normal lung tissues. There were statistically significant positive correlations, in both smokers and non-smokers, between the adduct values of the matched tumour and normal tissue pairs by 32P-postlabelling (Spearman correlation coefficients r≥0.74, p≤0.0005) and by CIA (r≥0.37, p≤0.048), respectively. There was a strong correlation between the DNA adduct levels, determined by the two methods, in tumour (r=0.456, p=0.0076), however, there was no such correlation in normal lung (r=0.250, p=0.161). The lower DNA adduct levels in the tumour tissue may originate from the combined effect of rapid cell proliferation, lower metabolic capacity, altered DNA repair processes, and other cellular activities that may resemble multidrug resistance. The positive correlation between the two methods in lung tumour tissues, and the lack of correlation in normal tissue may suggest qualitatively different DNA adduct patterns in the tumour and the normal tissue, as well as differences in adducts detected by each assay. The research project has been supported by the Hungarian OTKA T 034616 research grant, and in part by the UICC International Cancer Technology Transfer Fellowship to B.S. (ICRETT No. 605/2002) with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract NO2-CO-91012.

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P55 The comet assay as a tool to determine genetic components of radiation sensitivity in families of young lung cancer patients Maria Gomolka*, Ute Rössler*, Heike Bickeböller§, Vera Zietemann#, H.-Erich Wichmann#, Karin Hetzl*, Thomas Jung*, and Sabine Hornhardt* Federal Office for Radiation Protection, Department SG Radiation Protection and Health Oberschleißheim, § Instiute of Genetic Epidemiology, University of Göttingen, #Institute of Epidemiology, GSF Research Center, Germany Lung cancer is one of the most frequent cancer death causes among males in Germany. 8% of all lung cancer patients are 50 years old or younger. In young lung cancer patients the early onset of the disease is most likely promoted by genetic components. Genes that are responsible for the integrity of the genome or DNA repair, like p53 or ATM, are not only strong candidate genes for susceptibility to any cancer including lung cancer. In addition, they also modulate radiation sensitivity of an individual, so that individuals bearing a radiation sensitive phenotype display an intermediate phenotype for which it should be more likely to develop cancer. In order to investigate in vitro radiation sensitivity we analyse the individual reaction to radiation and subsequent DNA repair by the comet assay. The comet assay is a single cell gel electrophoresis and an ideal tool to study in vitro DNA damage and DNA repair. We will investigate radiation sensitivity in lung cancer families from the LUCY study (lung cancer in the young). Inclusion criteria for the index patient are primary lung cancer, histological or cytological verified, with age of onset equal to or less than 50 years. Data about smoking habits, medical treatment and other life style factors were collected for index patients and their first degree family members using a questionnaire. So far lymphocytes of 270 young lung cancer patients, 122 parents, 259 siblings, 104 adult children and 52 partners were isolated. Lymphocytes were stored in liquid nitrogen. Our first aim to be presented here is as follows: We want to test whether in vitro radiation sensitivity as measured by the outcomes of the comet assay (e.g. tail moment) in young lung cancer patients differs significantly from radiation sensitivity of their healthy relatives. Family members can be categorized into four groups: patients (1), siblings (2), children (3) and non-related partners (4). At the current stage we will not consider parents due to their older age. Comparisons will be carried out between groups in pairs matched by family. The group of non-related partners can be considered as the group representing “general” population in terms of genetic susceptibility and radiation sensitivity. We will present the detailed study design including our automated comet assay system, as we are still in process to increase our sample size in order to achieve statistical power. This will be the first analysis to quantify radiation sensitivity in families of young lung cancer patients where it is expected that relatives are genetically more comparable to patients than members of the general population. Moreover, if in vitro radiation sensitivity correlates with lung cancer development, the comet assay could also function as a prognostic biomarker for lung cancer with a strong genetic component.

P56 Genetical predisposition to human stress expression F.Ingel. T.Ivaschenko, I.Sidorova, V.Safronov A.N.Sysin Research Institute of Human Ecology and Environmental Hygiene of Russian academy of medical Sciences, Moscow, Russia Previously we demonstrated that human stress expression correlates with level of chromosomal aberrations in blood cells, sensitivity of genome to environmental mutagenic load and with the level of toxic exposure. Therefore, we speculated that a genetical predisposition to stress expression could exist, and it could be located among the family of genes encoding enzymes of detoxification. We studied the GST super-gene family (GSTM1+/+ or GSTM10/0 and (GSTT1+/+ or GSTT10/0) and PON 54 gene polymorphism (L/L, L/M or M/M) as well as stress expression among workers of chemical plants and control populations (total 54 men at age of 43±3). The level of stress was tested using a block of standard psychological questionnaires (detected psychological depression, alarm, overfatigue and interpersonal relations). Both GSTM1 and GSTT1genes ensure the synthesis of the relevant products that belong to the Phase II detoxification enzyme system, responsible for biotransformation and degradation of certain electrophilic compounds. The PON 54 gene takes part in the Phase I detoxification enzyme system. GSTM1, GSTT1 and PON 54 gene polymorphism analysis was carried out in blood samples as described elsewhere. Results are presented in Table 1 and Table 2. Table 1. Number of people with different stress expression among men with minor allelic variants of GSTM1 and PON 54 genes.

Number of people with different stress expression Gene

Number of people

Alarm Overfatigue Psychological depression.

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(allele) with allelic variants

Deadap-tion

Norm Deadap-tion

Norm Deadap-tion

Norm

PON 54 (L/M orM/M) 29 23 6 21 8 12 17 GSTM1 (0/ 0) 30 22 8 21 9 14 16 Table 2. Number of people with different types of deadaptive stress expression and frequency of GSTM, GSTT1 and PON 54 allelic variants among them

Number of people with different allelic variants PON 54 GSTM1 GSTT1 Total for 3 genes

Psychological test

Number of people with deadaptive stress

L/M + M/M

L/L (norm)

0/0

Norm

0/0

Norm

Mutant alleles

Notm alleles

Psychological depression 15

9

6

11

4

7

8

13 (86.7%)

2

Alarm 36 22 14 21 15 6 30 29 (80.5%) 5 Overfatigue 16 10 6 9 7 3 13 13 (81.3%) 3 Interpersonal relations 20 9 11 9 11 5 15 16 (80%) 4 Total in deadaptive stress

22 15

7

14

8

4

18

18 (81.8%)

4

In addition, correlation between deadaptive stress expression and frequency of GSTM1 0/0 in the population (p≤0,05) was observed. Our results are in a good agreement with H.Selye hypothesis that stress is first and normal reaction of an organism to any influence. Depending on the power and/or duration of influences and adaptive potential of an organism stress expression is the norm (adaptive) or deadaptive. Minor alleles in genes, encoding enzymes of detoxification, bind with decreasing of detoxificative functions of their products. Thus, the real toxic load to the organism which posesses these alleles is increased and can be expressed as deadaptive stress reaction. P57 Genetic health and psychological status of children living in Aral Sea region. Comparative study. F.Ingel1, G.Baturina1, P.Eckl2, L.Erdinger3, Sh.Khussainova4 1A.N.Sysin Research Institute of Human Ecology and Environmental Hygiene of Russian Academy of Medical Sciences, Moscow, Russia;2 University of Heidelberg Department for Hygiene and Medical Microbiology, Heidelberg, Germany;3 University of Salzburg, Institute of Genetics and General Biology, Salzburg, Austria;4 Scientific Center of Pediatrics and Children Surgery, Almaty, Kazakhstan

In the Aral Sea region of Kazakhstan the health of children is reported to be generally poor, with high morbidity and mortality, high rate of chronic diseases and retarded mental and physical development. In our study children (boys and girls of 5-7 years old), living in Aralsk town (27 persons) and settlement Akchi – a region of comparison situated far from the Aral Sea (25 persons) were tested for the level of micronuclei (with cytokinetic block), cells with 2,3,4 or more nuclei, cells in apoptosis as well as mitotic indexes in blood cell cultures. In addition we measured radiosensitivity against all of the parameters to 1.0Gy of γ-irradiation and adaptive response to γ-irradiation of 0.05+1.0 Gy with a 5 hour interval. Subjects were assigned to groups following determination of the social status of the families and children’s stress expression (Lusher’ test). Results. Results of the cytogenetical study are shown in the table 1.

Table 1

Spectrum of dividing cells Cells with micronuclei Settle-ment

MI

Part of cells with the number of nuclei

Influence

2 3 4 More

A

B

C

D

E

Aralsk 45.4* 209* 26.5* 72.1* 3.8 3.0* 1.0* 0.8* 14.3* 1.8* 0.0 Gy Akchi 21.3 278 11.7 45.2 4.8 10.8 3.0 4.1 54.3 8.4 Aralsk 49.8* 216 28.8* 52.5* 4.3 2.8* 0.8* 1.3* 164.3* 24.4* 1.0 Gy Akchi 19.3 225 16.9 24.9 3.5 12.8 3.9 5.7 212.5 32.2 Aralsk 47.7* 210 19.8 34.7 2.7 3.9* 1.2* 1.3* 206.6 32.0 0.05+1.0

Gy Akchi 22.5 249 14.4 27.7 3.6 16.4 4.5 5.4 209.8 33.1 Aralsk 1.6 1.1 1.7* 1.1 1.3 1.2* 1.2 24.9* 17.2* Radiosen

-sitivity Akchi 1.4 0.9 2.5 0.9 0.9 1.9 1.6 16.2 11.0

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*) P ≤ 0,05; MI – mitotic index; A - Number of cells in apoptosis; B - % of cells with apoptotic nuclei among dividing; C - Mononuclear cells with micronuclei; D - Number of binuclear cells with micronuclei; E - Number of binuclear cells with multiple micronuclei As it is shown, in spite of the fact that among children from Aralsk the background level of binuclear cells with micronuclei was significantly lower, than for ones from Akchi, indexes of radiosensitivity, as well as data of cellular population spectrum demonstrates that genome instability among children from Aralsk was more expressed. Inasmuch as in drinking water and in soil were not determined neither toxic and genotoxic compounds, nor genotoxic activity in vitro, we concluded that genetical status of children from Aralsk town was determined by sociological and psychological factors. Because of groups for the study were formed using data of psychological estimation of stress reaction, they were not differed by part of children being in state of adaptive and nonadaptive stress. But stress expression in Aralsk group commonly was higher, then in Akchi one, as well as their families were poorest then in Akchi. Results of detailed analysis of sociological tests demonstrated that the main reason of high stress expression among children from Aralsk was poverty. So, the connection: poverty – nonadaptive stress expression – increased genome sensitivity to genotoxic influence was demonstrated. These data once more supported results obtained previously and the position that human nonadaptive stress expression is connected with high genome sensitivity to genotoxic influences. The study was supported by INTAS grant 1005 (2002).

P58 A comparison of the cytogenetic response to irradiation of resting peripheral blood lymphocytes, IL-2 stimulated lymphocytes and EBV-transformed lymphoblastoid cells A.Baeyens*, A. Vral*, H. Thierens° and L. De Ridder* Department of Anatomy, Embryology, Histology* and Medical Physics°, University of Gent, L. Pasteurlaan 2* and Proeftuinstraat 86°, 9000 Gent, Belgium. Ionising radiation induces chromosomal damage. An enhanced chromosomal radiosensitivity has not only been demonstrated in a large number of patients with cancer prone genetic diseases (e.g. Ataxia-Telangiectasia (A-T)) but has also been observed in a significant proportion of breast cancer patients and other cancers with no obvious family history (head and neck, colorectal ...). To investigate the chromosomal radiosensitivity of lymphocytes in cancer patients the MN-assay is often used. In this MN-assay blood samples are exposed to 2Gy 60Co γ-rays and the number of radiation-induced micronuclei is scored in 1000 binucleate lymphocytes. Several radiosensitivity studies with AT-patients are done in Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines. The advantage of EBV cell lines for this kind of tests in cancer patients is that the tests can be easily repeated without any further blood sampling. Another possibility to allow repeating testing is to work with IL-2 stimulated lymphocytes. The question is whether the radiation response of IL-2 stimulated lymphocytes and EBV-transformed lymphoblastoid cells is the same as in resting peripheral blood lymphocytes. In our study we have used peripheral blood lymphocytes and lymphoblastoid cells derived from 10 healthy individuals and 10 breast cancer patients with an increased radiosensitivity. The results obtained with these 3 different cell culture systems are compared.

P59 Chromosomal radiosensitivity in BRCA1 and 2 patients and healthy carriers A. Vral1, A. Baeyens1, H. Thierens1, K. Claes2, B. Poppe2, L. Messiaen2 and L. De Ridder1 1Department of Anatomy, Embryology, Histology and Medical Physics, University of Gent 2Department of Medical Genetics, University of Gent, Gent, Belgium. The chromosomal radiosensitivity of a selected group of familial breast cancer patients carrying a mutation in BRCA1 or 2 and a group of healthy mutation carriers was investigated and compared to a reference group of healthy women carrying no mutation. The chromosomal radiosensitivity was assessed with the G2 and the G0-micronucleus (MN)-assay. For the G2-assay lymphocytes were irradiated in vitro with a dose of 0.4 Gy 60Co γ-rays after 71h incubation and chromatid breaks were scored in 50 metaphases. For the MN-assay lymphocytes were exposed in vitro to 3.5 Gy 60Co γ-rays at a high dose rate (HDR) or low dose rate (LDR). 70h post-irradiation cultures were arrested and micronuclei were scored in 1000 binucleate cells. The results demonstrated that the group of breast cancer patients with a BRCA2 mutation (n=10) was on the average more radiosensitive than the reference group (n=86) and this as well with the G2 as with the HDR and LDR-MN-assay (P values < 0.05). Comparison between the BRCA1 patient group (n=8) and the reference group revealed significant differences in mean MN values obtained as well with the HDR and LDR protocol, but no significant differences between the mean G2 values. As it was the aim of this study to investigate if this enhanced radiosensitivity observed in BRCA1 and 2 patients is related to a mutation in one of the breast cancer genes, a group of healthy carriers was also analysed. The results however showed no significant difference in chromosomal radiosensitivity between the healthy carriers (nBRCA1=6, nBRCA2=6) and the reference population of healthy women carrying no mutation. In conclusion our results reveal that the chromosomal radiosensitivtity observed in breast cancer patients with a BRCA1 or 2 mutation is not due to the presence of a mutation in the known breast cancer genes, this although BRCA1 and 2 are both involved in DNA repair/signalling processes.

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P60 Comparison of the cytotoxic effect of 131I-lipiodol therapy and 188Re-lipiodol therapy in HCC patients Kim De Ruyck1, Anne Vral1, Bieke Lambert2, Rudi Dierckx2 and Hubert Thierens1 1Department of Anatomy, Embryology, Histology and Medical Physics, Ghent University, Gent, Belgium 2Division of Nuclear Medicine, Ghent University Hospital, Gent, Belgium Primary hepatocellular carcinoma (HCC) is one of the most common malignant tumours in the world. One of the approaches available for treating irresectable HCC is intra-arterial injection of 131I-lipiodol. Although the obtained clinical results are positive, the therapy can be improved using 188Re instead of 131I. As high energy beta-emitter 188Re is a more interesting radionuclide for therapy. Furthermore, 188Re has a shorter half life and has only low intensity γ-rays in its decay. The aim of the study is to compare the cytotoxic effect of the radionuclide therapy in HCC patients treated with 131I-lipiodol and 188Re-lipiodol. For this study, dicentric chromosomes were scored in metaphase spreads of peripheral blood. The equivalent total body dose was obtained using an in vitro dose response curve. The population consists of a group of 18 HCC patients treated with 131I-lipiodol and 6 patients treated with 188Re-lipiodol. Patients were treated by intra-arterial injection of a mean activity of 1.94 GBq 131I- or 3.54 GBq 188Re-lipiodol. For each patient a blood sample was taken before therapy. From the patients treated with 131I-lipiodol, a blood sample was obtained one week and two weeks after therapy. Taking into account the shorter half-life of 188Re, blood samples of the patients treated with this radionuclide, were obtained one day after and two days after therapy. Analysis of the data show that the mean number of dicentrics for the patients, treated with 131I-lipiodol, was respectively 0.013 per cell, 0.093 per cell and 0.147 per cell before therapy, one week after and two weeks after therapy. The whole body dose for this patient population, after background correction, was 1.19 Gy one week after therapy and 1.61 Gy two weeks after therapy. For the patients receiving 188Re-lipiodol, the mean number of dicentrics scored in the lymphocytes resulted in a number of 0.049 per cell before therapy, 0.116 per cell one day after and 0.138 per cell two days after therapy. This corresponds, also after background correction, to 1.11 Gy and 1.32 Gy. The study indicates that, for the administered activities, the cytotoxic effect of 188Re-lipiodol therapy is lower than for 131I-lipiodol therapy. P61 Chromosome instability in rubber tyre plant workers as measured by the lymphocyte cytokinesis-block micronucleus assay: Implications for cancer risk prediction and prevention E. Mirkova and E. Alexandrova National Center of Hygiene, Medical Ecology and Nutrition, Sofia, Bulgaria Human monitoring studies of occupational genotoxic exposure of Bulgarian rubber industry workers by using the lymphocyte cytokinesis-block micronucleus assay were carried out to assess the chromosome and genome mutations for purposes of cancer risk prediction and prevention. The study population consisted of 48 exposed workers in two departments of a rubber tyre manufacturing plant where butadiene-styrene rubber was used (exposed group 1, front processing department, 23 subjects; exposed group 2, component assembly and building department, 25 subjects) and 31 matched controls. Workers were exposed to a range of specific compounding chemical additives. The exposure measurements data showed that shift average air-concentrations of benzo(a)pyrene, TMTD and altax at the Banbery mixer area and callendering site respectively, as well as the maximum mineral oils air concentration at the milling site area in the front processing department were 2.5-3.5 fold higher the respective national occupational exposure limits. TWA, 8h air-concentrations of total hydrocarbons above the occupational exposure standard were detected at the assembly line, solvent tanks, presses, tyre trimming machines in the component assembly and building department. The frequencies of micronucleated binucleated cells (MN-BN) and micronuclei (MN) were recorded in 1000 BN cells, harvested 72h after mitogen stimulation. Highly significant, 3-4 fold increase in the frequencies (mean +/- SD) of MN-BN [exposed subjects, 39.7+/-12.5 (E1 group), and 49.0+/-18.7(E2 group); controls, 13.4+/-6.1] and MN [exposed subjects, 48.2+/-16.5(E1 group), and 66.1+/-27.5 (E2 group); controls, 16.7+/-8.6] was found in all studied workers. The MN frequencies were affected by the occupational exposure only. There was no significant effect of gender, age and smoking habits of the study subjects on the cytogenetic biomarker. On the basis of the MN data obtained an increased cancer risk was predicted for the rubber plant workers and cancer prevention program was elaborated.

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P62 Smoking status-dependent correlation between DNA adduct levels in lung tissues and peripheral blood lymphocytes in lung patients E. Győrffy1, L. Anna1, Z. Győri2, I. Soltész3, S. Kostič3, A. Csekeő3, J. Minárovits2, and B. Schoket1 1 József Fodor National Center for Public Health, Budapest, Hungary, 2 Béla Johan National Center for Epidemiology, Budapest, Hungary, 3 Korányi National Institute of Pulmonology, Budapest, Hungary Investigations examining biomarkers of carcinogen exposure in lung tissues as target tissue, and in peripheral blood lymphocytes considered as potential surrogate tissue will provide new information on the usefulness of the blood lymphocytes for environmental carcinogen exposure assessment. As part of an ongoing molecular epidemiological study in a lung cancer population, samples of histologically normal peripheral lung tissues and peripheral blood lymphocytes were obtained from a total of 62 Hungarian patients who underwent lung resection. Information on smoking history was obtained from the patients by self-reporting. In the present study those patients who were active smokers at the time of the surgery or had given up smoking less than a year before surgery were categorised as smokers. Those patients who were never-smokers or had stopped smoking more than one year before the lung surgery were considered non-smokers. Levels of aromatic DNA adducts were determined by the 32P-postlabelling method with nuclease P1 enrichment. Tests used for statistical analysis included the Mann-Whitney U-test and the Spearman correlation test. Smoking status was clearly reflected in DNA adduct levels in the lung, showing significantly higher lung DNA adduct levels in smokers (10.0 ± 6.2 adducts/108 nucleotides, N = 28) as compared to non-smokers (4.0 ± 2.9 adducts/108 nucleotides, N = 21, P ≤ 0.0003). The DNA adduct levels in the normal peripheral lung from smokers were on average 60% higher than DNA adduct levels in the lymphocytes from the same individuals (p ≈ 0.06). Smoking status did not influence significantly the adduct levels in the peripheral blood lymphocytes (smokers: 6.5 ± 4.2 adducts/108 nucleotides, N = 18; non-smokers: 5.1 ± 3.3 adducts/108 nucleotides, N = 16). In the total study population a statistically significant correlation was calculated between DNA adduct levels of individual pairs of blood lymphocytes and normal lung tissue samples (r = 0.44, P = 0.0265, N = 26). However, when selection was made for smoking status of the patients, the relationships changed remarkably. Whereas there was no correlation in smokers (r = 0.14, P = 0.62, N = 12), a statistically significant strong correlation was observed between the DNA adduct levels of the blood lymphocytes and normal lung in non-smokers (r = 0.72, P = 0.0082, N = 14). Lack of correlation between normal lung tissue and peripheral blood lymphocytes in smokers, and the positive correlation in non-smokers suggest, that the correlation between target and surrogate tissue may depend on the dose of exposure and on the metabolic capacity of the corresponding tissues. The research project has been supported by the Hungarian OTKA T 034616 research grant. P63 The role of CYP2A6 polymorphism in modulating smoking behaviour and lung cancer risk S. Morandi, A. Nunziata and C. Andreoli, Research Department, Eti S.p.A, Rome, Italy Nicotine is a major constituent of tobacco smoke and is responsible for establishing and maintaining tobacco dependance. Addiction to nicotine has been established as a psychopharmacologic mechanism that maintains cigarette smoking behaviour. Nicotine is mainly metabolised to cotinine by the family of cytochrome P450(CYP)2A6 which is also responsible for much of the metabolism of cotinine and for much of the activation of the potent tobacco smoke carcinogens NNN and NNK. In the last few years, large interindividual differences in nicotine metabolism have been described, that result in a lower level of cotinine in plasma. The basis for costitutive differences in activity has been associated with variant CYP2A6 alleles encoding inactive enzymes. In this work, we reviewed some recent studies to the aim to clarifying the role of poor nicotine metabolism in smoking behaviour and nicotine dependence. Recent papers have studied the relationship between the poor metabolism of nicotine and genetic polymorphism in the human CYP2A6 gene. Several CYP2A6 polymorphisms have been observed. The CYP2A6*1A is the wild type. The CYP2A6*1B arises from a gene conversion with CYP2A7 in the 3’-untranslated region. The CYP2A6*2 allele encodes an unstable and catalytically inactive enzyme. The CYP2A6*3 allele has gene conversion in exons 3, 6 and 8 and is inactive. CYP2A6*4, CYP2A6*5, CYP2A6*6, CYP2A6*7, CYP2A6*8 and CYP2A6*1×2 result in a defective or null enzyme activity. These alleles are present with a low frequency in populations. A possible role of CYP2A6 genotype in modulating smoking behaviour and lung cancer risk has been proposed in many papers. There is evidence that smokers adapt their smoking behavior (such as number of cigarettes smoked, inhalation depth, volume of each puff) to maintain peripheral and central nicotine levels. It may therefore be postulated that the level of nicotine in smokers with CYP2A6 mutant form is normally higher because of impaired nicotine metabolism, and, as a consequence, there is a lower intake of nicotine. In fact, it has been reported that the presence of the CYP2A6*1 and CYP2A6*2 alleles significantly decreases the number of cigarettes consumed by smokers, and has been suggested that individuals who carry at least one copy of either CYP2A6*A2 or CYP2A6*B2 may be protected against becoming tobacco dependent. Morever, because tobacco smoke contains nitrosamines which can be activated to carcinogens by CYP2A6, individuals who carry CYP2A6-null alleles may also be less efficient at activating tobacco-smoke procarcinogens to carcinogens. This fact, together with the protective effect of CYP2A6-null alleles against becoming tobacco-dependent and in decreasing consumption, has been suggested as a possible factor that reduce the risk of lung cancers in smokers who carry CYP2A6-null allels. Moreover, a study suggests that smokers with duplicated CYP2A6 genes may have an increased risk to develop lung cancer.

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P64 Comparison of the influence of GSTM1, GSTT1 and GSTP1 Ile105Val genetic polymorphisms on the levels of bulky DNA adducts in two human study populations exposed to polycyclic aromatic hydrocarbons Schoket, E. Győrffy, G. Papp, K. Lévay, L. Anna National Institute of Environmental Health, József Fodor National Center for Public Health, Budapest, Hungary Impact of metabolic genotypes on biomarkers of exposure has been studied extensively in various human study populations. A great complexity is reflected in the results depending on the types of exposure, the investigated biomarkers, tissues and genotypes. In the present work we compare the influence of GSTM1, GSTT1 and GSTP1 ile105val genotypes and their combinations on bulky carcinogen-DNA adduct levels in different tissues in two Hungarian study populations exposed to polycyclic aromatic hydrocarbons (PAHs) (main descriptives of the study populations in: Schoket et al, Mutation Res 482: 57-69, 2001). DNA samples were analysed for both bulky DNA adducts and genotypes from macroscopically normal bronchial tissue samples from 200 lung cancer patients undergoing lung resection, and from peripheral blood lymphocytes from 170 workers occupationally exposed to PAHs in pot-rooms of aluminium plants. DNA adduct levels were determined by the 32P-postlabelling method using nuclease P1 adduct enrichment. Genotypes were determined by PCR-based methods. Smoking status of the individuals was self-reported. A statistically significant strong positive linear correlation was observed between bronchial aromatic DNA adduct level and low cigarette doses (≤15 cigarettes per day) for current smoker lung patients with GSTM1 null and GSTT1 null genotypes, respectively (correlation coefficient r= 0.70, two-tailed p value p<0.0001 for GSTM1 null, and r=0.897, p<0.0001 for GSTT1 null, respectively), but there was no such association for GSTM1 positive or GSTT1 positive individuals. There was a statistically significant linear correlation between white blood cell DNA adduct levels and PAH exposure, measured by urinary 1-OHpy concentrations, for smoking potroom workers with GSTM1 null genotype (r=0.581, p=0.011). In smoking lung patients with GSTM1 null genotype, GSTP1 val105 allele reduced bronchial DNA adduct levels significantly compared to the wildtype GSTP1 ile105 genotype, if those genotypes were associated with selected CYP genotypes. Similarly, in smoking pot-room workers with GSTM1 null genotype, the GSTP1 val105 allele caused a trend of decrease of white-blood-cell DNA adduct levels, particularly in association with GSTT1 null genotype. The similar results obtained in the two different study populations for different tissues suggest that i) genotoxic PAH dose-response relationship may be affected by GSTM1 genotype significantly, and ii) GSTP1 val105 allele may act against bulky DNA adduct formation in PAH exposure situations. The results may initiate further studies to investigate whether the findings hold a broader relevance. The research project has been supported by the Hungarian OTKA T 034616 and ETT 003/2001 research grants. P65 Inhalation of arsenic induces micronuclei in lymphocytes and epithelial buccal cells of the copper smelteries workers Dobrosława Gradecka, Kalina Wyszyńska, Konrad Rydzyński Department of Toxicology and Carcinogenesis, Nofer Institute of Occupational Medicine, Łódź, Poland Inorganic arsenic compounds are considered human carcinogens. Epidemiological studies have shown that chronic exposure to arsenic may cause skin, liver, kidney, urinary bladder and lung cancer. The main source of exposure to inorganic arsenic compounds is contaminated drinking water, but there are also other mostly anthropogenic sources of arsenic in the environment. They include: smelting of nonferrous metals, burning arsenic-containing coal, production and use of arsenical pesticides and glass manufacturing. In Poland the highest exposure to arsenic appears during smelting of nonferrous metals ores in the copper smelteries. The aim of the study was assessment of the chromosome damage expressed as frequency of micronuclei (MN) in peripheral blood lymphocytes and buccal epithelial cells of workers occupationally exposed to arsenic. The study group consisted of 72 subjects employed in the copper smelteries at the departments, where the arsenic concentration ranged from 3 - 80 µg/m3. The control group (83 persons) was recruited from healthy men, non-exposed to arsenic or other known genotoxic compounds. All subjects were at similar age and the numbers of smokers in both examined groups were similar, about 58%. The venous blood, buccal epithelial cells and urine samples were taken from all participants. From venous blood samples of each donor the 72 hours lymphocyte cultures were set up. For estimation of MN frequency 1000 binucleated cytokinesis-blocked lymphocytes were analysed for each person. The buccal cells were collected by centrifugation and the microscopic slides were done from the pellet. For determination of the MN frequency, a total of 2000 cells for each person were analysed. The arsenic concentration in the urine samples of each individual was determined by hplc-aas. The results have indicated increase (p<0.001) of the frequency of MN in lymphocytes of exposed (7.96±4.28) in comparison to the controls (3.47±1.70). Statistically significant differences were found also between smokers and nonsmokers in both exposed and control populations. One fact is noteworthy, that only in lymphocytes of As-exposed the binucleated cells with two MN have been observed. These finding suggests that occupational exposure to As might affect

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chromosomes of worker`s lymphocytes causing more severe damage. However arsenic has not affected the proliferation rate of the lymphocytes as was indicated by the values of Nuclear Division Index (close to 2.00) in both studied groups. Similarly as in lymphocytes, in buccal epithelial cells the frequency of MN was higher in exposed than in the controls (1.96±1.51 and 1.00±1.04 respectively). The difference was statistically significant (p<0.05). The content of total arsenic in urine of exposed was almost 6 fold higher than in the control group (57.0±50.3 µg/l vs 12.3±19.4 µg/l). The unvariable regression analysis has not revealed the correlation between the level of exposure to arsenic expressed as total arsenic concentration in urine of subjects and the genotoxic effects in lymphocytes and buccal epithelial cells. Study supported by 5th Framework Programme of UE, Contract No.QLK4-CT –1999-01142. P66 Effect of occupational exposure to arsenic on DNA damage in leucocytes and buccal mucosa cells of copper smelters Jadwiga Palus, Elżbieta Dziubałtowska, Konrad Rydzyński Department of Toxicology and Carcinogenesis, Nofer Institute of Occupational Medicine, Lodz, Poland Chronic exposure to inorganic arsenic compounds (i-As) is responsible for the prevalance of various types of cancer, including lung, skin, liver, kidney and urinary bladder cancers, as well as of other diseases. The studies of people exposed to high concentrations of i-As in drinking water show its genotoxicity. In Poland, the main source of exposure to i-As are fumes and dust at the working environment of copper smelteries. The some studies especially in vitro suggest that it does not affect DNA directly but it may intensify toxic effects of other physical and chemical agents, particularly by DNA repair inhibition. The aim of this study was to assess the level of DNA damage in leucocytes and buccal mucosa cells of workers occupationally exposed to i-As by inhalation at the copper smelteries. Arsenic group consisted of 72 men employed at the departments where the high temperature processes have proceeded. Their mean age was 42.2±7.3 years and the mean duration of As exposure was 18.0±7.3 years. About half of them are smokers. The control group included 83 volunteers from the health services. Most of them are men and smokers, at the mean age 37.9±11.6 years. Blood, oral mucosa and urine samples were collected in the morning after night shifts, transported to the lab during 8 hrs and processed. DNA damage, including single strand breaks (SSB), alkali labile sites (ALS) before and after DNA repair process and oxidative DNA damage (using Fpg enzyme), were measured by the comet assay. An image analysis system (LUCIA Comet Assay) was used to determine DNA damage. Median values of tail moment were used as a indicator of DNA damage. The average median of tail moment for each person both in arsenic exposed and control groups were statistically analysed using the two-way analysis of variance test. The differences were regarded as a statistically significant if p<0.05. The study has revealed the significantly higher tail moment in leucocytes of arsenic workers (13.2±2.8x10-3 ) compared to persons in control group (2.1±0.6x10-3 ). The significant repair of damaged DNA was observed mainly in the smelters (13.2±2.8x10-3, 3.2±1.0x10-3 before vs after repair). However the levels of DNA damage after repair process were still significantly higher in smelters (3.2±1.0x10-3) than in controls (0.9±0.5x10-3). After using FPG enzyme, the level of DNA damage in leucocytes has significantly increased both in exposed and control groups (70.1±13.0x10-3 and 8.6±2.0x10-3 ) compared to the respective level of DNA damage before enzyme (13.2±2.8x10-3 and 2.1±0.6x10-3 ). The level of DNA damage in exfoliated cells of smelters was a little higher compared to controls (10.29±1.14 vs 8.32±0.94) and this difference was not statistically significant. The oxidative DNA damage in these cells was similar to the respective DNA damage without enzyme both in exposed and control groups. The concentration of total arsenic excreted with urine in exposed group (57.0±50.3 µg/l) was significantly higher than in control group (12.3±19.4 µg/l). However the univariable regression analysis has revealed the lack of correlation between the level of exposure to arsenic and the genotoxic effects in lymphocytes of individuals. This work was supported by 5PR/99/01142/UE project P67 Lack of protective effects of inhaled corticoids on oxidative damage of peripheral mononuclear cells in allergic asthmatics Gazdik, F., Dušinská, M., Jahnová, E., Pijak, M.R. and Gazdíková, K. Institute of Preventive and Clinical Medicine Bratislava, Slovak Republic Bronchial asthma represents a chronic inflammatory disease with pathogenesis not completely elucidated and a multifactorial origin has been proposed for the disease. There is increasing evidence for the participation of oxygen free radicals in inflammation and therefore the potential protective role for dietary antioxidants in asthma is discussed. The aim of the study was to compare oxidative DNA damage of peripheral blood mononuclear cells (PBMC) assessed in allergic asthmatics with healthy subjects. Forty one patients (29 females, 12 males, average age 45 yrs) with mild and moderate persistent asthma (according to criteria defined in GINA 2002) were enrolled in the study. They were matched

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for age and sex with 23 healthy subjects (16 females, 7 males, average age 45 yrs). Bronchial asthma was controlled by administration of optimal doses of inhaled corticoids as demonstrated by pulmonary function tests and stabilized clinical status of patients. Oxidative DNA damage in PBMC was assessed by the comet assay modified with lesion-specific endonucleases to detect oxidised purines (FPG sites) and pyrimidines (EndoIII sites) as well as strand breaks (SBs). DNA breaks, with and without oxidised purines or pyrimidines, were estimated as arbitrary units (au) and analyzed by Wilcoxon´s unpaired test. The values of oxidative DNA damage in allergic patients were significantly higher compared to those of healthy volunteers (SBs 352+/-40 vs. 134+/-48, SBs+ EndoIII 367±33 vs. 184.1±43, SBs+FPG 370±22 vs. 186.5±34; p=< 0.0001, p=< 0.0001, p=< 0.0001, respectively). The higher levels of oxidative DNA damage of PBMC in asthmatics suggest that inhaled corticoids did not protect PBMC from oxidative stress. However the doses were sufficient for clinical controll of asthma. In addition these results support the evidence of excessive oxidative stress in allergic asthmatics. This work was supported by the Ministry of Health of Slovak Republic. P68 Evaluation of DNA damage in leukocytes of G6PD-deficient Iranian newborns (Mediterranean Variant) by using comet assay Seyed A. Mesbah-Namin1, Alireza Nemati1 and Mohammad Taki1 1 Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common inherited red-cell enzymopathy to cause neonatal hemolysis and jaundice. Recent studies of our group showed that the Mediterranean variant of this enzyme (Gd-Med) is the predominant state in Iranian infants suffering from jaundice. G6PD deficiency is characterized by increased susceptibility of erythrocytes to H2O2 , a well-known oxidant agent. H2O2 can readily traverse lipid bilayers to sites that contain a reactive substrate and is able to damage DNA. Detoxification of H2O2 depends of a sufficient source of glutathione (GSH). Maintaining of GSH in the reduced state is important function of G6PD. GSH protects hemoglobin against oxidative damage. Considering the importance of GSH, we hypothesized that failure of detoxification of reactive oxygen species by GSH induces primary DNA damage in G6PD deficient white cells. For this purpose we analyzed mononuclear leukocytes of 20 male newborns suffering from Gd-Med variant by using alkaline single cell gel electrophoresis (SCGE) or comet assay. The level of DNA damage was compared with the level of basal DNA damage in the control group, represented by healthy donors, and compared with normal WBCs treated with H2O2. Visual scoring was used for evaluation of damage. The results showed that the comet tail length at individual level was found in the range of minimal (class 1) to maximal (class 4) damage in all of Gd-Med samples. This is the first report about using comet assay for evaluating DNA damage in Gd-Med samples and we conclude and confirm that G6PD plays an important role in protecting oxidative DNA damage. P69 Evaluation of DNA damage by the comet assay in newborns suffering from hyperbilirubinemia after phototherapy treatment Nemati1 and S. A. Mesbah-Namin1

Faculty of Medical Sciences, Tarbiat Modares University, Tehran-Iran Based on various clinical evidence and correlation studies, it has been suggested that phototherapy of hyperbilirubinemia in newborns infants is a safe and efficient form of therapy. However, some reports indicated that bilirubin acts as a photosensitizing agent enhancing the level of DNA damage in cells exposed to phototherapy light. Some of in vitro investigations have shown that irradiation of bilirubin solution with phototherapy lights, resulted to form long-lived toxic photoproducts. In order to investigate the presence of DNA strand breakage in mononuclear leukocytes of newborn infants with neonatal hyperbilirubinemia undertaken phototherapy treatment, we used single cell gel electrophoresis (SCGE) or comet assay, which provides a very sensitive method for detecting single strand breakage of DNA. The level of DNA damage was compared with the level of basal DNA damage in control group represented by healthy donors and compared with normal WBCs treated with H2O2 as an internal standard. Visual scoring was used for evaluation of damaging effects. For this purpose, we utilized the comet assay to analyze mononuclear leukocytes from two groups of newborns suffering from jaundice, with phototherapy (no: 20) and without one (no: 15). Results showed that, in spite of some variability of comet tail length, DNA damage in newborns undergoing phototherapy was greater than damage observed in newborns that had not undergone the treatment. We conclude and confirm that bilirubin may induce DNA damage in phototherapy treatment of neonatal hyperbilirubinemia. This is the first report about evaluation of DNA damage in hyperbilirubinemia subjects.

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P70 DNA damage and repair in breast cancer J. Blasiak1, K. Wozniak1, M. Zadrozny2, R. Krupa1, J. Rykala3, A. Kolacinska3, and Z. Morawiec3 1 University of Lodz, Lodz, Poland, 2 Polish Mother’s Memorial Hospital, Lodz, Poland, 3 N. Copernicus Hospital, Lodz, Poland, 4 Medical University of Lodz, Lodz, Poland Impaired DNA repair may fuel up malignant transformation, due to accumulation of spontaneous mutations in target genes and increasing susceptibility to exogenous carcinogens, including anticancer drugs, which target DNA. Moreover, the effectiveness of DNA repair may contribute to failure of chemotherapy and resistance of breast cancer cells to drugs. An individual ability to remove DNA damage may be, at least in part, determined by the genotype of DNA repair genes. It is generally accepted that the breast cancer genes BRCA1 and BRCA2 are involved in DNA repair. To evaluate further the role of DNA repair in breast cancer we determined: 1) the kinetics of removal of DNA damage induced by hydrogen peroxide, anticancer drugs and nickel chloride 2) the level of basal, oxidative and alkylative DNA damage before and after chemotherapy 3) polymorphisms of DNA repair genes: ERCC1, RAD51, hOOG1, XRCC1, XRCC3, XPD in the peripheral blood lymphocytes of breast cancer patients and healthy individuals. Because we employed lymphocytes rather than tumor cells, our findings reflect systematic differences in DNA repair genes and are not a reflection of somatic mutations within the tumors. We observed slower kinetics of DNA repair after treatment by doxorubicin in lymphocytes of breast cancer patients compared to control individuals. The level of basal, oxidative and alkylative DNA damage was higher in breast cancer patients than in the control. When patients were examined after chemotherapy, the level of DNA damage was out of the range of our measurement system. We found associations between the genotypes of repair genes and appearance of breast cancer. Our results indicate that constitutive disturbances in DNA repair effectiveness as well as polymorphisms in some DNA repair genes may contribute to the appearance of breast cancer. This work was supported by the grant 505/431 from the University of Lodz. P71 European network on children’s susceptibility and exposure to environmental genotoxicants Lisbeth E. Knudsen, Peter Vinzents & Louise Grave-Larsen Institute of Public Health, University of Copenhagen, Denmark This Concerted Action (CA) will form a European network providing input and data for risk assessment at the national and European level. The Children Genotoxicity Network will focus on gene-environment interactions during the foetal, neonatal and infancy developmental periods, concentrating on genotoxic exposures and environmental factors with focus on air pollution (traffic and tobacco). The aim of the Concerted Action is to collect, validate and review the available, though few, European and other studies and study materials on children’s exposure and susceptibility to genotoxicants, with focus on air pollution. A further aim is to prepare protocols to conduct feasibility studies with perfused placenta and with mothers and their children (two siblings), where research will be financed by national or other sources. Differences in exposure to genotoxic compounds coming from the environment, both during foetal life or during childhood, may induce damage to children’s health, and thereby increase risks of childhood cancers and other diseases. The network will form the basis for future projects within Europe, including new partners and with weight on exchange and mobility of young researchers. For this project 6 relevant research areas (work packages) have been chosen that are accessible to investigation today. 1) Literature study of (cyto) genetic data on children and protocol for establishment of pooled database; 2) Mother Child cohorts/survey; 3) Placental exposure; 4) Impact of air pollution on pregnancy outcome and child development; 5) Risk assessment; 6) Ethics. To cover all aspects of these areas 16 research groups with appropriate expertise have been selected. The concerted action will arrange several conferences and workshops, openly announced and in collaboration with relevant EU institutions. www.pubhealth.ku.dk/cgn project no QLK4-CT-2002-02198 P72 Genotyping of stainless steel welders, who participated in cytogenetic biomonitoring 1987 Lisbeth E. Knudsen, Michala Tved, Steffen Loft, Jesper Nielsen and Thorkil Boisen

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Institute of Public Health, University of Copenhagen, Denmark (l.knudsen@pubhealth.ku.dk) In 1987, 226 men participated in biomonitoring study with measurements of environmental exposures to dust and metals, concentrations of chromium and nickel in blood and urine, cytogenetic testing of chromosomal aberrations and sister chromatid exchanges and score of semen quality. An increased level of chromosomal aberrations was found in welders compared with reference persons and especially manual metal arch welding implied high exposures and high levels of damage. The cohort is part of European Study Group on Cytogenetic Biomarkers and Health (ESCH), which found a positive association between high levels of chromosomal damage and increased cancer risk. A follow up study with more detailed exposure estimation suggested individual factors of genotypes of metabolism important predictors of high levels of chromosomal damage (1). A study with isolation of DNA from 198 old samples and genotyping was performed, analysing for members of the GST family of enzymes, GSTM1, GSTP1 and GSTT1 and N-Acetyltransferase. In general linear modelling statistically significant effects of the combination GSTM1 null genotype, welder and smoker showed higher levels of chromosomal aberrations, sister chromatid exchanges and high frequency cells. Stefano Bonassi Lars Hagmar, Ulf Strömberg, Alicia Huici Montagud, Håkan Tinnerberg, Alessandra Forni, Pirjo Heikkilä, Saskia Wanders, Peter Wilhardt, Inger Lise Hansteen, Lisbeth E. Knudsen, Hannu Norppa for the European Study Group on Cytogenetic Biomarkers and Health (2000): Chromosomal aberrations in lymphocytes predict human cancer independently of exposure to carcinogens. Cancer Res 60, 1619-1625. The study was supported with a grant from achieved from the Danish Research Centre for Environmental Health. P73 The influence of the 137Cs internal contamination caused by the Chernobyl accident on the level of DNA damage evaluated by the comet assay in lymphocytes of Ukrainian children Omar García, Tania Mandina. Centro de Protección e Higiene de las Radiaciones. Calle 20 No.4113 e/ 41y 47 Miramar. AP 6195 C. Habana Cuba The influence of the internal contamination caused by the Chernobyl accident on the level of DNA damage was evaluated by the comet assay on lymphocytes of 30 Ukrainian children. The study was performed at the beginning of the 2003 year on children with demonstrable 137Cs internal contamination caused by the food consumption. The children were selected for the study immediately after a 137Cs whole body counter measurement of the internal contamination. The minimal detectable amount (MDA) of 137Cs was 75 Becquerel. The control group included 20 children without a detectable internal contamination, while in the exposed group 10 children with the internal contamination over the MDA were included. Blood sample was taken by fingerprick, the alkaline version of the comet assay was used, in a combination with silver stained comets and arbitrary units for comet measurement. Factors like children disease, medical treatment, surface contamination of children living location, etc were considered in the study. Non significant differences (p<0.05) on DNA damage in control (9.6 arbitrary units) versus exposed (7.6 arbitrary units) group were found. These results suggest that low doses of 137Cs internal contamination are not able to produce detectable DNA damage under the conditions used for the comet assay in this study. P74 Cadmium and radiation induced oxidative DNA damage in lymphocytes from patients with Alzheimer’s disease Mandla Mlotshwa, Neil de Villiers, Jenny Hind, Heidi Abrahamse, Technikon Witwatersrand, Department of Biomedical Technology, School of BioSciences, Faculty of Health Sciences, P.O Box 17011, Doornfontein, 2028, Gauteng, South Africa. Although genes are known to play a critical part in the aetiology of Alzheimer’s disease (AD), it has been suggested that it could result from a multifactorial process involving both a genetic predisposition and an exposure to environmental factors modulated by the biological aging process (McGeer et al., 1996). Recent studies have shown markers of oxidative stress to be elevated in the brains of patients with Alzheimer’s disease (AD). Although the brain is the most affected organ in AD, studies have also detected structural and functional alterations in peripheral tissues. In the present study, we have investigated the contribution to DNA damage by oxidative stress induced by cadmium chloride and cobalt irradiation in peripheral lymphocytes isolated from patients with AD and age-matched control subjects. The alkaline comet assay modified with lesion-specific dna repair endonucleases (endonuclease lll and formamidopyrimidine glycosylase - fpg) and stained with DAPI (4’,6-diamine-2-phenylindol dihydrochloride) was used to

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assess the DNA damage. Visual scoring was done with an Olympus BH2-RFCA epifluorescent microscope using arbitrary units. The results were recorded with bar graphs with the Sigma Plot programme. Cadmium chloride induced DNA strand breaks and oxidised bases in a dose-dependent manner in both AD and control subjects with more effects observed in lymphocytes from AD patients. The level of DNA strand breaks induced after radiation showed a dose dependent increase but was not significant in both test groups. Radio-adaptation (Adaptive Response – AR) of the lymphocytes in patients with AD was observed. Induction of oxidised purines by radiation showed an AR suggesting a priming of the activities of antioxidant enzymes and DNA repair mechanisms. In addition, induction of oxidised pyrimidines by radiation showed a dose dependent increase in both AD patients and control subjects with more effects observed in AD patients. These results suggest that lymphocytes from AD patients are sensitive to cadmium chloride reflecting a condition of oxidative stress as a result of a high rate of disease-related free radical production. Although AD patients seem to have an AR to radiation, their lymphocytes are more sensitive to pyrimidine damage than age, gender matched controls. P75 Comet assay assessment of enzyme repair after mobile phone frequency and low dose cobalt irradiation J. Hind1, N. de Villiers1, B. Muissa Mangaya2 and H Abrahamse1 1 Faculty of Health Sciences, 2 Faculty of Engineering, Technikon Witwatersrand, P. O. Box 17011, Doornfontein, 2028, Gauteng, South Africa People living in modern industrial societies are exposed to ionizing Low and High Linear Energy Transfer (LET) irradiation and non-ionizing radiofrequency (RF) electromagnetic fields. The water in Gauteng gold mining areas contains, among other mutagens, low levels of cobalt (low LET irradiation). In South Africa undamaged antennae of mobile phones emit 900MHz. This does not take into account the additional RF emitted from base stations which will constantly vary in distance and therefore frequency depending on the distance an individual is from the station. Cobalt has been shown to cause DNA damage and the increase in the usage of mobile phones (electromagnetic frequencies between 800 and 1800 MHz) continues to receive attention. The debate continues over RF as well as cobalt as possible causes of cancer and degenerative diseases whose pathogenesis involves direct damage to cellular DNA by free radicals compromising enzyme repair mechanisms. Enzyme Repair times after 15, 30 and 60 minutes exposure to 900MHz RF and low doses (below 0.5 Gray) of cobalt irradiation were compared on lymphocytes suspended in RPMI- 1640 medium/fetal calf serum/DMSO. During the test the thermal effect generated by the RF was simulated. Enzyme repair times allowed were 30, 60, 90, 120 and 150 minutes. Longer repair times ranging from 3 hours to 24 hours are being investigated. DNA damage was assessed using the alkaline comet assay modified with lesion-specific DNA repair endonucleases (endonuclease lll and formamidopyrimidine glycosylase - fpg), stained with dapi (4’, 6-diamine-2-phenylindol dihydrochloride) and scored with an Olympus BH2-RFCA epifluorescent microscope using arbitrary units. The results were recorded with bar graphs with the Sigma Plot programme. Baseline controls of DNA damage were obtained by allowing repair times on non-irradiated lymphocytes and by nil repair time on all RF and cobalt doses. The non-irradiated control showed an increase in cellular stress up to 60 minutes with a decrease back to baseline levels over the next 90 minutes. Maximal damage for RF showed at 150 minutes with cobalt at 30 minutes. The Arbitrary Unit levels caused by RF and cobalt show a constant kinetic process of repair and damage prompting our current investigation into longer enzyme repair times. These results will be reported at the workshop. P76 Is 8-oxoGua (or 8-oxodGuo) a reliable biomarker of oxidative damage to DNA? R. Collins and ESCODD (European Standards Committee on Oxidative DNA Damage) Institute for Nutrition Research, University of Oslo, PO Box 1046 Blindern, 0316 Oslo, Norway Methods of estimating oxidative damage to DNA in human cells have come under scrutiny as it has become clear that the most commonly measured oxidation product, 8-oxo-7,8-dihydroguanine (8-oxoGua), is readily produced during preparation of samples for analysis by GC-MS or HPLC. This artefact probably largely accounts for the discrepancy (covering at least 3 orders of magnitude) between highest and lowest estimates of background damage levels. ESCODD was set up to examine the problem, to devise methods for minimising the adventitious oxidation of guanine, and to reach a consensus on the real level of oxidation.

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ESCODD has operated for the last 3 years as an EC-supported Concerted Action with 25 members. Member laboratories have received and analysed samples of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), oligonucleotides containing specified amounts of 8-oxoGua, calf thymus DNA with and without additional oxidation, pig liver, and HeLa cells with and without additional oxidation. Finally, lymphocytes were collected from volunteers in several of the member countries and analysed for 8-oxoGua/8-oxodGuo. In addition to HPLC and GC-MS, we have used an enzymic approach, in which 8-oxoGua in cellular DNA is converted to breaks by digestion with formamidopyrimidine DNA glycosylase (FPG). The breaks are then measured using the comet assay, alkaline unwinding or alkaline elution. HPLC with electrochemical detection is capable of accurate and precise measurement of experimentally induced 8-oxoGua, but in spite of strenuous efforts to eliminate spurious oxidation during DNA extraction and processing, estimates of background levels remain very variable. The enzymic methods, with little opportunity for additional oxidation to occur, generally measure lower levels of background damage (median values being several times lower than for HPLC), but they, too, give variable results on identical samples. Also, calibration of these methods is indirect and so estimation is approximate. However, from the latest results with human lymphocytes, we can conclude that the background level of damage is somewhere close to the range of 0.3 to 4 8-oxoGua per 106 Gua (the median values for FPG-based and HPLC methods respectively). Supported by EC contract QLK1-1999-00568 P77 Activity of human MutT homologue (hMTH1) protein in non small lung cancer patients Katarzyna D. Lichota-Arczewska1, Daniel Gackowski2, Agnieszka Siomek2, Barbara Tudek1, Jarosław T. Kuśmierek1 and Ryszard Oliński2

1Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5a Pawińskiego Str., 02-106 Warsaw, Poland 2Department of Clinical Biochemistry, The Ludwik Rydygier Medical University in Bydgoszcz, 24 Karłowicza Str., 85-092 Bydgoszcz, Poland Bases in deoxynucleotide-5'-triphosphate (dNTP) pool can be damaged by mutagens similarly to bases in DNA. Modified dNTPs are mutagenic, because they can be incorporated into DNA by DNA polymerases. MutT proteins are enzymes which sanitise dNTP pool of 8OH-dGTP by its hydrolysis to 8OH-dGMP and inorganic pyrophosphate. Cigarette smoke, implicated in development of 90% of lung tumours induces oxidative DNA damage in humans. hMTH1 protein is known to be overexpressed during oxidative stress. Therefore we carried out study concerning activity of this protein in lung tumour and healthy surrounding tissues from NSCLC patients. We compared obtained parameters with the level 8-OH-dG in DNA. We found that the activity of hMTH1 is significantly higher in lung tumour versus non-affected surrounding tissue, what is consistent with the earlier result that 8-OH-dG level is significantly lower in tumour than in healthy surrounding tissue. This is the first study showing involvement of these two parameters with NSCLC. P78 A comparative study of mitochondrial and nuclear genome instability in colorectal tumours. Fradley S1., Lewis P.D.2, Griffiths P.3, Beynon J.4, Parry J.M. 1 1Human Molecular Pathology Group, University of Wales Swansea, Singleton Park, Swansea, UK. 2Institute of Medical Genetics, Heath Hospital, Cardiff, UK. 3Department of Histopathology, Morriston Hospital, Swansea, UK.

4Department of

Surgery, Singleton Hospital, Swansea, UK. A number of recent investigations have revealed that a proportion of colorectal adenocarcinomas harbour mitochondrial genome (mtDNA) mutations, not present in normal surrounding mucosa and are classified as tumour-specific. Tumour specific mtDNA mutations have also been observed in many other tumours including lung and bladder and it has been suggested that these mutations may serve as diagnostic markers for cancer. MtDNA contains a short hypervariable, non-coding control region known as the D-Loop. This region is highly mutable and may be rapidly and cost-effectively scanned for mutations. Using PCR-SSCP and DNA sequencing we have undertaken a comprehensive survey of the D-Loop in colorectal adenocarcinomas, normal colonic tissue and where possible, intermediate stages such as metaplastic polyps and adenomas, as well as lymph node tissue and liver biopsies of 31 patients to gauge the level of mutation of the mitochondrial genome and search for possible molecular markers for colon cancer. Of the colorectal adenocarcinomas, 19% showed tumour specific mutations which where not present in the normal mucosa. As the majority of the mutations were within either the G tract (22%) or the C tract (44%), to further this study we assessed the samples for nuclear microsatellite instability (nMSI). Nuclear microsatellite instability (nMSI) is detected in 15% of sporadic colorectal adenomas and is of clinical importance as tumours with MSI have distinctive clinical features and therefore these patients may require a different follow up from those without MSI. Four nuclear genes containing microsatellites were

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studied using fragment analysis and direct sequencing to assess the samples for MSI. All of these samples were found to be microsatellite stable with no deletions or insertions for all of the marker sites. This research has confirmed previous findings that in colorectal cancer, unlike gastric cancer, there is no link established between mtMSI and nMSI. P79 Association between genetic polymorphism in biotransformation regulating genes and organochlorine levels in humans living in a PCB contaminated area M. Dušinská1, M. Pavuk2, A. Horská1, A. Kočan3, J. Petrík3, J.R. Cerhan4 1Institute of Preventive and Clinical Medicine, Bratislava, Slovakia, 2University of Texas School of Public Health, Dallas Regional Campus, Dallas, Texas, USA; 3National Reference Center for Dioxins and Related Compounds, IPCM, Bratislava, Slovakia; 4Health Sciences Research, Mayo Clinic, Rochester, USA; We carried out a molecular epidemiological trial in eastern Slovakia in the area highly contaminated with polychlorinated biphenyls (PCBs). PCBs were produced in the Michalovce district of eastern Slovakia from 1959 to 1984. During the decades of the production, considerable amounts of these compounds were released into the environment and caused a contamination of soil, sediments, biota, and through the food chain, humans. One of the highest PCB levels in Europe has been documented in the environment and in humans in this area. PCBs were found to be toxic in animal studies and have suspected carcinogenic, hormone-disrupting and developmental effects in humans. The aim of this study was to assess the possible association between the levels of PCB congeners and different genetic polymorphisms in biotransformation regulating genes. Altogether 200 subjects, males and females, were investigated, 66 subjects living within 10 km from the former PCB factory and 134 living more than 10 km away. Subjects were selected by systematic random sampling from the physician databases, which represent an almost complete population list for the polluted area. Fifteen mono-ortho and di-ortho PCB congeners (28, 52, 101, 105, 114, 118, 123, 138, 153, 156, 157, 167, 170, 180, 189) were measured in blood samples by high-resolution gas chromatography. Genetic polymorphisms in glutathione-S-transferase (GST) genes, GSTT1 and GSTM1 (deletions), GSTP1 (Ile105Val), and in microsomal epoxide hydroxylase genes EHPX3 (His113Tyr) and EHPX4 (His139Arg) were analysed in lymphocyte DNA. People who lived within 10 km distance from the former PCB factory had significantly higher levels of total PCBs compared to those living farther from the plant (4210 versus 2630 ng of PCBs per g of lipids; p=0.001). No association with the variant alleles of all investigated polymorphisms was observed in subjects living more than 10 km from the plant. In contrast, in persons living within 10 km of the factory, we found a significant association between total PCBs and GSTT1 genotype. GSTT1(-) subjects had higher levels of PCBs than GSTT1(+) (5580 versus 3530 ng/g; p=0.015). Similar results were observed for potentially estrogenic, mono-ortho PCBs and di-ortho PCBs; subjects with GSTT1(-) had higher levels compared with GSTT1(+) (643 versus 351 ng/g of lipids, p=0.004; 4670 versus 3080 ng/g, p=0.027, respectively). In contrast, lower levels of mono-ortho PCB congeners were found in subjects with the GSTM1 deletion (361 versus 543 ng/g of lipids, p=0.047). Results were not significant for other PCB congener groups. No consistent pattern was seen for other genotypes for any of the organochlorines, such as DDT, DDE, and hexachlorobenzene. No confounding effect of smoking or alcohol consumption was observed in these analyses. Results of our study show that levels of PCBs in humans can vary partly due to the differences in genetic polymorphism in biotransformation regulating genes. It seems that from five investigated polymorhisms especially those in GST genes may contribute to the individual susceptibility to toxic effects of these chemicals. The study was funded by the Fogarty International Center (NIH, Department of Health and Human Services, 2 D43 TW00621-06). Germ Cell Genotoxicity P80 The effects of combined X-ray-vincristine treatments on the reproduction ability of male mice M.M. Dobrzyńska and U. Mikulska Department of Radiation Protection and Radiobiology, National Institute of Hygiene, Warsaw, Poland Vincristine (VCR), an alkaloid extracted from leaves of Catharanthus roses, is applied together with radiation or with other chemotherapeutic drugs in therapy of human malignancies. The aim of the study was to investigate the effects of combined X-rays-vincristine treatments on the spermatozoa quantity and quality in the comparison to results obtained to each agent acting alone. 8-10 weeks old Pzh:SFIS male mice were whole body irradiated with X-rays (0.25 Gy, 1.00 Gy), or treated to VCR (1 mg/kg bw, 2 mg/kg bw), or exposed to the combination of both agents (0.25 Gy+1 mg/kg bw VCR, 1.00 Gy+2 mg/kg bw VCR). 5 weeks after treatments, males were killed, testes and epididymes were removed. Spermatozoa were counted and examined for incidence of sperm head abnormalities. Both testes were weighted, and one of them from each animal was used for Comet assay.

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Exposure to VCR alone as well as to combination of X-rays and VCR decreased testes mass. Reductions in sperm count were observed in all experimental groups. The lowest results were noted after combined exposure to 1.00 Gy+2 mg/kg bw VCR (23% of control values). Frequencies of abnormal sperm heads for X-rays were similar to natural incidence. Exposure to VCR twice increased percent of morphologically abnormal sperm. Results for combined treatments were 2.5 times higher in the comparison to control values. After exposure to 2 mg/kg and 1.00 Gy+2mg/kg bw VCR, teratozoospermia was observed. In Comet assay slight increases in DNA migrations were observed in all groups. The most clear effects were noted after exposure to 2 mg/kg bw VCR alone as well as in combination to 1.00 Gy. Combined treatments to X-rays and vincristine did not cause infertility of male mice, but decreased fertilization ability. P81 The modulation by flavonoids of DNA damage from oestrogen-like compounds in human lymphocytes and sperm in the Comet assay. E.Cemeli1, T.E.Schmid1 and D.Anderson1. 1Department of Biomedical Sciences, University of Bradford, Bradford, UK. 2School of Public Health, University of California in Berkeley, USA. Reactive oxygen species (ROS) have been shown to cause a broad spectrum of damage to biological systems. ROS are widely produced by exogenous chemicals and metabolic processes. As a consequence, ROS react with DNA, among many other biological targets, disrupting its structure and function. Oestrogen-like compounds have been recently shown to mediate DNA damage by ROS generation, and their effects can be modulated by several antioxidants, such as, catalase, SOD and vitamin C [Anderson et al., in press]. Flavonoids, which are natural constituents of our diet, have been investigated at clinical, epidemiological and molecular levels. However, their properties remain controversial. An influence of oestrogens in the susceptibility to hormone-dependent cancer has been reported [Kirk et al., Biochem Soc Trans 2001, 29:209-16; de Lemos, Ann Pharmacother 2001, 35:1118-21], as have beneficial features such as their function as antioxidants [Sierens et al., Mutat Res, 2001, 8:1245-55]. It may be that properties as pro-oxidant and/or antioxidant are dependent in their concentration. It is clear that flavonoids have an affinity for oestrogens receptors, explaining the stimulatory effect on certain genes. Furthermore, flavonoids interact with cellular signal pathways controlling the cell cycle, differentiation and apoptosis [Gee et al., Curr Med Chem, 2001, 8:1245-55]. Therefore, we examined human lymphocytes and sperm after treatment with four oestrogen-like compounds (β-oestradiol, diethylstilboestrol, daidzein and genestein) and their modulation by flavonoids (quercetin and kaempferol) in the Comet assay. Heparinised blood samples were obtained from a healthy donor. Lymphocytes were isolated and treated at 37° C for 30 min with the oestrogenic-compounds and the positive control (hydrogen peroxide, H2O2), either on their own or combined with the flavonoids. Viability was checked by trypan blue exclusion and only those concentrations giving a viability over 80% were accepted. Semen was obtained from a healthy donor. Ten µl sperm were made up to 1 ml RPMI 1640 medium in an Eppendorf tube. Each was treated at 37° C for 1 h. The procedure was then carried as for the lymphocytes, however, just before the slides were placed in lysis solution, 0.05 mg/ml proteinase K was added. The cells were checked for their viability before the start and after the completetion of the experiment using trypan blue dye. Preparation of the slides for the Comet assay and subsequent electrophoresis and staining were carried out according to standard methods [Singh et al., Exp Cell Res 1988, 175:184-91; Tice et al., Environ Molec Mutagen 2000, 35:206-21] All oestrogenic-compounds as well as the positive control, induced significant increases in tail moments in the case of lymphocytes and a decrease in the % head DNA in the human sperm. When combined with the highest concentration of flavonoids (500 µM), the DNA damage was reduced significantly. However, the lowest concentration of flavonoids (100 µM) did not affect the cells consistently. The parallel/similar responses of the oestrogenic compounds and H2O2 and the fact that kaempferol and quercetin produce similar reductions in response, confirms our hypothesis that diethylstilboestrol, $-oestradiol, genestein and daidzein, generate ROS. Furthermore, it also clarifies that quercetin and kaempferol possess antioxidant activities at a concentration of 500 µM. However, an explanation for the lack of homogeneity at the lowest concentration of flavonoids could be due to the cell membrane, which may have a role in limiting the access of these “double-edged antioxidants” [Szeto et al., Mutat Res 2002, 500:31-38].

EC was supported in part by a Leonardo da Vinci Scholarship from CAEB of the Balearic Islands.

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Genotoxic and carcinogenic effects of mineral fibres and particles P82 The genotoxic effects of urban particulate matter – size matters! K. Healey1, J.N.N. Lingard2, A.S. Tomlin2, K.L.M. White1, A.W.M. Hay1, C.P.Wild1, M.N.Routledge1 1 Molecular Epidemiology Unit, Academic Unit of Epidemiology and HSR, Algernon Firth Building, University of Leeds, Leeds, LS2 9JT 2 Dept. of Fuel and Energy, University of Leeds, Leeds, LS2 9JT Exposure to airborne particulate matter with an aerodynamic diameter of less then 10µm (PM10) has been associated with a number of health effects including lung cancer, asthma and cardiovascular disease. Recent studies have suggested that PM2.5 is primarily responsible for these effects. In urban environments up to 90% of PM2.5 is accounted for by diesel exhaust particles (DEP). DEP comprise carbonaceous cores with many organic and inorganic compounds adsorbed to their surface including polycyclic aromatic hydrocarbons (PAH) and transition metals. The toxicological mechanisms by which the particles act are likely to be numerous and complex. This study aims to assess the influence of particle size upon the levels of DNA strand breaks induced in the single cell gel electrophoresis assay (comet assay). We have collected size segregated samples of urban particulate matter (UPM) using an 8 stage Anderson Cascade Impactor. Samples were collected from a roadside location in central Leeds where passing traffic is predominately diesel fuelled. Two sets of samples were collected; the first onto quartz fibre filters and the second onto foil. Size ranges were defined between 10µm and <0.43µm. UPM was extracted from quartz filters by vortexing filter sections in PBS. A549 cells, a lung epithelial cell line, were exposed to UPM/quartz fibre extracts for 24 hours. UPM collected on foil was brushed into distilled water and dried to determine the mass collected. Each sample was re-suspended in PBS and 500µg UPM was used in 24 hour cell exposures. The comet assay was employed to determine the level of single strand breaks generated. The number of comet cells per one hundred cells counted was determined to give the comet%. Each size range of particles induced single strand breaks, and the levels of damage varied across size ranges. The pattern of damage induced by different size ranges varied for different batches of samples. For UPM extracted from quartz fibre filters the increase in comet%, as compared to control levels of comets (3.6%), was 4-7 fold for particles >2.1µm and 6-10 fold for particles <2.1µm. Subsequent experiments using a known mass of UPM extracted from foil demonstrated higher comet% for smaller particles. For one batch of samples the comet% increased from 6% for particles of size 9-10µm to 33% for particles <0.43µm. These results show that the finer size UPM induce higher levels of genotoxic damage compared to the coarse UPM, in this in vitro system. P83 Micronucleus formation in pulmonary macrophages and epithelial cells from rats exposed to quartz dust R. Schins1, G. Cakmak2, C. Albrecht1, A. Knaapen3, A. Becker1, R. Duffin1, and P. Borm1 1 Institut für Umweltmedizinische Forschung (IUF), Heinrich Heine University Düsseldorf, Germany, 2 Gazi University, Ankara, Turkey, 3 Maastricht University, The Netherlands Chronic exposure to crystalline silica (quartz) dust causes tumour formation in rat lungs. Although the mechanisms involved in this process remain to be elucidated, particle surface properties are considered to play an important role. Recently we demonstrated acute DNA damage (alkaline comet) in the lung epithelial cells from rats upon intratracheal instillation of quartz, but not in rats instilled with quartz that has been coated with either polyvinylpyridine-N-oxide (PVNO) or aluminium lactate (AL). In the present study, micronucleus formation was comparatively evaluated in alveolar and interstitial macrophages and in epithelial cells from rats (female SPF-free Wistar), 3 months after a single intratracheal instillation of phosphate buffered saline (control), DQ12 quartz (2mg/rat) or DQ12 coated with either PVNO or AL. The proportion of micronucleated cells was determined in alveolar macrophages obtained by brochoalveolar lavage (BAL), and in the interstitial macrophage fraction as well as in the lung epithelial cells, both obtained following lung digestion and epithelial cell isolation. A significantly higher micronucleus frequency was observed in the alveolar macrophages from rats treated with DQ12, but not in the alveolar macrophages from rats treated with coated quartz. Treatment of rats with uncoated quartz also tended to increase micronucleus frequency in the interstitial macrophages, although these values did not reach statistical significance. In contrast, differences in micronucleus frequency were absent in epithelial lung cells, which are considered as target cells for quartz-induced neoplastic changes. In conclusion, increased mutagenicity of quartz as observed in the respiratory tract of rats appears to be associated with its particle surface properties. The effects of the coatings likely relate to observed differences in kinetics of inflammation and particle clearance from the respiratory tract, as well as to contrasting particle-cell interactions between phagocytes and epithelial cells. Our data also further indicate that for cancer-risk assessment of poorly soluble particles such as quartz, in vivo genotoxicity tests should be interpreted in relation to the cell type evaluated herein.

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P84 Genotoxic and inflammatory effects in the human lung epithelial cell line A549 after exposure to different wood dust species J. Bornholdt, A.Thoustrup Saber, U.Vogel and H. Wallin. National Institute of Occupational Health, Denmark Sino-nasal adenocarcinoma is rare. However, furniture makers and employees who are exposed to wood dust have an up to 200-fold increased risk of this cancer. Wood workers frequently develop respiratory symptoms like astma, chronic bronchitis, extrinsic allergic alveolitis and rhinitis. Despite efforts to establish the cause of these effects on wood workers, no specific causal agent has been identified. It is currently debated whether hardwood, exotic wood and softwood confer different risk of respiratory tract problems. This study is a part of the WOODRISK European collaboration (Project No. QLRT-2000-00573), which aims to establish data for the risk assessment of wood dust. We exposed the human lung epithelial cell line A549 to 8 different species of well defined wood dusts; oak, pine, beech, birch, teak, medium density fibreboard, and heat-treated pine. The cells were exposed to 5 different concentrations; 10, 30, 100, 300 or 1000 µg/ml medium for 3, 6, 24 and 48 hours. The DNA damage was measured as strand breaks by the comet assay and interleukin-6 mRNA expression was used as marker for inflammation. In the samples collected after 24 and 48 hours a cytotoxic effect was observed. We therefore chose to focus on the samples exposed for 3 and 6 hours. From the preliminary data the wood dusts seem to have a small DNA damaging effect to the cells exposed to the higher concentrations of wood dust. Preliminary results indicate that all the wood species except oak and beech induce an inflammatory response. The data indicate that the hardwoods induce a less severe inflammatory response compared to the rest of the wood species. P85 Relationship between inflammation and genotoxicity of diesel exhaust particles in mice after a short-term exposure M. Dybdahl, L. Risom1, A. Saber, H. Autrup2, S. Loft1, and H. Wallin National Institute of Occupational Health, Copenhagen, Denmark 1Institute of Public Health, University of Copenhagen, Denmark, 2 Department of Environmental and Occupational Health, University of Aarhus, Denmark To investigate the mechanisms that may contribute to the development of lung tumours after diesel exposure, we characterized the effects of a short-term inhalation of diesel exhaust particles (DEP) on inflammation, cytokine expression, DNA damage, and mutagenesis. Briefly, BALB/c mice were exposed to 5 or 20 mg/m3 DEP by inhalation for 90 min on 4 consecutive days. One and 22 hr after exposure, inflammation and DNA damage were examined in bronchoalveolar lavage and lung tissue. As markers of lung inflammation, we characterized the expression of cytokines and the recruitment of macrophages and neutrophiles, and the DNA damage was assessed by the level of DNA strand breaks, 8-oxodG, and DNA adducts. In parallel to the BALB/c study, the mutation frequency (MF) after DEP exposure was examined using the transgenic Muta™Mouse and a similar exposure regimen. MF was measured in lung tissue 28 days after DEP exposure. With respect to the BALB/c mice, exposure to 20 mg/m3 DEP resulted in a significant induction of inflammation, DNA strand breaks, and DNA adducts. The response was not associated with an increase in MF, indicating that host defenses (e.g. antioxidant activity, DNA repair enzymes) were sufficient to “neutralize” the inflammatory mediators. In contrast, the only effect of 5 mg/m3 DEP was an increase in DNA strand breaks, which probably reflects an increased repair activity following the exposure to DEP. Currently we are investigating the relationship between inflammation and genotoxicity of DEP in the TNF-α and OGG1 knock-out mice. P86 Oxidative DNA damage and gene expression response after short-term inhalation of diesel exhaust particles in the lungs mice L. Risom1, M. Dybdahl2, J. Bornholdt2, U. Vogel2, H. Wallin2, P. Møller1, and S. Loft1*

1Institute of Public Health, University of Copenhagen, DK-2200 Copenhagen N, Denmark, 2National Institute of Occupational Health, DK-2100 Copenhagen Ø, Denmark Exposure to diesel exhaust particles (DEP) is suspected to contribute to lung cancer and cardiopulmonary diseases. The underlying mechanisms behind the genotoxic effects of particles need still to be clarified. Recent years of investigations

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seem to relate the carcinogenic potential of particles to the generation of reactive oxygen species capable of inducing cellular oxidative stress. The aim of this study was to characterise the effects of short-term inhalation to DEP in lungs of mice and demonstrate alteration in expression of key response genes towards oxidative stress. Mice were exposed to either 20 or 80 mg/m3 DEP inhaled as either a single dose, or four lower doses (5 and 20 mg/m3) inhaled on four consecutive days. The results indicate that HO-1 mRNA expression in lung tissue increased after both types of DEP exposures, whereas OGG1 expression was only increased after the repeated inhalations. The level of oxidative DNA damage in terms of 8-oxodG was increased in lung tissue after a single inhalation, whereas increased levels of strand breaks was observed in bronchoalveolar cells after repeated DEP exposures. The level of 8-oxodG and OGG1 mRNA level in lung tissue was mirror-imaged, indicating that the unaltered 8-oxodG after repeated inhalations was due to increased repair of this lesion. In conclusion, this study indicates that single high dose of DEP generate 8-oxodG in lung tissue, whereas the same dose inhaled as four low-exposures may evoke the antioxidative defence system and protect against generation of 8-oxodG. Presently, on-going investigations of DEP effects in OGG1 knockout mice are in progress. P87 Lung multinucleate cells after inhalation of amosite dust combined with cigarette smoke by rats M. Beno1, M. Hurbankova1, M. Dusinska1, K. Volkovova1, M. Staruchova1, S.Cerna1, M. Barancokova1, A.Kazimirova1, P. Bobek1, M. Horecky1, M. Mikulecky1 and S.A. Kyrtopoulos2

1 Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic, 2 Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece This study was part of a research project aimed at looking for biomarkers useful for the experimental comparison of the biological effects on laboratory animals of various fibre-dusts, alone or in combination with cigarette smoke. Multinucleated cells (MNC) may arise by fusion of macrophages engaged into cleaning foreign material from tissue. Experimental data support the hypothesis that, after exposure to fibrogenic dust, increased numbers of MNC appear as a result of monocyte or macrophage immigration into the lung tissue, followed by proliferation and fusion of these precursor cells into multinucleate entities. The present study gives information about the dose-response relationships governing the induction of lung MNC after inhalation by rats of two concentrations of amosite asbestos combined with daily exposure to cigarette smoke. Concomittantly, lung inflammation parameters have been followed. Six groups of male Fisher 344 rats inhaled amosite asbestos fiber dust of South African origin in a nose-only inhalation device every second day for one hour per exposure. The groups exposed to combined noxae breathed daily also diluted main-stream tobacco smoke at 30 mg of total particulate matter (TPM)/m3 air for one hour, corresponding to smoke arising from three cigarettes. The exposures lasted 175 days. The dosage groups were: 1) 60 mg/m3 amosite fibres plus cigarette smoke; 2) 60 mg/m3 amosite fibres; 3) 30 mg/m3 amosite fibres plus cigarette smoke; 4) 30 mg/m3 amosite fibres; 5) cigarette smoke; 6) no dust no smoke - control. MNC were counted in lung cell suspensions prepared after lung digestion with trypsin. Inflammation was monitored by the changes in the BAL fluid inflammatory parameters. The following parameters showed the presence of lung inflammation in exposed animals at sacrifice. A dose-dependent rise in the proportions of BAL lymphocytes, from 2.9% in the controls to 4.6% and 7.6% in the 30 and 60 mg/m3 amosite inhaling animals, respectively, was found. Furthermore, an increase was observed in the proportions of BAL PMN, from 0.4% in the controls to 1.4% and 2.3% in the 30 and 60 mg/m3 amosite plus cigarette smoke inhaling animals, respectively. Finally, the total BAL protein rose from 2.1 4g/ml to 2.8 g/ml and 3.0 g/ml in the 30 and 60 mg/m3 amosite plus cigarette smoke inhaling animals, respectively. Under these conditions, an increase was observed in the proportions of BAL MNC, from 0.7% in controls to 2.4% and 3.0% in the 30 and 60 mg/m3 amosite plus cigarette smoke inhaling animals, respectively. As regards changes in MNC in lung tisue, a dose-dependent rise of the proportion of MNC in lung cell suspensions was found, from 1.5% (95% confidence limits - CL = 1.3-1.7%) in control lungs to 2.1% (CL=1.9-2.3%) in the sole smoking group. The proportion in sole 30 mg/m3 amosite inhaling group was 11.7% (CL=11.3-12.2%), after sole 60 mg/m3 amosite inhaling group 12.4% (CL 11.9-12.9%), after combined 30 mg/m3 amosite plus smoking 15.2% (CL=14.6-15.8%) and after combined 60 mg/m3 amosite plus smoking 15.4% (CL=14.9-15.9%), respectively. These data suggest an additive, rather than synergistic, effect of combined amosite and tobacco smoke exposure for the induction of lung MNC at the exposure levels used. A positive significant correlation of the proportions of MNC in the BAL fluid to that in the lung cell suspensions has been found (r=0.46, p<0.05). It is concluded that, in the experimental assessment of effects of fibrogenic dusts, the number of MNC in lung cell suspensions and in BAL may serve as a useful semiquantitative biomarker. The work was supported by an E.U. grant, contract, No. QLK4-CT-1999-01629 (FIBRETOX project). P88 Cytotoxic parameters of bronchoalveolar lavage fluid after inhalation exposure to amosite and wollastonite fibrous dust combined with cigarette smoke in rats 1S. Černá, 1M.Hurbánková, 1M. Beňo, 1Z. Kováčiková, 1P. Bobek, 2S.A. Kyrtopoulos

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1Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic, 2 Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece Inflammation may play a role in the induction of lung genotoxicity and cancer by mineral fibres. Tobacco smoking is known to act synergistically with exposure to asbestos in the induction of lung cancer, but the mechanism of this effect is not known with certainty. The aim of this study was to evaluate the combined noxae effect (fibrous dust + cigarette smoke) on BALF cytotoxic parameters, as well as the dose response and the sensitivity of selected parameters. The fibrous dusts used in this experiments were amosite (a naturally occuring silicate of the amphibole asbestos group) and wollastonite fibers (a naturally occuring silicate used as a substitute of asbestos in some industrial applications). Fischer 344 rats were exposed to wollastonite and amosite asbestos fibers using a nose-only inhalation device. The duration of exposure was 175 days. Two doses of dust aerosol (60 mg/m3 air and 30 mg/m3 air) were used for one hour per exposure. Exposure to dusts proceeded every second day, including all holidays. Saturday and Sunday were hold free of exposures. The animals were exposed to fibres only or, following the daily fibre exposure, they inhaled mainstream cigarette smoke from 3 cigarettes at 30 mg TPM/m3 for 1 hour. Six groups, each of 11 animals were exposed to: 1) 60 mg/m3 fibres combined with exposure to cigarette smoke; 2) 60 mg/m3 fibres only; 3) 30 mg/m3 fibres combined with exposure to cigarette smoke; 4) 30 mg/m3 fibres only; 5) exposure to cigarette smoke plus immobilization stress as for animals exposed to dust; 6) immobilization stress as for animals exposed to dust. After 6 months animals were sacrificed and the following cytotoxic parameters in bronchoalveolar lavage fluid were investigated: phagocytic activity of AM, viability of AM, lactate dehydrogenase activity (in the cell-free lavage fluid), acid phosphatase activity (in the cell-free lavage fluid and in the BAL suspension), alkaline phosphatase activity (in the cell-free lavage fluid), cathepsin D activity (in the cell-free lavage fluid and in the BAL suspension). Results and conclusions: Amosite: The lower dose had no significant effect on cytotoxic parameters, while the higher dose significantly decreased cell viability and increased cathepsin D activity in BALF cells. Exposure to cigarette smoke alone significantly decreased the phagocytic activity and increased cathepsin D activity in BALF cells. The combined effect of amosite and cigarette smoke was stronger than the effect of amosite or smoking exposure alone. Most parameters were significantly changed. Dose dependence was not explicit. The parameters exhibiting highest sensitivity to exposure were phagocytic activity, viability of AM and cathepsin D. Wollastonite: Wollastonite alone (both doses) did not have any effect on the cytotoxic parameters, apart from a significantly increased activity of cathepsin D in BALF cells. Cigarette smoke alone significantly decreased the phagocytic activity and increased the activity of alkaline phosphatase in BALF. The combined effect (wollastonite + cigarette smoke) was weaker than the combined effect of amosite + smoking. However, no dose dependence was observed. The parameter exhibiting the highest sensitivity to exposure was cathepsin D activity. The work was supported by an E.U. grant, contract, No. QLK4-CT-1999-01629 (FIBRETOX project). P89 Evaluation of histological findings after exposure to asbestos substitute mineral fibres E. Tatrai1, M. Hurbánková2, É. Six3, Z. Kováčiková2, S. A. Kyrtopoulos4

1Department of Pathology, Fodor József National Center for Public Health, National Institute of Occupational Health, Budapest, Hungary, 2Respiratory Toxicology, Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic, 3Fodor József National Center for Public Health, Budapest, Hungary, 4Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece

In the context of an investigation of the relationship between lung inflammation, tissue toxicity and genotoxicity caused by mineral fibres, wollastonite, rockwool, glass fibers as well as amosite asbestos were intratracheally instilled into Fisher 344 rats at 2 doses (2 and 8 mg/animal). The dose of 8 mg was divided into 4 fractions of 2 mg instilled 1 week apart. After sacrifice (4 or 16 weeks after the last instillation), the animals were killed and lung morphology was examined by light microscopy and transmission electron microscopy. Results of histological findings in the lung (grade according to Belt-King scale): AMOSITE:

Fibre Dose 4 weeks post-exposure 16 weeks post-exposure 2 mg II III Amosite 4x2 mg III III- IV

Dose- and time-dependent effects were observed, consisting of serious interstitial fibrosis and chronic, progressive inflammation with complete destruction of pulmonary structure. WOLLASTONITE:

Fibre Dose 4 weeks post-exposure 16 weeks post-exposure 2 mg I I-II Wollastonite 4x2 mg II II

Mild, dose-dependent histological alterations were seen, including very moderate pulmonary fibrosis and chronic interstitial inflammation, without structural changes, as well as pulmonary fibrosis which did not show time progression. ROCKWOOL:

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Fibre Dose 4 weeks post-exposure 16 weeks post-exposure 2 mg I I-II Rockwool 4x2 mg I-II II

This kind of rockwool had a very weak fibrogen effect, similarly to that of wollastonite. The fibrosis observed showed a minimal tendency for progression. Ultrastructural studies confirmed the results of light microscopy. GLASS FIBERS:

Fibre Dose 4 weeks post-exposure 16 weeks post-exposure 2 mg I I Glass fibers 4x2 mg II II

Mild, dose-dependent histological alterations were seen. Conclusion: Sequential arrangement of examined fibrous dust according to their harmfulness from the point of view of histological findings after intratracheal instillation: AMOSITE > WOLLASTONITE > ROCKWOOL > GLASS FIBERS This work was supported by an E.U. grant, contract, No. QLK4-CT-1999-01629 (FIBRETOX project). P90 Inflammatory and cytotoxic parameters of bronchoalveolar lavage fluid after intratracheal instillation of asbestos and asbestos substitute mineral fibres 1M.Hurbánková,1 S. Černá, 1 Z. Kováčiková, 2 A. S. Kyrtopoulos 1Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic, 2 Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece Inflammation may play a role in the induction of genotoxicity and carcinogenesis by fibres. The aim of the present work was to examine the influence of asbestos and asbestos substitute mineral fibres (ASMF) on selected bronchoalveolar lavage fluid (BALF) inflammatory and cytotoxic parameters, as well as dose and time dependence of studied fibrous dust effect. Three types of ASMF (wollastonite, rockwool, glass fibers) as well as amosite asbestos were instilled at 2 doses (2 and 8 mg/animal) into Fisher 344 rats. Animals (number: 10 – 12 per group) were intratracheally instilled (noninvasively) with fibrous suspension (2 mg suspended in 0,2 ml of saline solution per animal) or only with 0,2 ml saline per animal (control group). The dose of 8 mg was divided into 4 fractions instilled weakly as 2 mg/0,2 ml saline solution. After sacrifice (4 or 16 weeks after last intratracheal instillation) the animals were anesthetised with thiopental), BALF cells were collected and the following parameters were investigated: a) Inflammatory response biomarkers: the number of BALF cells / ml, the number of alveolar macrophages (AM) / ml, the differential number of cells (% AM, neutrophils, lymphocytes), the levels of cytokines (TNF-alpha, IL-1alpha), the total amount of protein. b) Cytotoxic parameters: phagocytic activity of AM, viability of AM, lactate dehydrogenase activity (in the cell-free lavage fluid), acid phosphatase activity (in the cell-free lavage fluid and in the BAL suspension), alkaline phosphatase activity (in the cell-free lavage fluid), cathepsin D activity (in the cell-free lavage fluid and in the BAL suspension). Results and conclusions: Inflammatory parameters The inflammatory parameters were the most changed after amosite treatment. There were not many differences between two dose inflammatory effect. Changes were more explicit after 16 week exposure. The inflammatory effect of rockwool was comparable with amosite fibres, although more changes were induced by the 8 mg dose. After exposure to wollastonite and glass fibers, no effects on inflammatory parameters were observed at either dose or observation time. Cytotoxic parameters Cytotoxic parameters were the most changed after amosite exposure. The dose dependence was more evident than the time dependence. The order of the other fibrous dusts examined, according to their harmfulness from the point of view of cytotoxic parameters, was rockwool > glass fibres. With both fibres, a dose- but not a time- dependence was observed. Wollastonite exposure did not affect cytotoxic parameters the least at either dose or observation time. Sequential arrangement of examined fibrous dust according to their harmfulness from the point of view of inflammatory and cytotoxic parameters after intratracheal instillation:

AMOSITE > ROCKWOOL > GLASS FIBRES > WOLLASTONITE The work was supported by an E.U. grant, contract, No. QLK4-CT-1999-01629 (FIBRETOX project).

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P91 Genotoxic effect of asbestos and other mineral fibres in humans. M. Barančoková, A. Kažimírová, A.R.Collins, S. Kyrtopoulos, L.Wsólová, M. Dušinská Institute of Preventive and Clinical Medicine, Limbová 14 833 01 Bratislava, Slovakia We have measured a range of biomarkers related to DNA damage in three factories producing asbestos, rockwool and glass fibres. Asbestos factory was producing asbestos-based building materials until 1999. Sampling was carried out during the year following cessation of production. Workers exposed to asbestos were compared with a control group from the same factory who were employed as administrators, drivers etc. and who did not have direct contact with asbestos. In addition, residents of the town not employed at the factory were recruited as a further control group. DNA damage (strand breaks, oxidised and alkylated bases) was measured in lymphocytes with the comet assay (single cell alkaline gel electrophoresis) in combination with lesion-specific endonucleases. Micronuclei and chromosome aberrations were analysed in peripheral lymphocytes of whole blood. Exposed asbestos workers had significantly higher numbers of chromosomal aberrations compared with both control groups (P=0.0029). The same results were found comparing smokers or non-smokers. Exposed smokers had increased number of aberrant cells in comparison with both exposed non-smokers (P=0.04) and control smokers (P=0.005). Women (exposed and factory-control) had fewer aberrant cells than men (P=0.005) and the number of aberrant cells was influenced by exposure, smoking and sex (P=0.008). We did not find any differences in numbers of micronuclei between exposed and factory-control subjects. Micronucleus frequency was increased in town–controls. There were no differences between exposed rockwool and glass fibre workers and controls either in frequencies of aberrant cells or in micronuclei. Women (exposed and control) had significant elevated number of cells with micronuclei compared with men (P=0.001) in both factories. The project was supported by the European Commission (Fibretox, QLK4-CT-1999-01629). P92 The importance of DNA repair gene polymorphisms in people exposed to asbestos Z. Džupinková1,2, A. Kažimírová1, M. Barančoková1, A.R. Collins3, V. Harrington4, M. Dušinská1 Dept of Experimental and Applied Genetics, Institute of Preventive and Clinical Medicine, Limbová 14, 833 01 Bratislava, Slovak Republic Dept of Anthropology, Faculty of Natural Science, Mlynská dolina B-2, 842 15 Bratislava, Slovak Republic Institute for Nutrition Research, University of Oslo, POB 1046 Blindern, N-0316 Oslo, Norway Rowett Research Institute and Robert Gordon University, Aberdeen, Scotland

The genomes of all living cells are constant targets for DNA damage, induced by numerous chemical agents and various types of radiation. Asbestos belongs to the most dangerous mineral fibres. It is classified as a class 1 chemical carcinogen, but the mechanism of its effects on the living organism is not clear yet. At a former asbestos factory we investigated 61 exposed and 70 unexposed, altogether 131 people. PCR-restriction fragment length polymorphism (RFLP) assays were used to determine five polymorphisms: X-ray repair cross complementing group 1 (XRCC1, exon 10, G/A, Arg399Gln), xeroderma pigmentosum complementation group D (XPD, exon 10, G/A, Asp312Asn and exon 23, A/C, Lys751Gln), xeroderma pigmentosum complementation group A (XPA, 5′ non-coding region, 23A/G) and O6-methylguanine-DNA methyltransferase (MGMT, promotor-enhancer, 1099C/T). Individual genotypes were compared with biomarkers of DNA stability such as DNA damage (measured by the comet assay), micronuclei and chromosomal aberrations, and also with individual DNA repair capacity. Preliminary statistical analyses of 5 genotypes suggest a possible association between XPD-751, XPA-(-4) and MGMT polymorphisms and micronuclei. XPD-751 AA homozygous and AC heterozygous subjects had higher levels of micronuclei compared with CC homozygous (p=0.01). For gene XPA-(-4), heterozygous women (GA) had significantly higher levels of micronuclei compared with homozygous women (GG) (p=0.037). Exposed subjects with MGMT wild-type allele showed significantly lower micronuclei (p=0.093). We found an association between chromosomal aberrations and XPD-751 genotypes. Women with AA genotype had lower numbers of chromosomal aberrations than women homozygous for CC (p=0.012). Both the XRCC1 and XPA-(-4) polymorphisms were associated with oxidative DNA damage. People homozygous in the wild type allele GG for gene XRCC1 had significantly lower levels of oxidised purines than both heterozygous GT and homozygous TT (p= 0.003). Subjects with AA genotype XPA-(-4) from the group of controls had the lowest levels of oxidative damage (p=0.022). The XPA-(-4) polymorphism was also associated with changes in DNA repair capacity. Non-smokers with variant allele XPA-(-4) had higher repair rates then those with wild-type allele (p=0.04) Our results indicate that the polymorphisms in repair genes may contribute to inter-individual variations in DNA repair capacity and thus may play an important role in individual susceptibility to cancer. Funded by the European Union (contract no. QLK4-CT-1999-01629), Greek-Slovak bilateral project and Slovak contracts no. UK/73/2002.

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P93 Can genetic polymorphism of biotransformation enzymes modify risk for people exposed to asbestos fibres? Horská, J. Tulinská, M. Kuricová, A. Lišková, H. Petrovská, L. Wsólová and M. Dušinská Institute of Preventive and Clinical Medicine, Limbová 14, 83301Bratislava, Slovakia Inhaled asbestos dust in dependence on its size can persist in human lung tissue and start a cascade of biological processes potentially resulting in toxic effects such as inflammation, fibrosis or carcinogenesis. These alterations are due to the mechanic damage of lung cells or/and due to the bioactivation of asbestos fibres in cells, however the detailed mechanism is not fully discovered yet. We performed a molecular epidemiological study in asbestos cement factory in Slovakia. Altogether 130 subjects were investigated, 61 long term exposed workers, 48 town-controls, and 21 control subjects working in administration of the same factory. Control subjects were matched by smoking habits, lifestyle and sex. Genetic polymorphisms of biotransformation enzymes: glutathione S-transferases (GSTM1, GSTT1, GSTP1), microsomal epoxide hydrolases (EPHX3, EPHX4) and NAD(P)H quinone oxidoreductase (NQO1) were analysed. Using statistical ANOVA test we compared all analysed genotypes with biomarkers of exposure, effect and individual susceptibility such as DNA damage (measured by comet assay), cytogenetic parameters, cellular antioxidant defences, immune parameters and proinflammatory mediators. Frequences of analysed genotypes found in our study are in agreement with other European populations. Subjects with GSTP1 b/b show significantly less of alkylating DNA damage compared with the wild type and heterozygots (P<0.01). Exposed workers with mutant GSTP1 b/b genotype had significantly lower level of DNA breaks compared with normal genotype as well as with controls (P<0.05). Mutant GSTP1 b/b genotype in exposed smokers was associated with increased natural killer (NK) cells activity (P< 0.02). Both GSTP1 and GSTM1 genes modified phagocytic activities of immune response cells. Phagocytosis of polymorphonuclear neutrophils and whole leukocytes was significantly decreased in the group of subjects with GSTP1 b/b mutant genotype. Smokers with deficient GSTM1 gene had lower level of phagocyting granulocytes compared with non-smokers (P<0.02). Presence of mutant allele in NQO1 gene was associated with increased proliferation of memory lymphocytes (P<0.01). Possible explanation of great effect of glutathione S transferases on genetic stability and susceptibility markers is probably in their role in the processes of detoxification which is combined with the antioxidant power in scavenging of free radical species. Human body is a complex system and its priority is to keep homeostasis. If genetic predisposition decrease the capacity of cellular defences to neutralise toxic agents, organism have to compensate this disadvantage by the stimulation of other cellular pathways and by the involvement of alternative ways such as activation of immune response mechanisms which perform first line of defence. Supported by EC contracts QLK4-CT-1999-01629. P94 Evaluation of fibrous dust effects in alveolar epithelial type II cells and alveolar macrophages in vitro. Z. Kováčiková1, H. Petrovská1, E. Tátrai2, M. Dušinská1 1Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic, 2NIOH, Budapest, Hungary The bronchoalveolar region of lung consists of more than 40 different cell types. From the toxicological point of view the most important target cells are alveolar macrophages (AM) and alveolar epithelial type II cells. AM reside as a free cell population in alveolar spaces and their phagocytic activity is essential to the maintenance of clear and sterile alveoli. Tissue renewal following lung damage is carried out by type II cells. AM and type II cells were isolated from Fischer 344 rats and cultivated on 24-well tissue culture plates. After 20 h cultivation various concentration of fibres (amosite, wollastonite, rockwool or glass fibres) were added to the cells and the cultivation continued for another 24 h. After finishing the exposure the number of alkaline phosphatase positive type II cells was counted, the comet assay was used to detect DNA damage (strand breaks and oxidized bases), the activity of superoxide dismutase was estimated and ultrastructural changes were evaluated by transmission eletron microscopy. 1 µg.cm-2 amosite decreased the number of alkaline positive type II cells to about 50% and the higher concentration had a more pronounced effect. After exposure to other fibres the alkaline phosphatase positivity reached 50% between fiber concentrations of 5-10 µg.cm-2 . The activity of superoxide dismutase was decreased at most in type II cells exposed to amosite. The highest level of strand breaks (SBs) was observed after the exposure to amosite the lowest dose (1 µg.cm-2 ) enhanced the level of SBs three times in AM and two times in type II cells compared to the corresponding control. A dose response was found, the higher fibre concentration giving higher SBs level. In contrast, endogenous formamidopyrimidine glycosylase- and endonuclease III – sensitive sites, which are specific indicators of oxidative damage, showed only slight enhancement even after amosite treatment. Transmission electron microscopy showed that already 1 µg.cm-2 amosite caused destruction of AM while other fibres were phagocyted. Type II cells did not show any ultrastructural alterations. The study was supported by EC contract No.QLK4-CT-1999-01629, Hungarian Scientific Research Fund (OTKA T 033007/1999) and NKFP 1/008/2001.

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P95 Genotoxic effect of asbestos and its substitutes on alveolar macrophages and epithelial type II cells isolated from lungs Petrovská, Z.Kováčiková, A. Kažimírová, M. Barančoková, M. Hurbánková, M. Dušinská Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic Genotoxic effect of asbestos and mineral fibres was investigated in lung cells of Fisher 344 rats. Rats were exposed to four different types of fibres by intratracheal instillation. Amosite, rockwool, wollastonite or glass fibres were intratrachealy instillated in 2 doses (2 or 2x4mg/animal). Animals were sacrificed 4 or 16 weeks after the last instillation, alveolar macrophages and epithelial type II cells were isolated and examined for oxidative DNA damage (by the comet assay) and micronucleus formation. Wollastonite and rockwool did not increase significantly the number of micronuclei in alveolar macrophages or epithelial type II cells compared with their controls. Amosite induced micronuclei in alveolar macrophages in both concentrations compared to controls but elevated number of micronuclei was significant only when rats were sacrificed 16 weeks after the last instillation. Glass fibres induced statistically significant number of micronuclei in dose 8 mg/animal in alveolar macrophages isolated 4 weeks after last instillation compared with control and in rats which were treated with 2 mg/animal and isolated 16 weeks after last instillation. In epithelial type II cells we detected the significant increase of micronuclei in dose 2 mg/animal isolated 4 weeks after last instillation. The increase in micronuclei was at the marginal level of significance after 16 weeks exposure to 8 mg glass fibre/animal. Different kinds of DNA damage (strand breaks, oxidised purines, oxidised pyrimidines, and strand breaks induced by hydrogen peroxide in vitro) were investigated in each cell type. Elevated DNA damage was detected after amosite but also wollastonite treatment in both alveolar macrophages as well as epithelial type II cells. There were no signifficant differences between doses and length of exposure in both cells types. DNA damage was significantly enhanced in both cell types after 4 as well as 16 weeks exposure to both doses of glass fibres. Rockwool induced statistically significant number of DNA damage in epithelial type II cells compared with controls in dose 2 mg/animal isolated 16 weeks after last instillation. It seems that alveolar macrophages are slightly more sensitive than epithelial type II cells. This work was supported by the European Union (project no. QLK4-1999-01629), and Slovak Grant Agency for Science # 04.92.12.04 P96 Effect of rockwool exposure on oxidative damage M. Staruchová1, K. Volkovová1, K. Burghardtová1, J. Kadrabová1, Z. Kováčiková1, A.R.Collins2, V. Harrington3, M. Dušinská1 Institute of Preventive and Clinical Medicine, Limbova 14, Bratislava, Slovakia, 2) Institute for Nutrition Research, University of Oslo, POB 1046 Blindern, N-0316 Oslo, Norway; Rowett Research Institute and Robert Gordon University, Aberdeen, Scotland

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Oxidative damage to biomolecules (DNA, proteins, lipids) caused by free radicals is involved in the pathogenesis of different diseases such as cancer, atherosclerosis, inflammation, etc. With the aim of studying the effect of rockwool exposure on oxidative DNA damage and lipid peroxidation, an epidiemiological study was conducted in a rockwool factory in Slovakia. Altogether 141 subjects were investigated (21 to 58 years old), 98 exposed (75 men, 23 women; 61 nonsmokers, 37 smokers) and 43 controls (20 men, 23 women; 27 non-smokers, 16 smokers). The rockwool exposure lasted at least 5 years. We measured DNA strand breaks (SBs) in the lymphocytes using the comet assay, levels of vitamin C, ferric reducing ability of plasma (FRAP), ceruloplasmin oxidase activity (CPL), malondialdehyde (MDA) in plasma, activities of antioxidant enzymes in erythrocytes: glutathione peroxidase (GPX), glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT). Rockwool exposure induced elevation of SBs in the lymphocytes of investigated subjects. When analysed according to sex and smoking habit, this effect was apparent only in the group of non-smokers. The decrease in repair rate measured as incision at 8-oxoGuanosine was significant in the group of exposed men. We found higher MDA levels in the whole group of exposed workers (p=0.025) and in exposed non-smokers (p=0.003) which could have been the consequence of significantly suppressed activity of CPL-oxidase and CAT in these groups. The activity of GST was lower in exposed workers compared with controls (P=0.038). Dividing the group by sex, the effect of exposure was seen only in men (p=0.007). Dividing according to smoking status, exposure apparently affected only the non-smokers (P=0.032). Concentrations of vitamin C in plasma and FRAP were not affected by the rockwool exposure. There was a significant negative correlation between the activity of GPX and MDA in the whole group and in the exposed group, and between CAT activity and MDA in the group of exposed subjects. The presented results indicate that rockwool exposure induces an increase in oxidative damage of biomolecules especially in the group of male non-smokers. The work was supported by an EU grant, contract No. QLK4-CT-1999-01629

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P97 Lung multinucleate cells after inhalation of amosite dust combined with cigarette smoke by rats M. Beno1, M. Hurbankova1, M. Dusinska1, K. Volkovova1, M. Staruchova1, S.Cerna1, M. Barancokova1, A.Kazimirova1, P. Bobek1, M. Horecky1, Mikulecky M.1 and S.A. Kyrtopoulos2

1 Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic, 2 Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece This study was part of a research project aimed at looking for biomarkers useful for the experimental comparison of the biological effects on laboratory animals of various fibre-dusts, alone or in combination with cigarette smoke. Multinucleated cells (MNC) may arise by fusion of macrophages engaged into cleaning foreign material from tissue. Experimental data support the hypothesis that, after exposure to fibrogenic dust, increased numbers of MNC appear as a result of monocyte or macrophage immigration into the lung tissue, followed by proliferation and fusion of these precursor cells into multinucleate entities. The present study gives information about the dose-response relationships governing the induction of lung MNC after inhalation by rats of two concentrations of amosite asbestos combined with daily exposure to cigarette smoke. Concomittantly, lung inflammation parameters have been followed. Six groups of male Fisher 344 rats inhaled amosite asbestos fiber dust of South African origin in a nose-only inhalation device every second day for one hour per exposure. The groups exposed to combined noxae breathed daily also diluted main-stream tobacco smoke at 30 mg of total particulate matter (TPM)/m3 air for one hour, corresponding to smoke arising from three cigarettes. The exposures lasted 175 days. The dosage groups were: 1) 60 mg/m3 amosite fibres plus cigarette smoke; 2) 60 mg/m3 amosite fibres; 3) 30 mg/m3 amosite fibres plus cigarette smoke; 4) 30 mg/m3 amosite fibres; 5) cigarette smoke; 6) no dust no smoke - control. MNC were counted in lung cell suspensions prepared after lung digestion with trypsin. Inflammation was monitored by the changes in the BAL fluid inflammatory parameters. The following parameters showed the presence of lung inflammation in exposed animals at sacrifice. A dose-dependent rise in the proportions of BAL lymphocytes, from 2.9% in the controls to 4.6% and 7.6% in the 30 and 60 mg/m3 amosite inhaling animals, respectively, was found. Furthermore, an increase was observed in the proportions of BAL PMN, from 0.4% in the controls to 1.4% and 2.3% in the 30 and 60 mg/m3 amosite plus cigarette smoke inhaling animals, respectively. Finally, the total BAL protein rose from 2.1 4g/ml to 2.8 g/ml and 3.0 g/ml in the 30 and 60 mg/m3 amosite plus cigarette smoke inhaling animals, respectively. Under these conditions, an increase was observed in the proportions of BAL MNC, from 0.7% in controls to 2.4% and 3.0% in the 30 and 60 mg/m3 amosite plus cigarette smoke inhaling animals, respectively. As regards changes in MNC in lung tisue, a dose-dependent rise of the proportion of MNC in lung cell suspensions was found, from 1.5% (95% confidence limits - CL = 1.3-1.7%) in control lungs to 2.1% (CL=1.9-2.3%) in the sole smoking group. The proportion in sole 30 mg/m3 amosite inhaling group was 11.7% (CL=11.3-12.2%), after sole 60 mg/m3 amosite inhaling group 12.4% (CL 11.9-12.9%), after combined 30 mg/m3 amosite plus smoking 15.2% (CL=14.6-15.8%) and after combined 60 mg/m3 amosite plus smoking 15.4% (CL=14.9-15.9%), respectively. These data suggest an additive, rather than synergistic, effect of combined amosite and tobacco smoke exposure for the induction of lung MNC at the exposure levels used. A positive significant correlation of the proportions of MNC in the BAL fluid to that in the lung cell suspensions has been found (r=0.46, p<0.05). It is concluded that, in the experimental assessment of effects of fibrogenic dusts, the number of MNC in lung cell suspensions and in BAL may serve as a useful semiquantitative biomarker. The work was supported by an E.U. grant, contract, No. QLK4-CT-1999-01629 (FIBRETOX project). P98 Effect of exposure to amosite and wollastonite fibres on antioxidative parameters in bronchoalveolar lavage fluid and lung tissue of rats. Interaction with smoking K. Volkovová, M. Beňo, Z. Kováčiková, M. Staruchová, M. Hurbánková, M. Dušinská Institute of Preventive and Clinical Medicine, Bratislava, Slovakia The purpose of this study was to examine the effect of amosite asbestos, wollastonite fibres and smoking on selected antioxidant parameters in bronchoalveolar lavage fluid and lung tissue. Fisher 344 male rats (191.2 ± 10.4 g body wt) fed standard laboratory pellets and tap water ad libitum were used for the study. Groups of animals inhaled amosite asbestos or wollastonite fibres at two concentrations (30 and 60 mg/m3 of air) in a nose-only inhalation device for 1 hour every 2 days. The other group of animals was additionally exposed to mainstream cigarette smoke from 3 cigarettes/day following each day’s fibre exposure. After 6 months of exposure the animals were anaesthetised with thiopental and before removing the lungs were washed 3 times with 4 ml of saline solution. The activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), and ferric reducing ability (FRA) in bronchoalveolar lavage fluid (BAL) were measured. The same parameters were examined in the lung tissue together with the concentrations of malondialdehyde (MDA). Amosite inhalation alone induced a significant decrease in FRA of BAL at the higher dose only, while in combination with tobacco smoke the FRA of BAL was decreased in both doses along with the significantly lower activities of GPX and SOD compared with the corresponding amosite only inhalation group. There was a decreased

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activity of SOD and increased concentration of MDA in the lung tissue at both doses of amosite inhalation. In combination with tobacco smoke, only the higher dose of amosite inhalation increased the concentration of MDA in the lung tissue significantly. The activities of GPX and SOD remained unchanged when compared with the smoking control group of rats, but their activities were significantly higher when compared to the corresponding amosite only inhalation group. Wollastonite exposure did not induce any significant changes in antioxidant parameters in the BAL, either alone or in combination with tobacco smoke. A significant increase in SOD activity together with higher levels of MDA were detected in the lung tissue at both doses of wollastonite inhalation combined with tobacco smoke exposure when compared to the corresponding non-smoker groups. These results show that there is a synergistic effect of both amosite and wollastonite fibre exposure with tobacco smoke. Amosite exposure alone can induce profound alterations in antioxidative status of BAL and lung tissue, while wollastonite inhalation affects the pro- and antioxidative balance only in combination with tobacco smoke. This work was supported by FIBRETOX, EC contract No QLK4-1999-01629. P99 Effects of amosite and wollastonite mineral fibres, and exposure to tobacco smoke, on the immune response of fisher rats exposed via inhalation M. Kuricova1, J. Tulinska1, A. Liskova1, M. Beno1, S. Wimmerova1, S.A. Kyrtopoulos2 and M. Dusinska1 1Institute of Preventive and Clinical Medicine, Limbova 14, 833 01 Bratislava, Slovakia, 2National Hellenic Research Foundation, Athens, Greece While the lung is the primary target organ for asbestos and other mineral fibres toxicity, recent studies have shown that the immune system may also be altered by exposure to fibrous dust, something that may play a role in the induction of lung disease, including cancer. Cigarette smoking, a known modulator of immune response, may act as a modifying factor in this process. This study is a part of comprehensive project aimed at the investigation of the mechanisms of toxicity (including lung carcinogenesis and genotoxicity) of asbestos-substitute mineral fibres. Male Fisher rats were exposed to an aerosol containing 30 mg/m3 and 60 mg/m3 of amosite or wollastonite fibres. The aerosol was inhaled for one hour every second day for 6 months. Cigarette smoke was inhaled daily (x hours / day) at 30 mg TPM/m3. Cellular immunity was examined by phenotypic analysis of leukocytes (CD3, MHC II, CD4, CD8, CD161, B-lymphocytes) and by expression of adhesion molecules (CD11b, CD54) on leukocytes. Immune function assays included proliferative response of T and B-lymphocytes and phagocytic activity of blood leukocytes. Hematologic parameters were also evaluated. Exposure to amosite significantly increased the percentage of neutrophils and decreased lymphocytes in peripheral blood of exposed animals. The proliferative activity of T-lymphocytes and T-dependent B-cell response was significantly decreased. The immunosuppressive effect was more pronounced in low-dosed rats. A marked suppressive effect of amosite on phagocytic activity of leukocytes was also found. Inhalation of amosite fibrous dust resulted in a significantly increased percentage of B-lymphocytes and elevated expression of adhesion molecule CD11b on lymphocytes and CD54 on granulocytes of peripheral blood in non-smoking, high-dosed rats. Total white blood cell counts in peripheral blood were also decreased in rats given the high dose of wollastonite. All animals exposed to wollastonite fibres had suppressed phagocytic activity of leukocytes. The stimulative effect of smoking on the immune system was shown as significantly elevated percentage of some lymphocyte subsets (T-cytotoxic and T-helper lymphocytes, NK-cells and B-lymphocytes) in comparison with non-smoking animals. Moreover stimulation of the immune system was observed as increased proliferative activity of T-lymphocytes and phagocytic activity of leukocytes. Combined exposure to amosite and cigarette smoke altered the proliferative capacity of B-lymphocytes after in vitro stimulation with LPS. No effect on proliferative activity of B-cells of exposure to amosite fibres alone was seen in non-smoking rats, while a moderate enhancement was recorded in animals exposed only to smoking. However, a significant stimulation was found in rats exposed to fibres and smoking. The combined exposure to wollastonite and smoking caused a significant, dose-dependent increase of the percentage of cytotoxic cells, and enhanced expression of adhesion molecule CD11b on granulocytes in peripheral blood. In conclusion, inhalation of amosite and wollastonite mineral fibres resulted in marked changes in specific and non-specific immunity. Moreover, findings indicate mutual interference of mineral fibres and smoking in the modulation of the systemic immune response during combined exposure. This work was supported by the European Union (project no. QLK4-1999-01629) and Slovak Grant Agency for Science, # 04.92.11.05

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P100 Immune changes in Fisher rats intratracheally instilled with four types of mineral fibres Liskova1, J. Tulinska1, M. Kuricova1, M. Hurbankova1, S. Wimmerova1, S, A. Kyrtopoulos2 and M. Dusinska1 1Institute of Preventive and Clinical Medicine, Limbova 14, 833 01 Bratislava, Slovakia, 2National Hellenic Research Foundation, Athens, Greece This study was a part of comprehensive project aimed at the research of the mechanisms of toxicity of asbestos-substitute mineral fibres. Understanding the interactions between systemic and local immune changes is important in the investigation of mineral fibre-related disease processes (including genotoxicity and carcinogenesis). The systemic immune response was evaluated in male Fisher rats intratracheally instilled with 2 mg or 8 mg of amosite, wollastonite, rockwool and glass fibres. Dose of 2 mg was suspended in 0.2 ml of saline solution per animal or control group with 0.2 ml saline only. Dose of 8 mg was divided and instilled 4 times (weekly 2 mg/0.2 ml saline solution). Immunotoxic effect was examined using a panel of immune and haematological assays. The assays were performed 4 or 16 weeks after last instillation of the fibres. Phenotypic analysis of leukocytes (T-lymphocytes, activated T-cells, B-lymphocytes, NK-cells, T-helpers, T-cytotoxic cells) and expression of adhesion molecules (CD11b, CD54) were performed by flow cytometry. Immune functions were evaluated by proliferative activity of T and B-lymphocytes in vitro stimulated with mitogens and antigen and phagocytic activity of leukocytes. Basic haematology was also done. Amosite, rockwool and glass fibres caused altered total white blood cell counts in exposed animals. All fibres increased the percentage of neutrophils, while two (rockwool and glass fibres) also significantly decreased the percentage of lymphocytes in differential white blood cell counts, especially in high-dosed animals after 4 weeks. The effect of fibres on the percentage of T and B-lymphocytes in peripheral blood was most pronounced in the case of amosite and wollastonite. A significant decrease of B-cells was found in high-dosed amosite rats regardless of the period after exposure. In the same animals exposed to amosite, as well as in rats given the high dose of wollastonite, T cell were also elevated. T-helper lymphocytes were increased in animals exposed to amosite, wollastonite and glass fibres, although no clear time or dose-dependency was seen. Additionally, T-cytotoxic cells were elevated in high-dosed amosite rats. Although amosite seems to be most potent suppressor of T- and B-lymphocyte proliferation, especially in high-dosed animals, wollastonite and rockwool also interfered with lymphocyte proliferation and suppressed the response of T-lymphocytes. The opposite stimulative effect on proliferative capacity of B-cells was found in animals given a low dose of glass fibres. Phagocytic activity was dramatically affected by exposure to rockwool and glass fibres. A highly significant dose-dependent suppression was found in neutrophils and monocytes. Rats exposed to wollastonite fibres had also decreased phagocytic activity of peripheral blood phagocytes 4 weeks after instillation of either dose. Surprisingly, the phagocytic activity of animals exposed to amosite was affected only in high dosed rats. In conclusion, animal exposure to mineral fibres leads to alterations in the systemic immune response. Immune dysregulation consisted of changes of the main lymphocyte subsets. Moreover, the function of immunocompetent cells that are responsible for the specific immune response (T and B lymphocytes) and phagocytic cells was impaired. This work was supported by the European Union (project no. QLK4-1999-01629) and Slovak Grant Agency for Science, # 04.92.11.05 P101 The effect of glass fibres on the immune system in occupationally exposed workers S. Ilavska1, E. Jahnova1, J. Tulinska1, M. Horvathova1, M. Dusinska1, A. Liskova1, M. Kuricova1, L. Fuortes2

and S. A. Kyrtopoulos3 1 Institute of Preventive and Clinical Medicine, Bratislava, Slovakia, 2 University of Iowa, College of Public Health, Iowa City, Iowa, USA, 3 Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece The evidence for adverse health effects following exposure to asbestos has prompted widespread removal of asbestos-containing materials and led to banning of asbestos internationally. These changes have resulted in the increased use of substitutes consisting of naturally occurring as well as synthetic materials, including man made mineral fibres, which are thought to have lower toxicity. Many workers, as well as the general public, are exposed to glass fibres, which are among the most common man-made fibres. However, information related to the immunotoxicity and immunomodulation of these materials is still limited. Immunotoxicity may play an important role in the induction of fibre-related lung disease, including cancer. The aim of this study was the biological monitoring of exposure to glass fibres using immune markers in occupationally exposed workers. Immune and haematological parameters were examined in 80 workers chronically exposed to glass fibres and 36 in-factory unexposed controls. Cellular immunity was determined by phenotypic analysis of human peripheral blood leukocytes (PBMC): T-cells, T-helper and T-cytotoxic cells, NK-cells, B-cells and activated T-cells (CD3, CD4, CD8, CD16+56, CD19 and CD3/HLA DR). Humoral immunity was evaluated using the levels of immunoglobulins IgG, IgA, IgM, IgE, C3 and C4 component of complement. Concentrations of interleukins 1, 6, and 8 and soluble adhesion molecules ICAM-1, VCAM-1, and E-selectin were determined. Also expression of adhesion molecules CD11b, CD18, CD54, CD62l and CD49d on

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leukocytes was measured by flow cytometry. Expression of activation markers on eosinophils CD25, CD66b, CD81, HLA DR was assessed. Phenotypic analysis of PBMC showed significantly decreased expression of markers CD16+56 (NK cells) in people exposed to glass fibres in comparison with factory control. We found increased level of soluble adhesion molecule E-selectin and IL-8 in sera of people who manipulated with glass fibres. Exposed subjects had significantly increased expression of activation marker CD66b on eosinophils. Chronic exposure to glass fibres had immunomodulatory effects in occupationally exposed workers. Changes in lymphocyte subpopulation of natural killer cells in peripheral blood and findings of increased levels of soluble adhesion molecule E-selectin, proinflammatory cytokine IL-8 and enhanced expression activation marker on eosinophils may indicate negative effects of glass fibres on the immune system of exposed workers. This work was supported by the European Union (project no. QLK4-1999-01629), NIEHS Grant # 510205240 00000 and Slovak Grant Agency for Science # 04.92.11.08. P102 DNA damage induced in rats by intratracheal instillation of mineral fibres R.Stetina Purkynje Military Medical Academy, Department of Toxicology, Hradec Kralove, Czech Republic Amosite, wollastonite or rock wool fibres were administered intratrachealy to Fischer male rats at the dose 2mg/week for 4 weeks, total dose 8 mg. Animals were sacrified 1, 4 or 16 weeks after last instillation. One half of animals (10 of 20 in each group) was given benzo(a)pyrene (BaP) (2x40 mg) i.p. 48 and 24 h before killing. DNA damage (single strand breaks) measured in freshly isolated lymphocytes, alveolar macrophages and in lung cells (mixture of trypsinised lungs) was analysed by comet assay, while oxidised pyrimidines in the DNA were analyses after endonuclease III treatment of cells before electrophoresis. Amosite, wollastonite and rock wool induce DNA beaks in alveolar macrophages, lung cells and lymphocytes. Amosite was the most effective one. The induction is highest at about 4 weeks of exposition and decreases at 16 weeks. The induction of the damage in lymphocytes is slightly delayed, being highest after 16 weeks of exposition. There seems to be a synergism between effects of fibres and BaP, because while BaP itself does not induce a significant amount of the DNA damage, in combination with fibres the induction of DNA breaks is evident. The induction of DNA damage correlated with the ability of fibres to induce DNA repair. The higher induction of DNA damage, the higher repair capacity is found in lymphocytes by the fibre treatment. The repair of gamma rays-induced DNA breaks is slowed down in animals treated with BaP. This might be a mechanism of the potentiation of carcinogenic effect of fibres. P103 Effect of asbestos, wollastonite, rockwool and glass fibre on DNA of lung cells in vivo. Intratracheal instillation of fibres into Fisher 344 rats K. Burghardtová, M. Hurbánková, S. Černá, S. Wimmerová, K. Volkovová, M. Staruchová, M. Beno, M. Dušinská Institute of Preventive and Clinical Medicine, Limbová 14 833 01 Bratislava, Slovakia The unfavourable effect of naturally occurring fibres (such as amosite and wollastonite) as well as of man-made mineral fibres is known, but the mechanism of toxicity in the human body is not fully described. The proposed mechanism of fibre toxicity is associated with oxidative damage caused by free radicals generated by macrophages during phagocytosis. Fibres longer than 8 µm with diameter less than 0.25 µm are supposed to be more toxic. The length of 95 % of amosite asbestos fibres is 20–30 µm or more. We investigated oxidative DNA damage in mixed lung cells isolated from rat lungs exposed to four different fibres in vivo: amosite, wollastonite, rockwool and glass fibres. Two doses (2 or 2x4 mg/animal) of fibres were administrated by intratracheal instillation to Fisher 344 male rats. Animals were sacrificed 4 or 16 weeks after the last instillation. Oxidative DNA damage (oxidised purines and pyrimidines) was measured by the comet assay with lesion specific enzymes formamidopyrimidine glycosylase (FPG) and endonuclease III (Endo III) additionally to strand breaks (SBs). Alkylating DNA damage was detected with 3-methyladenine DNA-glycosylase II (AlkA). Sensitivity of lung cells to hydrogen peroxide ex vivo was also assessed. Lactate dehydrogenase activity (LDH) as a marker of nonspecific cellular toxicity was detected in the cell free bronchoalveolar lavage fluid of experimental animals. Amosite and wollastonite induced oxidative and alkylating DNA damage, though significant differences in DNA damage were seen only after the administration of 8 mg of wollastonite/animal and a shorter period of exposure (4 weeks). Oxidised pyrimidines (net Endo III sites) correlated with LDH in the wollastonite exposed group (r=0.421, p<0.05, n=26). Lung cells exposed to amosite (dose 8mg/animal, period 16 weeks) were more sensitive to hydrogen peroxide than those isolated from control rats which suggests a decreased antioxidant defence in amosite exposed lung cells. Rockwool did not induce oxidative DNA damage, nor did glass fibre. However, there was a significant positive correlation between

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alkylating DNA damage and LDH activities in rockwool exposed lung cells (r=0.432, p<0,05, n=30) and between sensitivity to hydrogen peroxide and LDH (r=0.415, p<0.05, n=30). Amosite and wollastonite fibres are more toxic to DNA in lung cells compared to rockwool or glass fibres. These findings differ slightly from observation on specific lung cells. It also seems that a mixed population of lung cells is less sensitive compared with specific target cell types such as epithelial type II cells or alveolar macrophages. This work was supported by the European Union (project Fibretox no. QLK4-1999-01629) and Slovak Grant Agency for Science, # 04.92.12.04 P104 DNA damage measured by the comet assay in rat lung cells after inhalation exposure to amosite and wollastonite fibres in vivo. Combined effect with smoking K.Burghardtová, M. Beňo, M. Hurbánková, K. Volkovová, M. Staruchová, P. Bobek, J. Horecký, S. Wimmerová, M. Dušinská Institute of Preventive and Clinical Medicine, Limbová 14 833 01 Bratislava, Slovakia There is an urgent need to substitute asbestos and other carcinogenic fibres with non-carcinogenic, less toxic fibres. The aim of this study was to investigate the effect of wollastonite, which should replace asbestos in industry. It is believed that the carcinogenic effect of asbestos fibres is caused by impairment of the DNA by oxygen radicals from cells involved in the removing of asbestos from lungs during the inflammation. Cigarette smoke is also known to cause lung cancer and therefore the combined effect of amosite and wollastonite fibres with smoking was also studied. Six groups of Fisher 344 male rats, each of 11 animals were exposed to 1/ 60 mg/m3 fibres (wollastonite or amosite) for one hour every two days combined with exposure to mainstream smoke from three cigarettes daily; 2/ 60 mg/m3 (wollastonite or amosite) for one hour every two days; 3/ 30 mg/m3 (wollastonite or amosite) for one hour every two days combined with exposure to mainstream smoke from three cigarettes daily; 4/ 30 mg/m3 (wollastonite or amosite) for one hour every two days; 5/ exposure to mainstream smoke from three cigarettes daily plus immobilization stress similarly as animals exposed to dust; 6/ immobilization stress, the same conditions as animals exposed to dust, /control group/. The alkaline comet assay was performed on lung cells isolated from homogenised lung tissues of exposed as well as control rats. Strand breaks (SBs), oxidised purines (using repair enzyme formamidopyrimidine, FPG), oxidised pyrimidines (by bacterial repair enzyme endonuclease III, EndoIII), and DNA alkylation (by bacterial enzyme 3-methyladenine DNA-glycosylase II, AlkA) were measured. Sensitivity of isolated lung cells to oxidative DNA damage by the ex vivo treatment with hydrogen peroxide was also studied. Both amosite and wollastonite induced DNA damage in lung cells in dose response manner, however the effect of amosite on DNA was more pronounced than effect of wollastonite. There was a signicantly higher number of SBs, SBs plus FPG, SBs plus Endo III and SBs plus Alk A detected in lung cells isolated from amosite-exposed groups, compared with controls. Wollastonite induced significantly elevated levels of strand breaks plus FPG after the higher dose only. Combination with cigarette smoke brought contradictory results. Strangely asbestos-exposed non-smoking rats had more DNA damage in their lung cells than asbestos-exposed smoking rats. These cells were also more sensitive to oxidative DNA damage than those from abestos-exposed smoking rats. A similar pattern was observed in lung cells isolated from rats after wolastonite inhalation. The elevated DNA damage was again significant in wollastonite-exposed non-smoking but not in wollastonite-exposed smoking rats. These results may suggest the hypothesis that cigarette smoke could exert a stimulating effect on the antioxidant defence system and thus induce slight protection against oxidative DNA damage. However, to confirm our results, more critical explanation and further experimental support will be needed. This work was supported by the European Union (project Fibretox no. QLK4-1999-01629) and Slovak Grant Agency for Science, # 04.92.12.04. P105 Combined inhalation exposure of rats to fibres and cigarette smoke. Genotoxic effect on alveolar macrophages and epithelial type II cells. Petrovská, Z. Kováčiková, A. Kažimírová, M. Barančoková, M. Beňo, M. Hurbánková, M. Dušinská Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic The ability to induce genotoxic effects in alveolar macrophages and epithelial type ii cells after inhalation exposure to amosite and wollastonite was studied. Fisher rats were exposed in an inhalation chamber to 30 or 60 mg.m-3 of amosite or wollastonite every second day for an hour. Half of each group was exposed daily to the mainstream of 3 cigarettes (1r1). One group was exposed only to cigarette smoke and one group represented unexposed control. The exposure lasted for 6 months. Statistical analysis (non-parametric Wilcoxon test) was performed to compare treated and untreated animals, different doses, length of exposure, and to compare fibres with each other. Both doses of amosite increased the frequency of micronuclei in alveolar macrophages comparing to the control groups. However, the enhancement was statistically significant only after the lower dose, both in the smoking and the non-smoking group. In epithelial type II cells

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the micronucleus induction was significant only in the non-smoking rats. There was no increase in frequency of micronuclei either in alveolar macrophages or in epithelial type II cells after inhalation of wollastonite. The comet assay modified with lesion-specific enzymes was used to measure strand breaks, oxidative and alkylating DNA damage. The sensitivity of both cell types to hydrogen peroxide ex vivo was also tested. Elevated DNA damage was observed after inhalation of amosite in both alveolar macrophages and epithelial type II cells, in both smoking and non-smoking rats. Sensitivity to hydrogen peroxide was also enhanced, especially in the group of non-smoking amosite-exposed rats. Inhaled wollastonite also induced oxidative DNA damage in both alveolar macrophages and epithelial type II cells compared with non-smoking or with smoking rats. In aleveolar macrophages, hovewer, the elevated DNA damage was significant only at the lower dose of wollastonite in both smoking and non-smoking rats. In epithelial type II cells a higher level of DNA damage was detected only in the group of smoking rats. Our results show the genotoxic effect of amosite but not wollastonite. Positive results coming from the comet assay after inhalation of wollastonite imply that other mechanisms exist. The results presented are in concordance with our findings in the study with intratracheal application of fibres: alveolar macrophages are slightly more sensitive than the epithelial type II cells. This work was supported by the European Union (project no. QLK4-1999-01629), and Slovak Grant Agency for Science # 04.92.12.04 P106 Biomonitoring of asbestos exposed workers: frequency of chromosomal aberrations, lipid peroxides, epidermal growth factor, and basic fibroblast growth factor S. Korraa1 and A. A. Kamal2 1Department of Radiation Health, National Center for Radiation Research and Technology 2Department of Radiation Biology, National Center for Radiation Research and Technology, ***Faculty of Medicine Ain Shams University, Cairo, Egypt Asbestosis is a pneumoconiosis that results from the inhalation of asbestos fibers. Deposition of asbestos fibers in the lung is associated with pathologic changes in the small airways and the production of pulmonary fibrosis or mesotheliomas. There is a body of evidence through in vitro studies that implicates the alveolar macrophage in the pathogenesis of asbestosis and that pulmonary fibrosis may result from the persistent release of inflammatory mediatorsm (chemoattractants, lysosomal enzymes, toxic oxygen radicals, arachidonic acid metabolites, interleukins, and fibroblast growth factors) at sites of asbestos deposition. Thus the frequency of chromosomal aberrations (FCA) in circulating lymphocytes, plasma basic fibroblast Growth Factor (bFGF) and Epidermal Growth Factors (EGF) and lipid peroxides (MDA) were measured in the blood of 45 asbestos exposed workers versus 20 controls. Although none of the of the asbestos exposed workers showed x-rays signs of either cancer or fibrosis, yet there has been an increase in levels in the FCA (9.8 ± 3.5 vs. 3 ± 1.1 %, p<0.01), bFGF (8.6 ± 3.8 vs. 1.5 ± 1 ng/ml, p< 0.001) and EGF (12.6 ± 1.5 vs. 2.5 ± 0.2 ng/ml, p<0.0001) and MDA (6.9 ± 2.5 vs. 2.5 ± 0.9, p<0.0001). It can be concluded that occupational exposure to asbestos induces an oxidative stress that can consequently induce genotoxicity. Growth factors such as EGF and bFGF are expressed as a consequence of the elicited oxidative stress. Although the significant increase in both growth factors could be an adaptive and/or repair response induced as a consequence of asbestos exposure, the presence of such growth factors circulating in blood jeopardize such workers to malignant transformation. These results show that growth factors and protein carbonyls can be reliable biomarkers in monitoring early effects of pesticides as well as the frequency of chromosomal aberrations. P107 Effect of tocopherol administration on the frequency of chromosomal aberrations among asbestos exposed workers S. Korraa1 and A. A. Kamal2 1Department of Radiation Health, National Center for Radiation Research and Technology 2Department of Radiation Biology, National Center for Radiation Research and Technology, ***Faculty of Medicine Ain Shams University, Cairo, Egypt The frequency of chromosomal aberrations among asbestos exposed workers (n=36) were measured before and after a course of tocopherol administration. Workers were divided into 3 groups of 12 persons. They were given tocopherol at doses of 0, 400 and 600 mg per day for seven weeks. A control group of 15 workers was investigated. Initially, the exposed group showed higher frequency of chromosomal aberrations than that of the controls. These were in the form of dicentrics, rings, gaps, chromosome and chromatid breaks. After the period of tocopherol administration, the occurrence

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of chromosomal aberration was re-evaluated, and the results will be discussed in terms of the prophylactic effect of tocopherol. P108 Immunotoxic effects in workers occupationally exposed to asbestos and rockwool E. Jahnova1, S. Ilavska1, J. Tulinska1, M. Horvathova1, M. Dusinska1, A. Liskova1, M. Kuricova1, L. Fuortes2 and S. A. Kyrtopoulos3 1Institute of Preventive and Clinical Medicine, Bratislava, Slovakia, 2University of Iowa, College of Public Health, Iowa City, Iowa, USA, 3Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece Occupational exposure to asbestos is widely believed to pose an increased risk for a range of pulmonary diseases, cancer, and immunotoxic effects. Both systemic and local immunity may play an important role in the pathogenesis of these events. Immune cells appear to be influenced by asbestos exposure, either through direct effects or as a result of the host’s protective response to exposure. Man-made vitreous fibres (MMVFs) have some structural features similar to those found in asbestos. This has led to concern that exposure to MMVFs could increase the risk of diseases similar to those caused by asbestos. Based on the above, we have assessed the immune response in workers with at least 5 years’ exposure to asbestos or rockwool fibres, at 2 industrial plants. In each plant, 97 or 61 exposed workers and 42 or 21 control clerical subjects, respectively, were recruited. In the case of the asbestos-exposed subjects, an additional town-control group of 47 people was included. Subjects were matched for sex, age, smoking habit and alcohol use. In the area of humoral immunity we evaluated levels of immunoglobulins IgM, IgG, IgE, IgA, C3 and C4 components of complement, concentrations of IL-1, IL-6, IL-8 and soluble adhesion molecules ICAM-1, VCAM-1 and E-selectin. Cellular immunity was determined by phenotypic analysis of human peripheral blood leukocytes: B-cells, NK-cells, T-cells, activated T cells, T-helper and T-cytotoxic cells. Expression of adhesion molecules on leukocytes (CD18, CD11b, CD54 and CD62L) and identification of surface molecules associated with activation of human eosinophils (CD69, CD66b) were also assessed using flow cytometry. Workers exposed to asbestos fibres had significantly increased levels of immunoglobulin E in comparison with two controls. IgG was significantly different among asbestos plant and town control groups. Significantly increased levels of IgA were found in both plant groups in comparison to the town controls. Significantly increased concentrations of IL-6 and IL-8 were found in the asbestos group in comparison with controls. The levels of soluble adhesion molecule ICAM-1 were higher in the exposed group in comparison to the town controls. Evaluation of the expression of adhesion molecules on lymphocytes, monocytes and granulocytes by flow cytometry showed significant increases in monocytes and granulocytes of the class of selectins CD62L (L-selectin, which mediates the homing of leukocytes to inflammatory sites). Moreover, significantly increased expression of marker CD66b on eosinophils was found among those exposed to asbestos when compared to control groups. Workers exposed to rockwool had significantly increased levels of immunoglobulin E, IL-8 and the soluble adhesion molecule ICAM-1 in comparison with controls, as well as of marker CD66b on eosinophils. However, they had significantly lower levels of VCAM-1.In conclusion, exposure to asbestos fibres and rockwool was found to have several negative effects on the immune system. Alterations of these immune parameters may indicate hypersensitivity and an elevated inflammatory status in exposed workers and may play a role in the induction of cancer and other types of lung disease by these fibres. This work was supported by the European Union (project no. QLK4-1999-01629), NIEHS Grant # 510205240 00000 and Slovak Grant Agency for Science # 04.92.11.08.

Inducible Responses P109 Classification of chemical carcinogens by gene expression profiling: discrimination of genotoxic versus non-genotoxic carcinogens Agen E van, Kleinjans JCS, Delft JHM van Department of Health Risk Analysis and Toxicology, University of Maastricht, Netherlands The screening of chemical compounds for hazardous properties relies on a few in vitro assays and on acute to sub-chronic studies with animal models. Despite their frequent use, the reliability, relevance and effectiveness of these methodologies are continuously questioned. Powerful technologies for functional genomics may provide new, mechanism-based, assays with a high predictive value for humans. It is envisaged that toxicology will benefit enormously from the application of DNA microarray technologies to analyse chemically induced alteration of gene expression. Possible topics include genotoxicity screening and identification of modes of action of carcinogens. Here, we investigated if gene expression profiling can be used for mechanism-based classification of chemical carcinogens. Specific aims are:

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1) discrimination of genotoxic from none-genotoxic carcinogens based on mRNA profiles, 2) identification of discriminating gene expression profiles, and 3) biomarker genes. HepG2 human hepatoma cells were treated for 24 hr with test compounds. 19 chemicals were chosen: 9 genotoxic carcinogens, 7 non-genotoxic carcinogens, and 3 negative controls. Two dose levels were applied; with the highest dose based on limited cytotoxicity (MTT-test), limit of solubility or otherwise 2 mM. For each treatment, control and test samples were hybridised to Phase-1 microarrays in duplicate (second with dye-flip). These arrays contain 600 toxicologically relevant human genes in quadruplicate. Scans and data are analysed by ImaGene and GeneSight, respectively, which include among others clustering analyses. Large variation in number and ID of responsive genes are found between the compounds. Clustering analyses with all genes appears poor for genotoxic versus non-genotoxic carcinogens. However, if a sub-set of genes is selected, almost perfect discrimination is obtained of genotoxic versus non-genotoxic carcinogens.

P110 Expression of intercellular adhesion molecule 1 (ICAM-1 / CD54) in invasive breast cancer Milićević Z1, Bajić V2, Petrović M3., Bogojević D3 1Institute of Nuclear Sciences ‘’Vinča’’, Lab. Mol. Biol Endocrinology 090 Belgrade 2ICN-Galenika, Pasterova 2, Belgrade 3Molecular Biology Laboratory, Institute for Biological Research ‘’Siniša Stanković’’, 29 Novembar 142, 11060 Belgrade, Serbia and Montenegro The basic concept that neoplastic transformation of cells and their progression to the metastatic state may be a result of the abrogation of control mechanisms of cell proliferation, intercellular adhesive and invasive behaviour of normal cells has been in vogue for over two decades. Metastatic ability is a heritable property; therefore, the progression of tumors to the metastatic state implies changes in the expression of genes. These differences in biological behaviour are not transient but are attributable to genetic alterations. The precise nature of the genetic changes undergone by tumors during progression in general or in the acquisition of metastatic potential are poorly understood. The evolution of cellular diversity within tumors is a significant feature of the development and progression of neoplasms. Breast cancer is a heterogeneous disease. Tumors are highly variable in their growth rates, patterns of metastases, and other biological characteristics. The identification of molecules that are differentially expressed in benign and malignant tumors can be an important step towards understanding the biochemical basis for the distinct clinical behaviour of these two lesions. ICAM-1 (CD 54) is an inducible cell adhesion molecule that can be expressed by many different types of cells and which mediates interactions with leukocytes via the integrin ligands lymphocyte function-associated antigen-1 and Mac-1. In clinical investigations, some researchers have reported that ICAM-1 expression in cancer cells was related to negative tumor growth and metastasis and resulted in a good prognosis for the patients. Other reported that ICAM-1 expression in cancer cells reflected aggressive tumor potential and poor clinical course. ICAM-1 expression by tumor cells has been considered of central importance to host/tumor interactions including lymphocyte–mediated tumor cytotoxicity and metastatic progression. The function of ICAM-1 which leads to malignant cell lysis during the process of tumor progression is not understood clearly. For analysis we used invasive breast carcinoma of common types with a different differentiation and stages as well as a normal breast tissue from patients with benign and malignant tumors. ICAM-1 expression was estimated and quantified by immunohistochemistry (APAAP and SABC) on fresh frozen sections and by Western-blott analysis using anti human ICAM-1 Mab (RR1/1). Our data show that breast cancer cells express not only membranous but also a cytoplasmic form of ICAM-1, differently from normal cells. The expression of ICAM-1 isoforms was found to be correlated with the invasive potential of breast tumor cells. The comparison of staining patterns on cryostat sections of highly metastatic breast cancers revealed a more membranous ICAM-1 localization, suggesting that mobilization of ICAM-1 to the cell surface, rather than mere synthesis, may be crucial determinant for impact on metastatic potential. P111 GeneChip analysis on transcriptional changes induced in human lymphoblastoid (TK6) cells by six genotoxic chemicals. T. Suzuki, P. Rajaguru, J. Kanno, H. Sakamoto, M. Hayashi, T. Yamaguchi, and M. Honma National Institute of Health Sciences, Japan Identification of genes that respond to toxic agents may provide useful information regarding their mode of action. Microarray analysis offers the unique potential to identify unknown targets of toxic agents, as transcriptonal responses of the entire genome can be measured in parallel. We report here the transcriptional changes induced in TK6 cells by the 4 hour treatment of six different genotoxic chemicals (Furylfulamide (AF2), AraC, EMS, MNNG, Monocrotaline (MQ) and Paraquat (PQ)). The dose was selected to give approximately 50% survival. To select the differentially expressed genes, the genes were divided in to groups based on their GeneChip (Affymetrix) intensity data and different levels of ‘significance’ were assigned – lower level of fold change to higher intensity and higher level to lower intensity (step-wise selection). The results showed that out of the 22,286 total feature sets present on the GeneChip arrays, 1,664 were either induced or repressed in at least one or more chemicals. In all six chemicals more genes were down regulated than

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up regulated. The number of genes altered by the chemical treatment was found to be, 53 genes in the case of EMS, 58 in PQ, 240 in MNNG, 421 in AF2, 470 in AraC and 796 in MQ. We did not find any common mode of gene alteration by all six chemicals. Maximum commonness for the up-regulation occurred among three agents: p21, GADD45a and ATF3 in AF2, AraC and MQ; piwi-like 1 in EMS, MNNG, and AraC; GPR88 in EMS, AF2, and MQ. Maximum commonness for the down-regulation occurred among four agents, that is solute carrier family 11A2 for AF2, AraC, MQ and PQ, and capillary morphogenesis protein 1 for AF2, EMS, MQ and PQ. These findings suggest that the molecular mechanisms and genes involved in chemical induced genotoxicity may be different and chemical specific at the earlier stage (4 h). It may be required to have different and/or multiple sampling times to detect more common changes for genotoxicity. P112 Model in vitro screening assay for detecting genotoxicity using engineered HepG2 cells Catherine C Smith1, Anthony M Lynch2, Nigel J Gooderham1 1Molecular Toxicology, Faculty of Medicine, Imperial College London, SW7 2AZ, UK. 2GlaxoSmithKline, Genetic Toxicology, Ware, Hertfordshire, SG12 0DP, UK. A new generation screening assay has been developed to rapidly detect genotoxic damage following treatment with chemical or biological DNA damaging agents. HepG2 cells were engineered to contain a single copy of the tetracycline repressor (tetR) gene and multiple copies of the tetracycline operator (tetO) gene fused to green fluorescent protein (GFP). In the resulting cell line, which we have named ‘HepGlow”, the tetR protein represses transcription of the tetO and GFP but upon mutation of the tetR gene the tetO and GFP genes are transcribed and fluorescence can be measured by flow cytometry. Here we report the results following treatment of the HepGlow cell line with chemical (MMS, AraC, Bleomycin, EMS and D-mannitol) and biological transducing agents (adenoviral vector, retroviral vector, ExGen 500 and Effectene). In parallel to analysing expression of GFP, the in vitro micronucleus (MN) assay using flow cytometry was also performed. There was high concordance between the fold changes of GFP and MN induction following the varying treatments at the lower doses. GFP showed increased sensitivity compared to MN induction in a number of chemical treatments at high doses where cell growth arrest was observed. MMS gives a historic fold change in fluorescence of 3.43±0.44 (31ug/ml 24h dose, n=4) and 4.55±0.96 (31ug/ml 24h dose, 24h wash, n=6). Treatment of HepGlow cells with biological agents incorporating red fluorescence protein (RFP) gene as a reporter of transduction (5-80%), allowed fluorescence to by gated in sub-populations to determine the percentage of GFP in the transduced cells. Since HepGlow cell line incorporates a fluorescent reporter gene (GFP) and the transducing biological agent also incorporates a fluorescent reporter gene (RFP), discrete cell sub-populations can be discriminated, enhancing the sensitivity and selectivity of the assay. Thus our system allows rapid and sensitive detection of genotoxic damage immediately after treatment by chemical DNA damaging agents and also by biological DNA damaging agents that vary in their transduction efficiencies. P113 Examination of Caucasian and Asian diabetic patients in the Comet assay Wyatt, N.P;1 Whitelaw, D2; Kelly, C2 & Anderson,D.1

1Biomedical Sciences: University of Bradford, UK and 2Department of Diabetes and Endocrinology; Bradford Royal Infirmary, UK. Within different population groups there may be altered susceptibility to oxidative damage from endogenous agents. This can be assessed using a variety of in-vitro assays. Diabetic patients show altered levels of DNA damage compared with controls in the Comet Assay1. A significantly higher level of basal and stimulated reactive oxygen species (ROS) in non-insulin-dependent diabetes mellitus (NIDDM) subjects in comparison with non-diabetic controls has been reported2, and a recently published paper3 has also demonstrated that mitochondrial ROS from H2O2 suppresses glucose-induced insulin secretion (GIIS) from the pancreatic β-cells; Therefore, higher concentration of ROS in diabetics may be implicated in both the pathogenesis and complications of diabetes.

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To further examine if there are differing susceptibilities, lymphocytes have been obtained from control (non-diabetic) and diabetic patients of different racial origins (Asian and Caucasian), and treated in-vitro with the oxygen radical hydrogen peroxide (H2O2). 1Anderson, D; Yu.T-W; Wright, J. & Ioannides,C. (1998) An examination of DNA strand breakage in the comet assay and antioxidant capacity in diabetic patients. Mut Res 398.151-161. 2Orie,N.N; Zidek, W. & Tepel, M. (1999) Reactive Oxygen Species in Essential Hypertension and Non-Insulin-Dependent Diabetes Mellitus. AJH.12.1169-1174. 3Sakai, K; Matsumoto, K. & Nishikawa, T. (2003) Mitochondrial reactive species reduce insulin secretion by pancreatic β-cells. Biochem Biophys Res Comm. 300(1). 216-222. P114 Activation of c-Jun N-terminal kinase is required for ultraviolet b induced apoptosis of murine peritoneal macrophages Gautam Sethi, Ajit. Sodhi and Rajesh Kumar Sharma

School of Biotechnology, Banaras Hindu University.Varanasi-221 005, U.P, India The mechanism of UVB induced apoptosis and the role of c-Jun N-terminal kinase (JNK) mitogen activated protein kinase (MAPK) was investigated in murine peritoneal macrophages. UVB dose (100 mJ/cm2) that induced apoptosis also caused a sustained activation of JNK and mitochondrial cytochrome-c release leading to caspase-3 activation. Late into the apoptotic process, UVB also induced a caspase-mediated cleavage of Bid. Caspase inhibitors N-Benzylocarbonyl-Val-Asp-fluoromethyl ketone and N-Acetyl-Asp-Glu-Val-Asp-aldehyde suppressed the UVB irradiation induced apoptosis without preventing the release of cytochrome c and JNK activation. The inhibition of JNK MAPK prevented apoptosis, cytochrome-c release and caspase-3 activation but had no effect on caspase-8 activation. These results indicate that activation of JNK MAPK upstream of caspases might play an important role in the apoptotic process of macrophages exposed to UVB irradiation. P115 Adaptive response to alkylating agents in E. coli Janion, J. Nieminuszczy, A. Sikora, and E. Grzesiuk Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Potentially mutagenic and genotoxic alkylating agents are widely spread in nature. They are present in our environment and in the living cells, where they arise during cell metabolism, mainly by nitration of aminoacids. It is widely accepted that the mutagenic effect of the alkylanes results from alkylation of bases in DNA. All organisms possess mechanisms that protect cells against the harmful effects of alkylanes. In E. coli these defence systems include tag-and ogt-genes, expressed constitutively, and ada, alkB, alkA and aidB, expressed after induction of the adaptive response. Both tag- and alk- encoded enzymes are N3meA DNA glycosylases (N3meA DNA glycosylase I, and N3meA DNA glycosylase II, respectively). Both remove N3meA from DNA and leave the abasic site. However, the specificity of DNA N3meA DNA glycosylase II is wider than that of N3meA DNA glycosylase I; besides N3meA, DNA glycosylase II removes also many other alkylated bases. Gene ogt- codes for O6meG DNA methyltransferase (MGT), a suicide enzyme that transfers methyl group from O6meG on its cys321 residue resulting in restoration of the G residue and irreversible loss of methyl transferase activity. Ada (ada-encoded) protein has multiplex properties: (i) 39 kDa Ada protein acts as a sensor of alkylation damage in DNA; (ii) It can accept the methyl group from the Sp-diastereoisomer of DNA methyl phosphotriester to its cys38 residue (or be methylated directly by SN2 methylating agents) and become the transcriptional activator of all genes induced under the adaptive response; (iii) 39kDa protein Ada or its19 kDa C-terminal domain may acts as MGT and rescue G. In fact the 19kDa Ada protein and the product of the ogt gene are functionally and structurally the same; and (iv) It can destroy MMS directly. The role of AlkB (alkB-encoded) protein has been established recently. AlkB is a dioxygenase that oxidatively demethylates N1meA and N3meC in DNA in a reaction involving ketoglutarate, O2, and Fe II, resulting in recovery of the natural A and C bases. AidB possesses the activity of isovarelyl CoA dehydrogenase and it most probably destroys the methylating agents. It is amazing that the system defending cells against the alkylanes involves so many ways for repair of modified bases. Presently, we will show that N1meA and N3meC, when present in DNA, are potent inducers of mutations. In E. coli AB1157 defective in alkB the frequency of argE to Arg+ reversions induced by MMS is 1,000 -fold higher than in the AB1157 alkB+ strain, and the reversions are mainly due to AT - TA transversions. These reversions are totally dependent on umuC encoding DNA polymerase V. In E. coli AB1157, a double mutant in alkB and umuC36, reversions to Arg+ are dramatically reduced. The high mutagenic activity of MMS in response to the defect in alkB seems to indicate that there are more (besides N1meA and N3meC) methylated bases, which serve as a substrate for AlkA protein, and/or that unrepaired methylated bases induce an error-catastrophe.

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Notice that the reaction catalysed by AlkB resembles that involved in the Krebs and fatty acid oxidation cycles. In both cases ketoglutarate is oxidised to succinate with the exception that in the Krebs cycle, coenzyme A becomes attached to succinate and NAD+ serves as an electron acceptor. P116 E.coli BW535, xth nth nfo - a triple mutant for the DNA repair genes chronically induces the SOS response E.Grzesiuk, A. Sikora and C. Janion Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland The SOS response is a bacterial defense system enabling survival of cells whose DNA is damaged and replication is arrested. The SOS system in E. coli is regulated by the LexA-RecA* repressor-activator proteins, respectively and forms the SOS regulon involving an increase in transcription of more than 50 (up to 66) genes that encode proteins which participate in DNA repair, recombination and translesion synthesis across the modified bases. Among mutagenic agents there are many chemical and physical inducers that form lesions able to induce the SOS response, e.g. UV-C, mitomycin C (MC), methylmethane sulfonate (MMS) and many others. Since1985 there is an increasing body of information showing that mutation in certain genes chronically induces the SOS response without any exogenous inducers. These genes include dam, that disturbs the dam-directed mismatch repair system, mutD (dnaQ) causing defective 3'-5' exonuclease (proof-reading) activity of DNA pol III, uvrD causing defects in DNA helicase II required for nucleotide (NER) and mismatch repair priA encoding a protein required for primosome assembly, polA-encoding DNA polymerase I, a main repair polymerase which also takes part in processing of Okazaki fragments; and some other genes involved in metabolism of DNA. All the bacteria showing chronic SOS induction exhibit also mutator activity, suggesting that SOS mutagenic polymerases might have a greater role in "spontaneous" mutagenesis of mutator strains than has so far been supposed. Here we show that E. coli BW535 strain, a triple mutant in xth, nfo, nth and hence severely defective in repair of apurinic sites in DNA chronically induces the SOS system. This was shown here by visualization of filamentous growth of the BW535 strain and by measuring the level of β-galactosidase expressed in the BW535/pSK1002 vs. the AB1157/pSK1002 strain. The plasmid pSK1002 bears an umuC::lacZ fusion in which lacZ is under control of the umuC promoter and regulated under the SOS regulon. Increase in expression of β-galactosidase occurs in BW535 without any external SOS inducer. However, not all the mutators show this property since chronic induction of SOS is not observed in mutT or mutY mutators. MutT and MutY proteins, when active, protect bacteria from mutations induced by 8-oxoG lesions in DNA. This may suggest that accumulation of apurinic sites, but not 8-oxoG residues in DNA, induce the SOS response. P117 Nitric oxide and nitric oxide donating agents as the complex stress response regulators in E.coli S. Vasilieva 1, E. Moschkovskaya 1, and A.Vanin 2 1 Institute of Biochemical Physics RAS, Moscow, Russia, 2 Institute of Chemical Physics RAS, Moscow, Russia Cells adapt to changing and sometimes stressful environments through specific genetic responses. Biologically generated free radical nitric oxide (NO) signals these extracellular and intracellular changes in pro-and eukaryotic systems. We studied complex DNA repair responses as a reaction of E.coli cells to treatment with NO, GSNO and dinitrosyl iron complexes (DNIC) with natural (glu, cys and N-acetyl-DL-penicillamine) or synthetic ( thiosulphate, heterocyclic thiol , etc.) ligands. The last (stable) compounds were synthesized at the Institute of Problems of Chemical Physics, Russia. We have reported at the first time an ability of NO and NO-donors to induce the SOS DNA repair response in E.coli PQ66 (sfiA::lacZ) uvrA. DNICgly and DNICcys were the most potent SOS-inducers. E.coli cells PQ65 (sfiA::lacz) uvrA+, isogenic to PQ66 uvrA, were equally sensitive to cytotoxic and much less sensitive to sfiA- gene expression activity, induced by DNIC thio. It appeared that the UvrABC-dependent NER-excision pathway influenced the process of DNA lesions repair induced by the NO-donating agent. Pretreatment of the cells with the iron-chelating agent o-phenanthroline (OP) prevented induction of the SOS response by all agents used. To elucidate the underlying molecular mechanisms of signal transduction via nitrosylation of Fe-S centers in SoxR protein transcriptional activity we compared the ability of pure NO and the above NO-producing agents to activate the soxRS regulon in E.coli, bearing a soxS::lacZ operon (promoter) fusion Unlike the results in the experiments with sfiA-gene expression, namely DNICthio was the most effective soxS-gene expression at the equal toxic concentrations of NO and NO-donors tested. EPR-spectroscopy of the whole cells has been used to monitor the formation of inducible protein-DNIC-like complexes. A treatment of intact E.coli with NO or GSNO used in equal dose of 150 mcmole showed the appearance of a single EPR-detectable DNIC-like signal with g= 2,03. Pretreatment of the cells with OP leads to a decrease or even disappearance of the DNIC that corresponded with the attenuation of NO and GSNO biological activities. So we proposed that combination of cell non-heme iron with NO or GSNO leading to formation of low molecular DNIC really ensures of NO and GSNO- activation of SOS and SoxRS regulons. Analysis of the molecular mechanisms of NO reactivity in E.coli reveals a dual role of the iron ions: on the one hand they ensure DNIC formation from NO that protects NO from oxidative action of superoxide; on the other hand NO combination with iron provides its ionization to NO + active state.

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Thus the data obtained allow us to hypothesize that not free NO molecules but rather their complexes with non-heme iron reveal really biological activity. At present the source of endogenous NO generation in E.coli remains obscure. It cannot be excluded that not mammalian type of NO- synthase but rather the enzymatic activity similar to plant nitrate reductase or even animal xanthine oxidase functions in E.coli cells as an NO generator. Industrial Genotoxicity and Regulatory Guidelines P118 Measures of cytotoxicity in the Mouse Lymphoma Assay (MLA): Implications for data comparison with in vitro cytogenetic assays M.M. Moore1 and M. Honma2 1 National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR, USA, 2 National Institute of Health Sciences, Division of Genetics and Mutagenesis, Tokyo, Japan. The MLA and in vitro aberration analysis are considered to be acceptable alternatives for in vitro mammalian cell evaluation of potential genotoxicity. The attainment of sufficient cytotoxicity is a key parameter for the proper conduct of these assays. The choice of chemical concentrations is based upon agreed required ranges for cytotoxicity. Unfortunately, these two assays do not use the same cytotoxicity measure. Historically most in vitro mutation assays have used cloning efficiency immediately following treatment to assess cytotoxicity. The mouse lymphoma assay was originally developed using a new concept, the relative total growth (RTG). The RTG was developed to take into account the toxicity that occurs not only during treatment but also during the expression and mutant selection phases of the assay. While other measures (including plating efficiency immediately after treatment) have also been used with the MLA, recent discussions of the MLA Workgroup of the International Workshop for Genotoxicity Testing (IWGT) have resulted in a consensus that the RTG should be the standard cytoxicity measure for the MLA. Cytogenetic assays generally do not include plating efficiency as a measure of cytotoxicity. In recent years, it has become the practice to use the relative cell count approximately 24 hrs after treatment. Over the past several years, there has been a lively debate concerning the appropriate level of required cytoxicity. While the MLA assay requires attaining 10-20% RTG to determine a chemical to be negative, the general consensus for in vitro cytogenetic assays is to require a 50% reduction in day 1 cell count. In order to evaluate the impact of the use of these different measures, we have graphically compared the day 1 relative cell growth and the RTG for a wide variety of chemicals analyzed in the MLA. The relationship between these two measures varies dramatically among the chemicals evaluated. On one extreme, 2-amino-hydroxyadenine- treated cultures showed no toxicity at day 1 (growing approximately 100% as well as the negative control) yet yielding RTG values as low as 1%. t the other extreme, trichloroacetic acid-treated cultures demonstrated day 1 relative growth and RTG values that were approximately the same. For the majority of the chemicals, cultures showing approximately 50% day 1 relative growth had RTG values from 40-10%. This analysis demonstrates that a “properly conducted” MLA and a “properly conducted” in vitro cytogenetic assay can easily involve testing over different concentration ranges. The magnitude of the difference will vary depending upon the chemical under evaluation. It is to be expected, therefore, that there will be situations where the results of the two assays will differ and that difference may be solely due to the concentrations of test chemicals used for the assays. P119 Collaborative Assessment of the Yeast Genotoxicity Screen. P M Collins 1, P A Weale 1, M G Barker 2, P Cahill 2 and R M Walmsley 2. 1 Sequani Limited, Ledbury, Herefordshire, UK; 2 Dept of Biomolecular Sciences, UMIST, Manchester, UK. The Yeast Genotoxicity Screen has been developed to provide a rapid genotoxicity assessment of compounds with interesting pharmacological or chemical properties. The objective of this collaborative study was to establish the reproducibility and reliability of the DNA damage reporter system in an independent laboratory. Yeast is an attractive model system for the detection and evaluation of carcinogens, as being eukaryotic, it is useful for the testing of highly bactericidal compounds, which preclude the use of the regulatory or screening Ames Test. The yeast has been genetically engineered (incorporation of reporter gene RAD54) to produce a fluorescent protein in proportion to the activation of their DNA repair systems. The harder its DNA repair systems work the brighter and stronger the fluorescence. The yeast cells, serial dilution of test article solutions, genotoxic (MMS) and cytotoxic (methanol) controls and diluent (2 % DMSO) were manually mixed in 96-well microplates, then incubated overnight at 25 oC. Cell density and fluorescence measurements provided quantitative measures of cytotoxicity (positive, weak or negative, proportional to proliferation, lowered by toxic analytes) and genotoxicity (positive, weak or negative, proportional to fluorescence, increased by genotoxic analytes). During the course of the study, the plating procedure was improved so increasing the number of test chemicals that could be plated per run, along with an improvement in the data capture program making it more user-friendly and the data easier to interpret.

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The test was performed with rapid end-point determination, with good concordance of results, confirming the lack of inter-laboratory and inter-personnel variation, therefore supporting the use of this screen as a useful and rapid genotoxicity assessment tool. Considering the original plating procedure was the first trial outside of the developer’s laboratory, the results gained were pleasing. The assay can be taught to skilled technicians within a single day, increasing its ease and frequency of use as a screening tool. Twelve test compounds can be set-up using the manual protocol by a single operator in 2/3 hours, which is a marked improvement over the standard regulatory Ames test or screen. P120 Assessment of a screening experience with the Ames II test and future prospects Véronique Gervais , Didier Bijot and Nancy Claude

Drug Safety Assessment, Servier, Orléans-Gidy, France, IRIS, Servier, Courbevoie, France

Most pharmaceutical companies look for miniaturized genotoxicity tests which require a minimum amount of drug candidates for an early selection in the discovery process.

The prerequisites for the choice of a miniaturized genotoxicity test are its consumption of small amounts of compound, its possibility to automate, its rapid achievement of results and its good concordance with other genotoxicity tests.

For these reasons, the Servier Drug Safety Department has selected the Ames II test, a liquid fluctuation version of the Salmonella mutagenicity assay, provided by Xenometrix GmbH.

This test is composed of the TA7000 series of tester strains (TA7001, TA7002, TA7003, TA7004, TA7005, and TA7006), which revert by a specific base substitution in the histidine operon. This mixture of six base-specific Salmonella typhimurium strains (also called “mix”) is used as if it was one single strain. In addition to the “mix”, the frameshift tester strain TA98 is also used. The treatment performed in microtiter plates allows partial automation, and consequently it requires less test substance than the standard Ames test (about 60-fold less).

Three hundred and fifty compounds were tested, including molecules issued from our own chemistry department, known non- or genotoxicants, or molecules producing equivocal results. The concordance, between the results achieved in this Ames II test and those reported in the literature or in the standard Ames test performed in our company, ranged from 79 to 85 %. No false positive results were obtained with known non-mutagenic substances. But false negative results with the “mix” may arise when chemicals revert only specific strains like TA1535 or E. coli WP2 uvrA (pKM101), which have no equivalent in the “mix”.

All these results supported the Ames II test as a reliable screening tool. However, we are still exploring ways to reconcile the Ames II test product consumption required (typically 50 mg) with much lower substance amounts supplied by chemistry without lowering the prediction of the test.

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P121 Polyorgan micronucleus test L.P. Sycheva and A.N.Sysin Research Institute of Human Ecology and Environmental Health, RAMS, Moscow, Russia The necessity of an assessment of the organ- and tissue-specific mutagenic еffect of chemicals for correct identification of mutagens, their hygienic regulation, prediction of carcinogenic properties and identification of target organs is justified. We elected to use the micronucleus test in the cells of several organs (polyorgan micronucleus test- PMT) as most informative and adequate to problems of environmental health . The schedule of experiment for PMT are recommended. А subacute experiment for mutagens assessment are carried for 1-2 weeks under conditions of repeated everyday administration doses 0,1; 0,02 and 0,04 LD50 with 24-h interval and can be combined with toxicological study. The route of exposure is applied same, as to the man. The methods of slides preparation of the separately laying cells of different organs (lung, forestomach, stomach, colon, bladder, kidney and others) after fixation by 10% formalinum and alkaline dissociation (50% KOH for 14-16 h) of tissues for an microscopic counting of micronuclei frequency are used. The cells are stained with acetoorcein and light green. One or two thousand cells with good morphology are assessed for micronuclei per slide. This approach was allowed to received a new data. The spontaneous level of frequency of cells of different organs with micronuclei did not exceed 2‰, for lung cells - 3‰; the frequency of hepatocytes with micronuclei after 2/3 partial hepatectomy varied from 3 to 7‰. The qualitative and quantitative assessment of the mutagenic effect of benzo(a)pyrene, 1,2-dimethylhydrazine (DMG), cyclophosphamide, N-nitrosodiethylamine (NDEA), 1,2-dibromo-3-chloropropane (DBCP), 1,2-dibromopropane (DBP) and 1,1,3-tribromopropane (TBP), benzamide, chrisotile-asbestos, of drinking water with iodine, by-products of a chlorinating of cyclohexene or butanol in the cells of different organs were studied. The most informative parameters of a quantitative evaluation of organ specificity mutagens and identification of target organs were: minimally-reacted doses, doubling doses and quotients of the regression "dose - effect" equations. The dependencies of organ specificity of the mutagens on a species, sex of animals and strain of mice are defined. The specific, sex and strain sensitivity to an investigated mutagens differed quantitatively (on a level of effect), but is not qualitative (on presence of effect). All three possible variants of assessment of species specificity are noted: the mice are more sensitive to DMG, DBCP and TBP than rats; the rats are more sensitive to DBP than mice; the sensitivity of mice and rats to NDEA is approximately identical. The sensitivity of the different sexes to the same mutagen can differ at an animal of a different aspect: the sensitivity of liver and colon cells of male and female mice to DMG was identical, whereas cells of female rats were more sensitive to DMG in comparison with cells from male rats. The spontaneous frequency of micronucleated cells of different mice organs in some strains can differ from 2 up to 12 times. A doubling dose of a mutagen was the most informative and sensitive parameter for comparative assessment of species, sex and strain specificity with the count of effect in different organs. The mutagenic effect of all investigated carcinogens and target organs of carcinogens were revealed. We recommend to carrying of the systemic study of chemicals with the PMT for more correct evaluation of their mutagenic and a prediction of carcinogenic properties. P122 Evaluation from existing data of mutagenic potential of compounds in mainstream cigarette smoke F. Flamma, A. Martino, A. Nunziata and C. Andreoli. Research Department, Eti S.p.A., Rome, Italy. Toxicological studies in the field of tobacco smoke compounds is of great interest for human health protection both for smokers and non smokers. Cigarette smoke contains about 4800 substances, 60 of them have been reported to be tumorigenic to several rodent species and mutagenic in different in vitro systems. The carcinogenic compounds present in cigarette smoke are listed by Hoffmann. Among the most potent carcinogens in this list are polycyclic aromatic hydrocarbons (PAHs) and nitrosamines (NAs). The reduction of toxicity of mainstream cigarette smoke is one of the most important goals to be achieved in order to reduce the risk associated with smoking.Therefore, removing biologically active compounds from cigarette smoke is a logical step towards reducing toxicity. The question then becomes “which compounds have the highest priority for removal?” Because mainstream smoke is a complex mixture, an initial step to reduce the toxicity of mainstream smoke is to develop a rational scheme for ranking the organic compounds in mainstream smoke by their effective toxicity. The present study is focused on estimating the mutagenic potential of some compounds belonging to these chemical classes: four PAHs (benz(a)anthracene, benzo(a)pyrene, dibenz(a,h)anthracene, dibenzo(a,i)pyrene) and four NAs (nitrosodimethylamine, nitrosopyrrolidine, nitrosonornicotine, 4-methylnitrosamino-3-pyridyl-1-butanone), at the concentration present in the cigarette smoke, by extrapolating mutagenic activity in the Ames test from data published in the literature. To this aim, scientific publications, applying Ames test in TA98 and TA100 Salmonella typhimurium strains in presence of microsomal fraction S9, were selected. The papers have been reviewed on the basis of the following criteria: compliance of experimental protocols to the guidelines codified by international organisations (OECD, ICH, etc), publication year, journal kind and research centre.

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As reported in literature, a clear dose-related mutagenic activity was found for all compounds in TA100, while nitrosonornicotine and 4-methylnitrosamino-3-pyridyl-1-butanone did not show any mutagenic effect on TA98 strain. Moreover, the role of metabolic activation in modulating the mutagenic response has been highlighted. In fact, both different animal sources and hepatic inductors (arochlor, phenobarbital etc.) influenced the magnitude of the effect of microsomal fraction on the mutagenic response. However, it is important to point out that the lowest dose employed experimentally (that in some cases did not induce an increase in the number of revertants) was approximately 1000 times higher than the concentration detected in cigarette smoke. These results underline the importance to define both a testing strategy for tobacco products and biologically significant criteria in carrying out new cigarette designs aimed at producing safer cigarettes, with a reduced or removed amount of hazardous compounds. P123 Genotoxicity investigations of a pharmaceutical compound presumed to be acting by an oxidative mechanism W. Muster1, AA. Chételat1, S. Kirchner1, S. Albertini1, G. Speit2, and E. Gocke1 1Pharma Research Nonclinical-Development–Safety PRNS, F. Hoffmann-La Roche Ltd. CH-4070 Basel, Switzerland 2University of Ulm, Department of Human Genetics, D-89069 Ulm, Germany Mutagenicty testing of a pharmaceutical development candidate (Ro 63-1908) revealed weak genotoxicity in the Ames test in strain TA98 (+/-S9) and more prominent effects in the chromosomal aberration test with human lymphocytes (HCA) and the Mouse Lymphoma (ML/TK) test. The in vivo MNT test (initially performed with mice by p.o. treatment, later repeated with i.v bolus and with continuous infusion in rat), as well as an ex vivo UDS assay in rat (iv, bolus), proved to be negative. In order to understand the mechanism of action we tested more than 40 structural analogues in a screen with strain TA98, and about a dozen analogues in the HCA and ML/TK tests. There was a very good concordance of the test results between the ML/TK and HCA test and a clear structure activity relation could be recognised. Clear positive effects were seen at concentrations > 20 and > 40 µg/ml, respectively. The mouse lymphoma colonies were predominantly of small size. In contrast, the Ames test data did not correlate with the clastogenicity data and no structure activity relation was obvious. Altogether the responses in the Ames test were rather marginal (factor 1.5 to 2, at most) at dose levels in the mg/plate area, which hampered an unambiguous evaluation. A para-hydroxy phenoxy (phenylamino) moiety of the molecules appeared to be responsible for the observed in vitro clastogenicity effects. A mechanism based on the generation of reactive oxygen species (ROS) was postulated. To gain supportive evidence, eight structural analogues were investigated in the Comet assay in conjunction with incubation of the slides with bacterial formamidopyrimidine DNA glycosylase (FPG protein) to provide indirect evidence that the treatments lead to a pre-mutagenic oxidative DNA base modification. Ro 63-1908 showed positive results and an amplification of the DNA damage by post-treatment with FPG. Treatment conditions with increasing oxidative protection reduced the effect. DNA migration could be observed already after 5 minutes treatment. The cells had almost completed repair of the induced damages after 3 hrs. Such behaviour has previously been observed after exposure of the cells to H2O2 and the test results are taken as further indication of oxidative damaged DNA bases. In addition, as part of a general validation exercise, stress response assays were performed with Ro 63-1908 covering 13 mammalian and 13 bacterial reporter gene constructs. Contrary to expectation the mammalian cell systems showed no stress gene response while almost all of the bacterial stress genes except rec A gave a clear response. The Ames II assay, also included in the exercise, did not yield any indication of a genotoxic activity. Thus, no helpful information was obtained in this exercise. Based on these genotoxicity investigations a risk/benefit assessment is presented. P124 Optimisation of Comet Assay Methodology for the Stomach and Liver Williams L1, Howe J2, Clements J1

1 Covance Laboratories, Otley Road, Harrogate, HG3 1PY 2 GlaxoSmithKline, Ware, Hertfordshire The Single Cell Gel Electrophoresis Assay (Comet assay) is increasingly being used in genetic toxicology as a second in-vivo test and problem solving tool. One main advantage of this assay is the ability to assess DNA damage in any tissue from which single cells can be obtained. The oral route of administration is commonly used for delivery of substances in to the body. When a substance is delivered via this route the internal wall of the stomach is one of the preliminary sites of contact, and the Comet assay can be used to assess these cells for DNA damage. There are various methods available for obtaining single cell suspensions from the stomach and various rationales for selecting which cells to assess in the Comet assay, which will be discussed. Using histopathological assessment we have identified a suitable method to obtain relevant cells for assessment in the stomach Comet assay. The electrophoresis step of the Comet assay can introduce substantial variability in the Comet results. We carried out experiments with liver and stomach cells from untreated animals to establish the importance of the placement of slides in

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the electrophoresis tank, and also the effects of temperature on Comet results for both tissues. The standard 20 minutes was employed for unwinding and electrophoresis, and the current was set at 0.7V/cm and 300mA. Electrophoresis was carried out at 0, 4 and 7oC. The results from the stomach were more dispersed at 7oC, when compared to the data from the lower temperatures, and this will be discussed. P125 Predicting the carcinogenic potential of chemicals through Structure-Activity Relationships models: definitive results of the second NTP comparative exercise Benigni1 and R. Zito2 1Istituto Superiore di Sanita’ - Lab. TCE -Viale Regina Elena 299 00161 Rome Italy; 2Istituto Regina Elena – Via delle messi d’oro Rome Italy

The environment -in its broadest sense - is the major determinant of cancer; a commonly shared opinion is that the environment is responsible for at least 50–80% of cancers. A component of our environment is chemical. However, only a relatively small percentage of the chemicals currently in commercial use have undergone testing, so Structure-Activity Relationship (SAR) and Quantitative Structure-Activity Relationship (QSAR) approaches are of particular interest. This presentation will discuss the results of second comparative exercises on the prediction of rodent carcinogenicity held under the aegis of the US NTP, where different modeling approaches were challenged to predict the carcinogenicity of a common set of chemicals. The performance of the “general purpose” models of chemical carcinogenicity (the subject of the comparative exercises) will be contrasted with the applications where individual classes of carcinogens have been successfully modeled with the classical QSAR approaches. P126 Disinfection by-products: questions to be answered T. Grummt, H.G. Wunderlich, and R. Heinze Environmental Federal Agency, Bad Elster, Germany Although there are numerous research studies on DBPs, this is a complex area with many unanswered questions. Most health and occurrence information is on THMs. There is a need to evaluate the risks from further priority DBPs. Additional information on the comparative toxicity and relative potency of DBPs is need to assist regulatory process. We started with the development of a strategy for prioritising DBPs for further research and with identifying the data deficiencies. We have selected chemicals that represent important families of DBPs: haloacetonitriles, haloacetic acids, aliphatic aldehydes, and haloacetoaldehydes. Representative members of these families of chemicals were evaluated in the following short-term test systems: cytotoxicity tests (plating efficiency, neutral red assay, inhibition of RNA synthesis), Ames test, Comet assay, chromosomal aberration test, and apoptosis. In addition the chemicals were screened for potential immunotoxicity. We found that only the exposure of dibromoacetic acid and dichloroacetic acid to concentration levels approaching human exposures caused positive effects in the Ames test. We consider the positive findings in the chromosomal aberration test in vitro for all selected representatives of DBPs to be artificially positive findings. In all cases the dose-effect relationships were very steep and the cytotoxicity was also high. The assumption seems to be plausible that, in this case, indirect effects led to the occurrence of chromosomal aberrations. We measured a series of mechanisms that indirectly lead to damage, including apoptosis and inhibition of DNA repair. This finding is important for establishing standards with confidence, if we assume the existence of a threshold for these indirect mechanisms. Dibromoacetic acid was shown to cause significant immune effects. Based on the results of this preliminary study a mechanistic study of dibromoacetic acid is currently in progress. A complicating factor when assessing risk from DBPs is that they occur in complex mixtures that vary by a lot of different parameters. The observed mixture toxicities were in almost all cases stronger than what could be expected from the most potent component alone. The extent of interaction depends mainly on the stability of mixture composition. We anticipate that studies of DBP mixtures will receive increased attention in the future. The project was supported by the Federal Ministry of Education and Research. P127 Comparitive analysis of peripheral blood micronucleus frequencies in methotrexate treated rats using flow cytometry and manual scoring Geoffrey Hynes, Brian Burlinson and Anthony Lynch.

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Genetic Toxicology, GlaxoSmithKline, Park Rd, Ware, Hertfordshire, SG12 0DP, UK. The Micronucleus (Mn) test in rodents is an internationally recognised system for the detection of compounds that are potentially clastogenic and/or aneugenic. Micronucleus frequencies are usually determined manually by microscopy and this is both time consuming and labour intensive. The single-laser flow cytometer method (Dertinger et al 1996) has many advantages over manual scoring, including high speed analysis and improved throughput. As this is usually coupled with the analysis of larger cell numbers (20,000 cf. 2000 with manual scoring) flow cytometry analysis has the potential for improved sensitivity and consistency. Therefore, we have participated in a multi-laboratory validation study of the flow cytometry Mn test. The results of Phase 1 of the collaborative trial were published (Torous et al, 2001) and here we report our contribution to Phase II comparing Mn frequencies obtained in mouse peripheral blood by flow cytometry with manual scoring (using acridine orange staining). Rats were treated with methotrexate (0.5, 1, 2 and 4 mg/kg ip/po/iv) or vehicle daily for 3 days and peripheral blood sampled 24 hours after the final dose to determine Mn freqency. Methotrexate induced a dose dependent increase in reticulocyte Mn frequencies by flow cytometry and by manual scoring and overall, there was good concordance between both methods of analysis. These results support the conclusions of the Collaborative Study Group for the Micronucleus Test (Wakata et al, 1998) that the rat peripheral blood micronucleus test can be integrated into routine toxicology assays which assess the acute toxicity of chemicals.

Ecogenotoxicology P128 Long-term monitoring of chromosomal aberrations in the population of a large industrial region of Western Siberia V. Druzhinin, N. Mokrushina, V. Minina, A. Volkov and T. Golovina Kemerovo State University, Kemerovo, Russia Constant genetic monitoring of the population of the ecologically unsuccessful area of Russia, Kemerovo region at the Laboratory of Genetics of the Kemerovo State University, Russia, has been carried on since 1987. Analysis of chromosomal aberrations (CA), sister chromatid exchanges and the micronucleus assay in cytokinesis-blocked lymphocytes have been used as a biomarker for DNA damage in human populations. One of the important aspects is the investigation of cytogenetic effects in different groups of the population of Kemerovo region brought about local ecological circumstances. This problem is of great importance as Kemerovo region has the status of ecological disaster zone, because of a high concentration of chemical, machine-building, coal-mining and other plants. Most of them have out-of-date technologies and emit a great number of toxic, mutagenic and carcinogenic compounds. In the present paper the results of the CA study in the pheripheral blood lymphocytes of 925 inhabitants of the region (456 male and 469 female) aged 9 - 69 are presented. The proportion of metaphases with aberrations in this sample is 3.73 ± 0.1%. The separate types of aberrations have the following frequencies: single fragments – 2.49 ± 0.08; chromatid exchanges – 0.04 ± 0.01; double fragments – 1.21 ± 0.05; chromosomal exchanges – 0.14 ± 0.02. The modifying influence of the sex, age and the season of inspection were insignificant. Smoking caused an insignificant increase of the level of aberrations of all types in smokers in comparison with nonsmokers (P> 0.05). All donors were divided into 4 groups: 1 - inhabitants of village districts (n=110); 2 - inhabitants of miners' towns (n=157); 3 - inhabitants of cities with a high level of chemical pollution (n=378); 4 - workers of coke-chemical and aluminium plants (n=280). The average frequencies of metaphases with aberrations were the following: 1 – 2.86 ± 0.26%; 2 – 3.79 ± 0.24%; 3 – 3.29 ± 0.28%; 4 – 4.65 ± 0.21%. A method of the mapping the cytogenetic effects for a regional examination of the toxico-genetic conditions of the environment has been proposed. P129 The cyanotoxin cylindrospermopsin induces cell alterations without DNA damage visualized by the alkaline comet assay V. Fessard 1 and C. Bernard 2 1 Agence Française de Sécurité Sanitaire des Aliments, Unité de Toxicologie Alimentaire, BP 90 203, 35 302 Fougères Cedex, France, 2 Muséum National d’Histoire Naturelle, Laboratoire de Cryptogamie, USM 0505, Ecosystèmes et interactions toxiques, 12 rue Buffon, 75 231 Paris Cedex 05, France Eutrophication of brackish and freshwaters is commonly associated with blooms of cyanobacteria, most of them producing toxins. The cyanotoxin cylindrospermopsin has been involved in animal and human intoxications in different countries. Acute toxicity is due to liver necrosis but thymus and kidney are also affected. This toxic effect is caused by inhibition of protein synthesis. However, further research on its chronic toxicity is required. We focused our attention on

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the genotoxic potential of cylindrospermopsin. Induction of DNA damage by this toxin has been reported by some authors describing either a clastogenic or an aneugenic effect. We evaluated DNA damage using the alkaline comet assay coupled to various cell alteration measurements: mortality, growth inhibition, cell blebbing, apoptosis and cytoskeleton modifications. No induction of DNA strand breaks was detected on Chinese Hamster Ovary K1 cells after 24 h treatment with 0.5 and 1 µg mL-1 cylindrospermospin. However, inhibition of cell growth was noticed as well as cell blebbing and rounding. These morphological effects were not linked to apoptosis but attributed to cytoskeletal reorganisation, mainly microfilaments. We concluded that cylindrospermopsin does not obviously react directly with DNA even if the hypothesis that a genotoxic compound could be produced by metabolisation should not be discarded. P130 Biomonitoring environmental genotoxicity and mutagen sensitivity in individuals living/working on the banks of the highly polluted river Musi Vijayashree B. and Ahuja Y.R. Genetics Unit, Bhagawan Mahavir Medical Research Centre, India With increasing environmental pollution there is a growing interest in using biological markers to monitor populations for consequences of excessive low dose exposure to environmental toxicants in a complex mixture form. Musi River that flows through the city of Hyderabad in India has now become a dumping channel for a multitude of industrial and sewage waste. The liquid effluents from the industrial outfalls bring in a complex mixture of hazardous wastes, such as heavy metals and polyaromatic hydrocarbons and many other unknown compounds whose individual doses cannot be quantified. In addition the municipal waste-water, which include sanitary wastes, discharges from hospitals and combustion by-products from surface run-offs which, are being dumped into the river could also potentiate a major biological hazard (potent mutagens and carcinogens). Since Musi riverbank has been inhabited for over 30 years, the possible deleterious outcome from such exposures may range from subtle to irreversible damage to DNA. Hence, the present study was attempted to evaluate the risk for genetic damage and DNA repair deficiency in peripheral lymphocytes collected from exposed and control individuals using comet assay. Mutagen sensitivity assay (in vitro treatment with a known mutagen MNNG) was used as a biomarker of susceptibility, to screen 171 exposed and 202 control individuals for inter-individual variability in their DNA damage and repair deficiency which may become a precursor for increased susceptibility to neoplastic induction (Hus et al 1985). The results indicated a significant increase (p<0.05) in mean basal DNA damage and mutagen sensitivity (DNA repair deficiency) (p<0.05) in the exposed population when compared to controls. The ultimate goal of this study was to use these biomarkers to detect DNA damage and DNA repair deficiency to predict increased risk for development of long-term health effects. This study proves that constant low doses of exposure to complex mixtures of environmental toxicants, which is often overlooked, can cause DNA repair deficiency, which could become a precursor for potential long-term health problems. P131 The micronucleus and the comet assay for in situ monitoring of genotoxicity in freshwater environments M. Pavlica, G.I.V. Klobučar, A. Štambuk, R. Erben and D. Papeš Department of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, Croatia In this study the micronucleus assay and single cell gell electrophoresis assay (comet) were used on haemocytes of swan-mussel, Anodonta cygnea, to monitor in situ genotoxicity of alluvial, continental part of Drava/Danube basin in the eastern Slavonia (Croatia). Caged swan-mussels were exposed for three weeks at two polluted sites in the river Drava. One site was downstream from the main wastewater outlet of the city of Osijek. The other site was upstream of Osijek, in the town of Belišće. The site is receiving waste from chemical and paper-mill industry together with municipal wastewaters. The reference site was in the National Park Kopački rit. The results of micronucelus assay showed a positive response for the sites Osijek (1.3‰) and Belišće (1.1‰) in comparison with reference site (0.6‰). Statistically significant increases in DNA single strand breaks were observed after exposure at both sites in the river Drava (Osijek and Belišće). This study showed that both micronucleus and comet assays used on swan-mussel haemocytes are very valuable biomarkers for in situ biomonitoring.

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P132 Comet assay in genotoxicity evaluation of alachlor in multiple mouse tissues D. Zeljezic, V. Garaj-Vrhovac Institute for Medical Research and Occupational Health, Division for Mutagenesis, Zagreb, Croatia Pesticides of worldwide application are used in agriculture in vast amounts each year, with herbicides being the most prominent class. Alachlor is a chloroacetanilide, developed and introduced in 1967 by the Monsanto Company from the USA. It inhibits protein synthesis in young roots and is used for pre-emergence control of a broad spectrum of grass, sedge, and broadleaf weeds in corn, soybeans, dry beans, cotton, grain sorghum, sunflowers, peanuts and other crops Due to the extensive production and application of this chemical its potential detrimental effects on life should be known and minimized. In this study we applied the comet assay on blood and 4 mouse organs (kidney, liver, bone marrow, blood and spleen) to evaluate possible genome damage caused by pesticide formulations (Bravo) containing alachlor as its active compound. Five male CBA mice were assigned to each of 4 treatment groups and control group. Bravo was injected intraperitoneally once. Bravo was given at the concentrations of 15 mg/kg and 0.01 mg/kg. Mice were sacrificed 24 hours after treatment and the alkaline comet assay performed on blood, kidney, liver, bone marrow and spleen samples. A statistically significant (p < 0.01) increase of tail length for all 5 tissues examined compared to the control was found. DNA of kidney and liver showed largest increase in migration. Also, distribution of tail length values for all mouse tissues examined showed a shift to the right when compared to the controls. In conclusion, by using the alkaline comet assay on different tissue samples, genotoxic effects of pesticide in vivo can be detected and more knowledge on the specificity of its mutagenic action for different organs could be gained. Also, used on different organs in in vivo genotoxicity studies, the comet assay could provide a good assessment of potential pesticide carcinogenicity. P133 Genotoxic studies of okadaic acid, a marine toxin, using the in vitro and in vivo micronucleus test. L Le Hégarat1, V. Fessard1, J. M. Poul1, S. Dragacci2 and P. Sanders1

1AFSSA, Laboratoire d’Etudes et de Recherches sur les Médicaments Vétérinaires et les Désinfectants, Unité de Toxicologie Alimentaire, Fougères, BP 90203, La Haute Marche, 35302 Fougères, France, 2AFSSA, Laboratoire d’Etudes et de Recherches sur l’Hygiène et la Qualité des Aliments, Unité Toxines Microbiennes, 94704 Maisons Alfort cedex, France Okadaic acid (OA) is a phycotoxin produced by dinoflagellates which accumulates in the digestive gland of shellfish and is responsible for Diarrhetic Shellfish Poisoning (DSP) in humans. OA is a selective inhibitor of serine/threonine protein phosphatases and a potent tumor promoter in mouse skin and in rat glandular stomach. We showed that OA is an aneugenic compound after 24 h treatment in CHO-K1 cells using the cytokinesis-block micronucleus (CBMN) test coupled with a FISH technique. We also demonstrated that, after a shorter (4h) treatment with a metabolic activation system, the toxin induced micronuclei formation. In this study we investigated if the metabolite(s) formed have a clastogenic or an aneugenic effect. FISH analysis showed a dose-dependent increase of centromere positive (CEN+) micronuclei, 60 % of CEN+ at 30 nM and 75 % of CEN+ at 50 nM. Considering that the intestine is an organ likely to be in contact with this toxin during an intoxication, we chose to measure the in vivo genotoxic effect by the mouse gut micronucleus test. Preliminary experiments with the dose range of 0.05 -1 mg/kg given by gavage showed no induction of micronuclei. Higher doses are under investigation (1.25 -1.75 mg/kg). P134 Comet assay on gill cells from the blue mussel, Mytilus edulis, exposed in vivo to the genotoxic agents benzo(a)pyrene and sodium dichromate J Rank. Department of Environment, Technology and Social Studies, Roskilde University, Denmark Comet assay on cells from aquatic organisms has proved to be very useful in the assessment of genotoxic agents in the environment, and in vivo laboratory studies can be useful to confirm the findings of DNA damage in wild living organisms exposed to genotoxic pollution. The present in vivo study shows DNA-damage on gill cells from the blue mussel, Mytilus edulis in the alkaline single cell gel electrophoresis assay (comet assay). The mussels were exposed 48 h or 96 h. Four mussels were pooled as one sample in the comet assay. Exposure to sodium dichromate (1µM, 10 µM and 50 µM) showed significant DNA-damage but no clear dose-response relationship and the sensitivity was significant different for three different assays. Exposure to benzo(a)pyrene was carried out at 48 h and 96 h, respectively, and experiments were carried out to see if the DNA-damage could be optimised if the mussels were offered food during the exposure. 48 h exposure to 48 µM, 91 µM, and 166 µM benzo(a)pyrene showed significant increase in tail moments for the two highest concentrations, and highest for the experiment with food addition. When the experiment was repeated using 91 µM, 166 µM and 200 µM benzo(a)pyrene a significant increase in the tail moment was seen for all concentrations, but lowest for the experiment where food were added. The results from 96 h exposure to 91 µM, 166 µM and 200 µM benzo(a)pyrene showed significant increase in the DNA damage in the non feeding as well as in the feeding experiment. No clear dose-response relationship was seen with or without food. The conclusion of the present study was that it is possible to induce

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DNA damage in gill cells from mussels with sodium dichromate and benzo-a-pyrene by using in vivo exposure, and that no important effect from feeding was observed. P135 DNA adducts as a biomarker for exposure of small mammals to diffuse PAH-pollution Hamers12, ATC Bosveld34, FJ van Schooten5, AJ Murk1 1 Wageningen University – Toxicology Group, Wageningen, NL, 2 current address: Vrije Universiteit- Institute for Environmental Studies, Amsterdam, NL, 3 Alterra Green World Research, Wageningen, NL, 4 current address: SETAC-Europe, Brussels, B, 5Maastricht University – Health Risk Analysis and Toxicology, Maastricht, NL Diffuse pollution can be characterized as an omnipresent complex mixture of pollutants that (i) is emitted by numerous sources, (ii) has a heterogeneous composition in time and space, and (iii) contains a substantial fraction with an unknown chemical identity and toxicity. To assess the integrated environmental exposure to diffuse pollution, it has been suggested to measure biomarkers that specifically respond to major categories of diffuse pollutants. Polycyclic aromatic hydrocarbons (PAHs) make up one of such categories. In the present study, the applicability was determined of measuring bulky DNA adducts in small mammals as a biomarker for exposure to complex mixtures containing PAHs. Among other parameters, levels of bulky DNA adducts were determined in tissues from laboratory-bred shrews that were orally exposed to PAHs under laboratory conditions and from feral shrews and mice that were expected to be exposed to PAHs at (contaminated) field sites. Greater white-toothed shrews (Crocidura russula) were exposed in the laboratory to food contaminated with benzo[a]pyrene (BaP; 0-6-60 mg/kg food) or an artificial mixture of PAHs (PAHmix; 0-9-90 mg/kg food, including 0-1-10 mg BaP/kg). DNA adduct levels in lung showed a dose-dependent increase in shrew exposed to BaP, but not to PAHmix. As CYP-activity increased in both BaP- and PAHmix-exposed shrews, good correlation was found between DNA adduct levels and CYP-activity in the shrews exposed to BaP, but not to PAHmix. A possible explanation may be that exposure to the PAHmix leads to a relatively high EROD induction compared to the low BaP-dose, and a subsequent rapid and complete metabolization of BaP. To further study their applicability as a biomarker for PAH-exposure, DNA adduct levels were measured in common shrew (Sorex araneus) and bank vole (Clethrionomys glareolus) collected in a highway-gradient and in floodplains of the Biesbosch estuary, The Netherlands. On average, levels were lower than in control animals of the laboratory study, possibly due to species differences. Despite the low levels of DNA adducts, differences among sites were found within the highway gradient in lung from herbivorous C. glareolus. In contrast with our expectations, the results indicated an “opposite” highway gradient of PAH exposure, i.e. animals had higher DNA adduct levels at the background location (5 km away from the highway) than next to the highway (10 and 200 m away). No such results were found for carnivorous S. araneus, suggesting that herbivorous C. glareolus were mainly exposed via consumption of PAH-containing airborne particulates that were deposited on plant surfaces. No differences among sites were found in lung tissue from animals collected in regularly or in rarely inundated floodplains, suggesting that soil pollution and subsequent uptake by soil dwelling prey species is not an important route of exposure to PAHs for predatory shrews. Given the relatively low levels of DNA adduct, their age-dependent increase, and the probably not very relevant route of exposure through soil pollution, it is concluded that DNA adduct levels are not an appropriate biomarker for exposure of shrews to complex mixtures of PAH-containing soil pollution. DNA adduct levels in herbivorous voles may be an indicative biomarker for deposited airborne PAH pollution, but it should be stressed that levels are low and that differences among sites are small. P136 Development of in vivo assays to detect genotoxic, cytotoxic and behavioural effects of contaminants using spiny starfish, Marthasterias glacialis Sarah Leaney1, Rebecca Brown2 and Awadhesh Jha1 1School of Biological Sciences, University of Plymouth, Drake Circus, Plymouth 2 Brixham Environmental Laboratory, AstraZeneca, Brixham, Devon Echinoderms are known to be sensitive to a wide range of contaminants and they are often one of the first macro-invertebrate species to decline after a pollution incident in the marine environment. However, in contrast to other marine invertebrate species (e.g. molluscs, annelids and crustaceans), the echinoderms have not been extensively studied with respect to contaminant exposure responses. The spiny starfish, Marthasterias glacialis is an asteroid echinoderm with a wide distribution and a dominant role in the marine food chain, consequently its responses to environmental contaminants is of importance for ecosystem hazard and risk assessment. In the present study a suite of ‘biomarkers’ are being developed to evaluate responses at different levels of biological organisation which includes DNA strand break measurements (comet assay), induction of micronuclei, neutral red retention assay to measure cytotoxicity and ‘righting’ behaviour of the individuals. Following characterisation of the coelomocytes, single cell gel electrophoresis or the comet assay has been optimised and validated using a range of concentrations of hydrogen peroxide under in vitro conditions. Furthermore, following

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validation of the assay under in vivo conditions using a reference genotoxin (i.e. methyl methane sulphonate), it is planned to evaluate the toxic potential of a contaminant highly relevant to the marine environment (e.g. Tributyltin oxide, TBTO). The information obtained so far, is valuable in its own right, since these assays have not been attempted on any asteroid echinoderm species previously. The results of this investigation will be presented and discussed during the conference. P137 Evaluation of potential cytotoxic and genotoxic effects of Bisphenol A under in vitro conditions using rainbow trout gonad (RTG-2) cells Zoë J. Lyle, Christine A. King and Awadhesh N. Jha School of Biological Sciences, University of Plymouth, Drake Circus, Plymouth, PL4 8AA Bisphenol A (BPA) is a bicyclic aromatic compound leached from polycarbonate materials during the autoclaving procedure. The majority of domestically produced BPA is as an intermediate in the production of polycarbonate products and epoxy resins. The estrogenicity of this compound has recently been confirmed using in vivo and in vitro systems, however there is a lack of studies investigating its genotoxic potential in aquatic organisms, which might provide useful information on its short and long term consequences to human and ecosystem health. In the current study the genotoxic potential of BPA is being investigated in metabolically competent rainbow trout gonad cells (RTG-2) using the comet assay to determine DNA strand breaks and the micronucleus assay to detect clastogenic and aneugenic potential. Initially, the comet assay was optimised following which the assay was validated using a range of concentrations of hydrogen peroxide (10, 50 and 100 µM) as a reference genotoxin. Various concentrations of cytochalasin B (0.5, 1.5 and 3 µm/ ml) are being optimised in this cell line prior to its use for the micronucleus assay to inhibit cell division, therefore giving rise to binucleate cells. Direct (methyl methane sulphonate) and indirect acting [Benzo(a) pyrene] genotoxins will be used for the validation of micronucleus assay. Cytotoxicity of BPA is also being assessed using the neutral red retention assay, based on the retention of the cationic dye neutral red in the lysosomes of viable cells. There is a potential hazard to the aquatic environment from this compound and this study aims to elucidate the in vitro cytotoxic and genotoxic effects of this compound. P138 Evaluation of the DNA damage caused by the exposure of RTG-2 cells to selected biocides using a Fast Micromethod Assay Sanchez-Fortún S.* Llorente M.T. Garcia,P and Castaño A. *Dpto. Toxicología y Farmacología, Universidad Complutense de Madrid, Spain. CISA-INIA, Valdeolmos, 28130 Madrid, Spain. Aquatic organisms are exposed to a huge variety of genotoxic pollutants from different sources. The requirement for test methods to detect the genotoxic potential which are fast and easy to perform will contribute to the estimation of the genotoxicity for aquatic sentinel species. Thus, the establishment of versatile assays that can be applied as biomarkers of genetic ecotoxicology is needed to contribute to the generation of knowledge on the genotoxic effects for as many species as possible. Although DNA is a universal molecule, and it is assumed that genotoxicity damage is universal too, large differences in sensitivity have been observed among organisms, even between vertebrates. For instance, fish cells have shown to be more sensitive to some genotoxicants and this could be mainly due to the low efficiency of fish DNA repair mechanisms in comparison with mammals. In this work, we have adapted the simple and sensitive Fast Micromethod Assay, usually performed with suspension cells, to be used with anchorage dependent cells (RTG-2 cells derived from rainbow trout). The procedure is based on the ability of commercially available fluorochromes to interact preferentially with dsDNA in the presence of ssDNA, RNA, and proteins at high pH, thus allowing direct measurements of DNA denaturation without sample handling or stepwise DNA separations. The method has been applied to evaluate the genotoxicity of selected biocides employed in the disinfections processes of cooling towers: tetrakis-(hydroxymethyl)-phosphoniumchlorid (THPC), trichlorocyanuric acid (TCIC), monopersulfate compound (OXONE), benzalkonium chloride (BC) and sodium bromide (BrNa). To select assay concentrations the neutral red uptake (NRU) assay has been tested on the different biocides. The results obtained indicate that THPC is the most cytotoxic compund (IC50(48) = 0.017 pmb), followed by BC (IC50(48) = 2.71 ppm), TCIC (IC50(48) = 30.73 ppm), and OXONE (IC50(48) = 101.11 ppm). Sodium bromide, has not cytotoxic effect on RTG-2 cell (IC50(48) > 5000 ppm).

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To obtain the optimum output of the methodology in the detection of massive DNA damage, pH values between 11-12.4 have been assayed. The results indicate that the maximum fluorescent ratio is obtained at pH 11.6. The preliminary results indicate that this method can be used on fish cells as screening assay for estimation of massive DNA damage in the study of genotoxic effects of chemicals. P139 Comparison of RAPD Profiles generated from EPC cell line and common carp, Cyprinus carpio García P 1, Acevedo H 2, Becerril C 2 , Llorente, M.T. and Castaño A 1. 1 Division of Environmental Toxicology, CISA, INIA, E-28130, Valdeolmos, Madrid, Spain. 2 Toxicological Department, I.S. Carlos III, E-28220, Majadahonda, Madrid, Spain. In vitro assays are a commonly used tool in genotoxicity studies to provide faster, cheaper and easier methods to study the mechanistic effects of chemicals on the DNA. The use of fish cells provides a powerful alternative to the use of live fish for genotoxicity assays. Our earlier studies using random amplified polymorphic DNA (RAPD) analysis have shown a strong genetic similarity between rainbow trout (Oncorhynchus mykiss) and the RTG-2 cell line derived from this species. In addition the ability to detect genetic alterations caused in the RTG-2 cell line by genotoxic chemicals makes this in vitro system suitable for genetic ecotoxicological studies. Since common carp (Cyprinus carpio) is an ubiquitous species in Spanish rivers and is able to live in regions much more contaminated by environmental pollutants than Salmonidae species (like rainbow trout) the evaluation of in vitro system originated from this fish specie could be more appropriate for ecogenotoxicological studies. Here we have investigated the degree of similarity between common carp and the epithelioma papulosum cyprini (EPC) cell line. Genomic DNAs from 20 carp individuals collected from Tajo River and EPC cell line were amplified individually by RAPD analysis using six arbitrary primers. PCR products from RAPD were electrophoretically separated on agarose gels and the banding profiles were visualized by ethidium bromide staining. Comparison between all carp individuals showed high analogy in the banding patterns using all six primers, in contrast low analogy is observed when comparing carp and EPC cell line DNA fingerprinting. Because a great genetic similarity of the in vivo and in vitro systems is an essential requisite in predictive genotoxicity assays, our results with the EPC cell line indicate that this transformed cell line is not useful for this purpose. Therefore, there is a need to evaluate other cell lines derived from carp. P140 Genotoxicity of air pollution mixtures in vitro Gábelová1, Z. Valovičová1, E. Horváthová1, D. Slameňová1, B. Binková2, P. Farmer3 1Cancer Research Institute, Bratislava, Slovak Republic; 2Institute of Experimental Medicine of the Academy of Sciences, Prague, Czech Republic, 3University of Leicester, UK Epidemiological studies have consistently demonstrated that environmental air pollution has a negative impact on human health. It is supposed that polycyclic aromatic hydrocarbons (PAHs), which are associated with the respirable particulate matter (PM10, ∅< 10µm), are a major source of the genotoxic and embryotoxic activities of the air pollution mixture. In order to assess the genotoxicity of the complex mixtures of organic compounds associated with the airborne particulate in vitro, human metabolically competent cells (Hep G2) were used as a model. Air pollution mixture (extractable organic matter, EOM) was prepared by dichloromethane extraction of PM10 collected during winter and summer seasons by high volume samplers (HiVol) in three European countries, Czech Republic (Prague), Slovak Republic (Košice) and Bulgaria (Sofia). Concentrations of selected PAHs in each organic extract were determined by HPLC method with fluorimetric detection. For evaluation of biological activities of EOMs in vitro, EOMs were re-dissolved in DMSO. Both DNA breakage and oxidative DNA damage induction due to exposure to EOMs were analysed using the modified single cell gel electrophoresis (Comet) assay. Benzo[a]pyrene (BaP), a known carcinogen, was used as a positive control in this study. Hep G2 cells were exposed to EOM at equivalent concentrations ranging from 5 to 150 �g/ml. In general, two-hour exposure of cells to EOMs resulted in a significant dose-dependent increase of DNA migration (r = 0.86 to 0.97, p<0.001) regardless the season and locality. A 1.5- to 4-fold increase of the level of DNA breaks over the control level was determined. In order to compare the genotoxic potential of individual EOM in dependence on the locality and season, the data were expressed in relative values. It was found that EOMs from winter sampling from Sofia and two localities in Prague (PRG-SM, polluted area and PRG-LB control area) induced relatively higher levels of DNA damage in comparison with EOMs from summer season. No clear differences in genotoxicity of EOM between winter and summer sampling were found in the locality Košice. However, the comparison of the genotoxic activity of the ambient air expressed in terms of EOM �g/m3 revealed statistically significant differences between seasons and less significant differences among localities. No statistically significant increase of oxidative DNA damage due to exposure to EOMs was observed. This study was supported by the EC funded programme ‘Quality of life and management of living resources’ (QLK4-CT-2000-00091).

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P141 Genome-wide identification of expressed sequence tags in Mytilus galloprovincialis P. Venier1, C. De Pittà1, F. Marsano2, A. Pallavicini3, A. Viarengo2 N. Vitulo1 and G. Lanfranchi1 1 Department of Biology, University of Padua, Italy 2Department of Science and Advanced Technology, University of Piemonte Orientale, Italy, 3 Department of Biology, University of Trieste, Italy Marine bivalves of the genus Mytilus are model organisms whose importance is established by their ecological role, marketable production as sea-food, and long-standing use in coastal pollution bio-monitoring. However, limited knowledge is available on mussel genes and their expression in ordinary or stressing conditions. The systematic production and sequencing of the 3’-end cDNA clones (Expressed Sequence Tags or ESTs) allows rapid and large-scale identification of protein coding genes also providing information on the relative abundance of the most common mRNAs. Therefore, we have started the construction of 3’-end cDNA libraries from normal and pollutant-treated mussels in order to investigate genes basically expressed and specifically modulated by the exposure to toxic and genotoxic pollutants. This experimental approach represents the starting point for the identification and functional characterization of specific genes and for genome-wide expression profiling in Mytilus galloprovincialis. RNA purification from multiple tissues of normal adult mussels and massive sequencing of the corresponding 3’-end ESTs allowed us to assemble 829 sequences in 524 clusters (98 virtual consensuses and 426 singletons). Similarity searches performed against the non-redundant NCBI database, identified a small number of mussel transcripts (e.g. actin, twitchin, COIII) and revealed significant similarities with a number of mitochondrial and nuclear housekeeping genes. Marked abundance of mitochondrial mussel transcripts was also evident and, gene functions potentially recruited in stress responses were putatively identified (e.g. myticin A, heat shock proteins, methallothionein). A large fraction (59%) of the consensus sequences obtained from the unstressed mussels showed poor or no similarity with any listed entry and it is indicative of unknown mussel genes. Work is in progress to enlarge the 3’-end EST collection from normal and pollutant-treated mussels and to produce a new tool for studying mussel transcription profiles. At the moment, 5664 ESTs from multiple tissues of normal mussels and 1731 independent sequence clusters have been produced.

Aneuploidy and Chromosome Stability P142 Development of the in vitro micronucleus assay on an intestinal model, the Caco-2 cell line A.G. Jacquin and V. Fessard AFSSA, Laboratoire d’Etudes et de Recherches sur les Médicaments Vétérinaires et les Désinfectants, BP 90203, 35302 Fougères Cedex, France In genotoxicity tests, intestinal cells are not commonly used except with the in vivo micronucleus assay on rodent colon. However, this model is interesting for toxicological studies related to food contaminants as this cell type is certainly more representative for this purpose than lymphocytes or fibroblastic cell lines. We focused this work in developing the in vitro micronucleus assay on the Caco-2 cell line isolated from a human colon carcinoma. Various modifications of the protocol commonly used for cell lines were tested: recovery period, with or without cytochalasin B, Giemsa or fluorescent staining. The protocol which was finally adopted consisted of one day subcultures treated for 4 to 24 hours and followed by a recovery period of 24 hours without cytochalasin B. Cells are further trypsinized, treated with an hypotonic solution before fixation and spreading on a microscope slide. Results with a positive control, methyl methane sulfonate (MMS) in a dose range between 5 and 40 µg/ml showed a maximum of a two fold increase in the micronucleated cell percentage. This model will be further used for testing toxins found in contaminated food like okadaic acid, a phycotoxin involved in the human Diarrhetic Shellfish Poisoning. P143 Deletion, rearrangement, and gene conversion; the genetic consequences of chromosomal double strand breaks in human cells M. Honma1, M. Izumi2, M. Sakuraba1, S. Tadokoro1, H. Sakamoto1, W. Wang1, F. Yatagai2, and M. Hayashi1 1Div. Genet & Mutagen, NIHS, Tokyo, Japan, 2Div. Radio Tech, RIKEN, Saitama, Japan Chromosomal double strand breaks (DSBs) are usually repaired in mammalian cells through either of two pathways: end-joining (EJ) or homologous recombination (HR). To clarify the relative contribution of each pathway and the ensuring genetic changes, we developed a system to trace the fate of DSBs occurring in a single copy gene of the human genome. The lymphoblastoid cell lines TSCE5 and TSCER2 are heterozygous (+/-) or compound heterozygous (-/-) for the thymidine kinase gene (tk) respectively, and we introduced an I-SceI endnuclease site into the gene. EJ for a DSB

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occurring at the I-SceI site results in TK-deficient mutants in TSCE5 cells, while the HR between the alleles produces TK-proficient revertants in TSCER2 cells. We found that DSBs significantly stimulated EJ as well as HR, but EJ contributed to the repair of DSBs 270 times more frequently than HR. Molecular analysis of the tk gene revealed that EJ mainly causes small deletions limited to the tk gene. Seventy percent of the small deletion mutants analyzed showed 100 to 4,000 bp deletions with a 0 to 6-bp homology at the joint. Another 30%, however, were accompanied by complicated DNA rearrangements, presumably the result of sister chromatid fusion. HR, on the other hand, always resulted in non-crossing-over gene conversion without any loss of genetic information. Thus, although HR was important to the maintenance of genomic stability in the face of DSBs, almost all chromosomal DSBs were repaired by EJ in human cells. P144 Two chromosome aberrations types induced by solar UV in Crepis capillaris cells Ranceliene, K. Slekyte, K. Cieminis Institute of Botany, Vilnius, Lithuania Cyclobutyl-pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidones (6-4 PPs) are major and specific UV-induced DNA lesions, producing lethality and mutations in all living organisms, including plants. Both, CPDs and (6-4)PPs, are effectively and also specifically repaired by photoreactivation (PHR). So, all UV-induced biological effects, removed by PHR, must be attributed to pyrimidine dimers. In our fourth year investigations of solar UV action on Crepis capillaris the meristematical cells of root tips were exposed to solar UVB, UV(B+A) and PHR light in special chambers with UV filters and frequency of chromosome aberrations (CA) was determined. Two general conclusions were made: 1.Solar UV(B+A) was permanently more effective in CA induction as UVB. 2. Two types of chromosome lesions exist with regard to photoreactivation: PHR-repairable and PHR-unrepairable. Only about half of CA were removed by PHR. Unrepairable CA-type may be resulted by CPDs involved in DNA replication or by the other types of DNA lesions than pyrimidine dimers. The higher effectivity of UV(B+A) suggests on involvement of ROS (reactive oxygen species) in that effect. For confirmation of that presumption the pretreatment of roots with ascorbic (AA) and salicylic (SA) acids with following UV-irradiation were tested. Antimutagenic action was noticed for SA but the action of SA may be attributed not only to ROS, but also to plant stress-inducted PR-proteins. P145 Changes in chromosome markers in PhIP induced and spontaneous tumours Å. Andreassen, R. Vikse, A. Mikalsen, I-L. Steffensen, A-K. Olsen, N. Duale, H. Hjertholm, G. Brunborg and J. Alexander Norwegian Institute of Public Health, Oslo, NO. The Min mouse, which carries one mutated and one functional Apc allele, develops numerous small intestinal tumours. In previous studies we have shown that the food carcinogen 2-amino-6-phenylimidazo[4,5-b]pyridine (PhIP) strongly increases the number of intestinal tumours by causing truncation mutations or loss of heterozygosity (LOH) in the Apc gene. In this work we have used a Min mouse derived intestinal epithelial cell line (IMCE) that does not grow in soft agar. The cells were exposed to N-OH-PhIP, and soft agar colonies were picked. 18 different cell cultures were obtained. All the cultures were tested for LOH, none of them had lost the Wt Apc allele, but some had an increased amount of the ApcMin allele relative to the Wt Apc. Neither did any of them show a truncated protein in segment 2 and 3 of exon 15 of the Apc gene upon analysis by in vitro protein synthesis (IVSP). Seven of these transformed cell cultures have been transplanted on Balb/c nude mice to investigate tumourigenicity. Five cultures were tumourigenic. None of them have mutations in β-catenin (exon 3) or K-ras (codon 12 and 13). Moreover, none of the cultures were reverted back to the wild-type of SV-40T. Western blot analysis showed that four of the five cultures have reduced expression of β-actin and Raf-1, proteins involved in cytoskeleton formation and chromosome segregation. Does PhIP exposure of cells with a ApcMin background have any influence on chromosome number? To test our in vitro data we performed an in vivo study where C57Bl/6J-Min (B6-Min) mice were crossed with A/J or AKR/J mice. From the F1- hybrid mice small intestinal tumours were sampled. All samples were analyzed for LOH in Apc and for two different microsatelite markers on chromosome 18, D18mit19 and D18mit4. These microsatelites are situated close to the telomers on each end of the chromosome and they were choosen to keep track of the two chromosomes. In the non-LOH group from the B6-Min/AKR hybrid mice there were no changes in relative ratio of both markers between the two alleles after PhIP exposure when comparing normal tissue samples and tumour samples. In the LOH group for both markers the relative ratio increased by 1.5 in PhIP treated and untreated tumour samples compared to normal tissue samples. In the non-LOH group from the B6-Min/A/J hybrid mice there were no changes in relative ratio between the two alleles of both markers when comparing normal tissue samples and both treated and untreated tumour samples. In the LOH group the picture was more complicated. The relative ratio of the D18mit19 marker were increased from two to three in the normal tissue compared to untreated tumour samples respectively, while in the PhIP exposed the relative ratio was equal to the normal samples (two and two, respectively). The relative ratio of the D18mit4 marker was doubled from one in the normal tissue, to two in both PhIP treated and untreated tumour samples. The mechanism involved in tumour induction by PhIP seems to be different from the spontaneous in the B6-Min/A/J hybrid mice, however this in not the case in the B6-Min/AKR hybrid mice. Since all our samples contain both the D18mit19 and the D18mit4 alleles, our

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results indicate that none of the tumour samples have lost the whole chromosome carrying the wild-type Apc allele. However, we cannot exclude that somatic recombination have occurred. Tumours induced both spontaneously and after PhIP treatment, display a relatively higher number of the chromosome carrying the ApcMin allele, compared to normal tissue. The mechanism causing changes in chromosome number could be defect chromosome segregation, which can influence important cellular functions like DNA repair. Preliminary results indicate that DNA repair are reduced in human and mouse cell cultures with a mutated Apc gene when exposed to PhIP or UV-C radiation. P146 Characterisation of chromosomes and chromosomal fragments in human lymphocyte micronuclei by centromeric and telomeric FISH Ghita C.-M. Falck, Hilkka Järventaus, and Hannu Norppa Laboratory of Molecular and Cellular Toxicology, Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Helsinki, Finland The frequency of micronuclei (MN) in peripheral lymphocytes is increasingly used as a biomarker of human genotoxic exposure and effects. MN derive from chromosomes and chromosomal fragments lagging behind in anaphase. The two types of MN can be distinguished from each other by fluorescence in situ hybridization (FISH) using centromeric DNA probes. Centromere-positive (C+) MN should contain entire chromosomes and centromere-negative MN chromosomal fragments. The use of centromeric FISH is expected to increase the sensitivity of detecting exposure-related effects, when the exposure induces only one type of MN, and the difference in MN frequency between the exposed subjects and referents is small. In studies of clastogen exposure, the use of centromeric FISH will practically remove the influence of both age and sex, two major sources of variation, on MN frequencies. Age and sex primarily affect the frequency of C+ MN, due to the age-dependent micronucleation of sex chromosomes and high prevalence of the X chromosome in MN of women. The frequency of C- MN is not markedly modulated by age or sex. The contribution of various types of chromosomal fragments and chromosomes and chromatids to MN is poorly understood. More detailed information on the contents of MN may further improve the specificity and sensitivity of the MN assay. Combined telomeric and centromeric FISH could be used for differentiating MN with terminal and interstitial fragments and possibly also chromatid- and chromosome-type fragments from each other. The number of telomeric labels might also be used to distinguish whole chromosomes with both sister chromatids from single chromatids. We used telomeric and centromeric FISH to characterise 200 MN in cultured (72-h) binucleate lymphocytes of four unexposed, healthy human subjects. Most C- MN (87%) and C+ MN (90%) showed telomeric signals (T+), indicating that they contain, respectively, terminal deletions and entire chromosomes. Interstitial fragments, acentric (C-T-) and centric (C+T-), both appeared to constitute 6% of all MN. The majority (68%) of C-T+ MN had one (rather than two or more) telomeric signal, suggesting higher prevalence of chromatid breaks than chromosome breaks. Most MN with entire chromosomes (63%) contained one or two (rather than more) telomere signals, implying that chromatids are more frequently involved than whole chromosomes with both sister chromatids. This would agree with existing knowledge on chromosome lagging in anaphase. The present study suggests that combined centromeric and telomeric FISH is a practical method to improve the specificity of the MN assay. This approach could particularly have use in studying genotoxic effects in humans, especially in exposure to chemical clastogens which primarily induce chromatid-type breaks. P147 The mouse micronucleus test: past, present and future Annelies Vanhauwaert and Micheline Kirsch-Volders. Laboratory for Cell Genetics, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium. In a previous study the laboratory showed that for oral gavage to model aneugens and a clastogen, the in vivo mouse gut micronucleus test was more sensitive than the in vivo mouse bone marrow micronucleus test, the mandatory in vivo test in mutagenicity screening of new pharmaceuticals and chemicals (Vanhauwaert et al., 2001). Both bone marrow and gut cells prepared from the same animals were analyzed. The reference substances tested were colchicine (COL), carbendazim (CAR), tubulazole (TUB), and griseofulvin (GRI), all known aneugens, and 1,2-dimethylhydrazine (DMH) which is a colon carcinogen with clastogenic activity. Two doses of each compound were tested. COL and TUB induced micronuclei in both gut and bone marrow cells, while DMH, CAR and GRI did so only in gut cells. We proposed an in vivo gut (geno-)toxicity testing model for the detection of relevant clastogens and aneugens. This model includes the TUNEL assay, the micronucleus test (± FISH) and the Comet assay to assess cytotoxicity, chromosome and genome mutations. Methods to perform these tests on columnar epithelial cells of the mouse (and to obtain these cells from the colon) were developed and implemented in the laboratory.

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To allow extrapolation to the human species and to go from hazard assessment to risk assessment (and in the long end risk management and definition of ADI, i.e. acceptable daily intake), the laboratory also developed and ex vivo/in vitro cytokinesis blocked micronucleus test on mouse lymphocytes. A first series of in vivo experiments (exposure and sampling) was performed in 2002, testing the substances cyclophosphamide, nocodazole, ethylmethane sulphonate and bisphenol-a at 3 different doses (+ negative control). Animals (6 per dose group) were sacrificed at 24 and 48 hours after exposure. Analysis of the samples is ongoing. Ref.: Vanhauwaert, A., Ph. Vanparys and M. Kirsch-Volders (2001) The in vivo gut micronucleus test detects clastogens and aneugens given by gavage. Mutagenesis, 16(1), 39-50. Aknowledgements: EU-contract QLK4-CT-2000-00058: IWT-scholarship (Institute for Promotion and Innovation by Science and Technology in Flanders) P148 Clastogenicity of aniline in the rat bone marrow Eryl Jones1, Virginia Fox1, Barry Elliott1 and Ernst Bomhard2 1 Health Assessment, Syngenta CTL, Alderley Park, Macclesfield, Cheshire, SK10 4TJ, UK 2 Institute of Toxicology, Bayer AG, 42096 Wuppertal, Germany

Aniline does not induce chromosome aberrations in the bone marrow of the mouse although it increases the incidence of micronuclei under comparable conditions (Jones and Fox, 2003). As aniline induces spleen tumours in rats but not in mice, we conducted studies to investigate the clastogenicity of aniline in the rat bone marrow. Two studies were performed: a micronucleus test and a metaphase analysis test. Both studies used groups of 7 male PVG rats, which received a single oral dose of aniline HCl at dose levels of 0, 300, 400, or 500 mg/kg bodyweight. For the micronucleus study bone marrow smears were prepared 24 and 48 hours after the dose and stained with acridine orange. In the metaphase test bone marrow was sampled 18 and 30 hours after administration. Cyclophosphamide was used as the positive control in both studies. Clinical signs (e.g. cyanosis) indicated systemic toxicity in the dose range studied. A small but significant and dose-related increase in the incidence of micronuclei was observed at the 24 h sampling only. In the metaphase analysis study a small but statistically significant increase in the incidence of aberrant metaphases was observed at the 18 hour sampling time at the highest dose level. The relevance of this finding to the aetiology of the rat spleen tumours observed in the cancer bioassay will be discussed. P149 Aneugenic effects of trichlorfon on mouse oocytes in vivo F. Pacchierotti, G. Gambuti, M. Piscitelli ENEA, Section of Toxicology and Biomedical Sciences, CR Casaccia, Via Anguillarese 301, 00060 Roma, Italy Trichlorfon is an organophosphate pesticide which was suspected to have played a causal role in a cluster of Down syndrome cases in Hungary (Czeizel et al., Lancet, 341, 539, 1993). Subsequently it was shown to affect meiotic segregation on mouse oocytes in vitro (Yin et al., Chromosoma, 107, 514, 1998). To link the epidemiologic and the in vitro observations and further test the hypothesis of trichlorfon (TCF) being an environmental risk factor of aneuploidy, a study was undertaken to test the aneugenic effects of this chemical on mouse oocytes after in vivo administration. Four-seven week old MF-1 female mice received an intraperitoneal (i.p.) injection of 7.5 I.U. Pregnant Mare Serum (PMS) followed 48 hours later by an i.p. injection of 5 I.U. Human Chorionic Gonadotropin (HCG) to synchronize the estrous cycle and hyperstimulate ovulation. Mice were then assigned to four groups: the first received an i.p. injection of 400 mg/kg TCF dissolved in bidistilled water right after HCG treatment; the second group of mice was similarly treated 4 hours later; the third group was given 600 mg/kg TCF by oral gavage after HCG injection; finally, the fourth group received the same oral dose of the tested chemical 4 hours later. A fifth group of superovulated untreated animals was used as control. All treated animals showed clear symptoms of cholinesterase inhibition (loss of equilibrium, muscolar tremors) starting 20 minutes after TCF admnistration. Twentytwo and 4 % of orally and i.p. treated animals respectively died within 17 hours after treatment. All the other mice were sacrificed 17-19 hours after HCG injection; oocytes were then collected from the oviducts and processed for cytogenetic analysis by a mass harvest technique. So far, 102, 110, 34, 170 and 134 metaphase II oocytes have been analyzed in the four treated and in the control group respectively. Only one of these oocytes, harvested from the group of mice i.p. treated with TCF 4 hours after HCG, was hyperploid; in addition, in the orally late-treated group, 2 polyploid metaphase II oocytes and 1 metaphase I-arrested oocyte were observed and in the i.p. early-treated group 1 metaphase I oocyte was detected. Finally, in all treated groups metaphase II oocytes with extensive premature centromere separation, and even, in some cases, premature telophase II (untriggered by sperm penetration) were observed, with frequencies ranging from 0.6 to 3.7. Such oocytes were never observed in matched control mice and suggest that TCF exposure before the first meiotic division may affect also meiosis II. All these observations qualitatively match those collected by in vitro experiments, albeit with a much lower

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frequency of occurrence, and seem to confirm that indeed trichlorfon may disrupt oocyte meiotic progression and chromosome segregation. P150 The creation of a heterogeneous probe for FISH analysis of chromosomes in Multiple Myeloma H. Ruth Morse1, Julie E. Hayes2 and Ann T Doherty2. 1 Faculty of Applied Sciences, University of the West of England, Frenchay Campus, Coldharbour Lane, Bristol BS16 1QY. 2 Genetic Toxicology Safety Assessment, AstraZeneca Mereside, Alderley Park, Macclesfield, Cheshire. SK10 4TG. Karyotypes in multiple myeloma (MM) are characteristically complex, however several recurring abnormalities have been identified using fluorescence in situ hybridisation (FISH) and flow cytometry [1]. Most notable of these is total or partial deletion of chromosome 13, which is indicative of a poor prognosis [2]. Recent studies have indicated a minimal region of deletion overlap at 13q14, with deletion hotspots at the STS markers, D13S272 (70%) and D13S31 (64%) [3]. In an attempt to correlate chromosomal deletion with changes in gene expression, we have attempted to create fluorescently labelled probes by PCR, for analysis of newly diagnosed MM patients. However, whilst creating a probe for D13S272, we noted inconsistency in the generation of PCR constructs, with concomitant poor labelling during FISH. Further investigation of the D13S272 locus (Genbank accession number Z23374) revealed a microsatellite (CA repeat) polymorphism within the STS locus. DNA from 75 healthy platelet and bone marrow donors was analysed for presence and frequency of the microsatellite repeats, and this revealed the presence of 5 alleles. These alleles were present in the investigated population at the frequencies 0.5, 0.33, 0.11 and 0.06 for alleles 1 to 4 respectively. Allele 5 represented a rare polymorphism, being present in only one individual in the heterozygous state (freq = 0.006). We describe the creation of a heterogeneous probe incorporating all polymorphic types, for use during FISH analysis of the D13S272 locus and demonstrate use of the probe in control and patient lymphocytes and bone marrow. References: Sawyer JR, Waldron JA, Jagannath S, Barlogie B (1995). Cytogenetic findings in 200 patients with multiple myeloma. Cancer Genet Cytogenet 82; 41-49. Tricot G, Barlogie B, Jagannath S et al (1995) Poor prognosis in multiple myeloma is associated only with partial or complete deletions of chromosome 13 or abnormalities involving 11q and not with other karyotypes abnormalities. Blood 86; 4250-4256. Shaughnessy J, Tian E, Sawyer J et al (2000) High incidence of chromosome 13 deletion in multiple myeloma detected by multiprobe interphase FISH. Blood 96; 1505-1511. P151 Preantral follicle culture as new in vitro assay in aneuploidy testing in mammalian oocytes F. Sun1, I. Betzendahl1, R. Cortvrindt2, J. Smitz2, and U. Eichenlaub-Ritter1

1 Institute of Genetechnology/Microbiology, Faculty of Biology, University of Bielefeld, D-33501 Bielefeld, 2 Follicle Biology Unit, Centre for Reproductive Medicine, University Hospital and Medical School, Dutch Speaking Brussels Free University, Laarbeeklaan 101, B-1090 Brussels, Belgium Germ cell mutagens lead to heritable gene mutations, and heritable structural and numerical chromosome aberrations in germ cells. The consequences of germ cell mutations in sub-sequent generations include genetically determined phenotypic alterations, which may cause implantation failure, a reduction in fertility due to embryonic or perinatal death, spontaneous abortion, congenital malformations, and genetic diseases with various degrees of health impairment. The most common genetic disorder in humans, trisomy, is caused predominantly by errors in chromosome segregation during oogenesis, especially at meiosis I. The formation and development of oocytes begins in the mammal already during early embryogenesis. Primary oocytes then become meiotically arrested at dictyate stage, enclosed within a primordial follicle, for long periods. Recruitment and growth of the primordial follicle is initially hormone independent and may start at prepuberty while the later stages from the preantal stage onwards rely on gonadotrophic stimulation. During folliculogenesis oocytes dramatically increase in size (e.g. in humans by 40 fold), and only then acquire full nuclear and cytoplasmic competence to resume and complete maturation, and support early embryogenesis. Maturation from the preantral stage relies on timed and highly orchestrated interactions between oocytes, the follicular compartment and the neuroregulatory axis. During the follicular growth phase paracrine and autocrine factors may exhibit specific sensitivities to environmental chemicals. In vivo aneugen testing in females requires large numbers of experimental animals, is time-consuming and expensive. Obviously, for economic and ethical considerations, there is an urgent need to develop in vitro systems to detect mutagenic insults during female gonadogenesis quantitatively and qualitatively. When fully grown oocytes e.g. from the mouse are removed from the inhibitory influences of follicle cells and follicular fluid they spontaneously resume meiosis

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and mature in vitro to metaphase II. Such oocytes can be exposed to chemicals for dose-response analysis and identification of active metabolite, stage-specific sensitivities, mechanisms and targets of environmental chemicals affecting directly meiotic progression, spindle formation, and fidelity of chromosome segregation. However, an indirect and earlier influence of somatic cells on the oocyte’s developmental programme and its genetic constitution can not be explored in this system. To extend in vitro tests to earlier stages of oogenesis, and see whether follicle cells can protect from aneugenic exposures or rather potentiate the activity of aneugens, for instance by disturbing oocyte acquisition of meiotic competence, a novel in vitro test has been established. Preantral follicles from prepubertal mice are cultured in medium supplemented with gonadotrophin (recombinant follicle stimulating hormone, rFSH) for 12 days under oil. Each second day medium is replenished. During this time oocytes grow in vitro. Oestrogen production increases, and steroidogenic theca cells proliferate. Follicle cells differentiate into cumulus granulosa cells surrounding the oocyte and mural granulosa cells lining the periphery of the follicle such that an antral cavity is formed. Upon hormonal stimulation with rHCG (recombinant human chorionic gonadotrophin) and rEGF (recombinant epidermal growth factor) oocytes resume maturation and develop to metaphase II within 16h. Cumulus cells mucify and ovulation is trigged in vitro. We show that oocytes grown and matured in vitro possess normal spindles and well aligned chromosomes. Cytogenetic analysis reveals that chromosome constitution is normal in the in vitro grown and matured oocytes obtained from preantral follicle culture, which is an essential prerequisite to employ this in vitro model for aneuploidy studies. To further assess the potential of the method, oocytes grown in vitro were exposed to classic aneugens during the resumption of maturation in vitro. The follicle enclosed oocytes appeared to be more sensitive to aneugen-induced meiotic delay or meiotic arrest as well as induction of errors in chromosome segregation as compared to naked oocytes or somatic cells in culture. The initial method has been improved recently to culture without oil layer to omit segregation of lipophilic substances to the oil phase and replenish medium only each forth day. Since large cohorts of homogenous preantral follicles can be obtained from prepubertal mice and the yield of metaphase II oocytes is high, preantral follicle culture appears to represent an effective and reliable novel method for assessing the hazards and risks of exposures of female germ cells to environmental chemicals, and to drugs potentially affecting genomic integrity of oocytes and embryos, and fertility in mammals. Supported by EU (QCRT-2000-00058). P152 Antigenotoxic properties of selenium compounds on potassium dichromate in TK6 lymphoblastoid and whole blood using the micronucleus assay S. Cemeli1, J. Surrallés2, R. Marcos2 and D. Anderson1. 1Department of Biomedical Sciences, University of Bradford, Bradford, UK, 2Grup de Mutagènesi, Departament de Genètica, Universitat Autònoma de Barcelona, Barcelona, Spain. In all living organisms, heavy metals are generally present. They are essential for nutrition, but non-essential metals are found as contaminants as foodstuffs and are ingested daily. Thus in the human diet, there is presence of both essential and toxic metals. Selenium is an environmental metal that occurs ubiquitously and is produced throughout the world for various industrial activities. Selenium has been shown to have preventive effects in clinical and epidemiological studies. It also has anticarcinogenic effects since selenium supplements can inhibit chemically induced tumours. Selenium is not considered by IARC to be carcinogenic for man, but in contrast hexavalent chromium is considered as a known respiratory carcinogen, producing DNA damage through free oxygen radicals. Thus in the present study we examined the effects of three selenium compounds, sodium selenate, sodium selenite and selenious acid on potassium dichromate in the TK6 human lymphoblastoid cell line and lymphocytes from stimulated whole blood using the micronucleus assay (Fenech, 2000. 455:81-95). Depending on the selenium compound used different patterns of response where achieved. The TK6 cell line showed more sensitivity towards chromium than whole blood lymphocytes. These results will be discussed further. P153 The relative sensitivity of human chromosomes to the known aneugen 17-β oestradiol “the search for biomarkers” E.L Quick, E.M Parry and J.M Parry. Centre for Molecular Toxicology, University of Wales Swansea, Singleton Park, Swansea, SA2 8PP Oestradiol is a naturally occurring oestrogen secreted by the ovary; it is biologically significant in the development and characterisation of the female reproductive system and behaviour. At high doses17-β oestradiol has been implicated as an embryotoxin, tetratogen and carcinogen and it is now thought to be involved in the development of post-menopausal breast cancer. In breast cancer it has been shown that there is aneuploidy of specific human chromosomes (Heselmeyer-Haddad 2002. Hoglund 2002). We have investigated the aneugenic activity of 17-β oestradiol in a human metabolically active cell line, AHH-1, using Florescence in situ hybridisation (FISH). In our study we have been investigating the relative sensitivity of individual human chromosomes to the induction of aneuploidy by 17-β oestradiol. These studies aim to identify potential “biomarker” chromosomes as indicators of exposure to aneugenic chemicals. Our

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data suggests that these chromosomes that are most commonly aneugenic in post-menopausal breast cancer also show an elevated sensitivity to aneuploidy induced by 17-β oestradiol. P154 Cytogenetic analysis of the premature aging syndrome, Hutchinson-Gilford progeria Seager, A.L., Corso, C., Faragher, R., Parry, E.M., Parry, J.M. Centre for Molecular Toxicology, University of Wales Swansea, Singleton Park, Swansea, SA2 8PP Hutchinson-Gilford progeria (HGP) is a rare genetic disorder characterised by signs of marked premature aging. The median age of death is13 years and at least 90% of the HGP patients die from heart attacks or congestive heart failure. Cultured cells from HGP patients show premature senescence, both clinically and in cell culture. It has been proposed that the progressive accumulation of senescent cells contributes to the ageing process. In humans the main constitutive mechanism of fibroblast senescence appears to be telomeric attrition (Hayflick limit). In this study we analysed cytogenetically the chromosomal complement of three progeria clones that had been artificially transformed with a retroviral cassette encoding the gene telomerase and corresponding primary cultures, using dual-colour FISH (Fluorescent in situ hybridisation) and three-colour FISH. Chromosome 11 and chromosome 22 aberrations have been consistent between all of the cell lines analysed. The results imply important roles for both chromosome 11 and 22 in the HGP aging phenotype. Other P155 Growth advantage in stationary phase phenomenon in the mixture of Escherichia coli and Salmonella enteric a V. Bacun-Druzina, Z. Cagalj and J. Franekic Colic Faculty of Food Technology and Biotechnology, University of Zagreb, Croatia Natural selection could occur in cultures of Enterobacteriaceae maintained during prolonged stationary phase, namely under carbon starvation stress. The resulting mutants with increased fitness express Growth Advantage in Stationary Phase (GASP) phenotype enabling them to grow and displace the parent as the majority population. The occurrence of mutators among natural isolates of Escherichia coli and Salmonella enterica is up to 10%. The aim of our investigation was to analyse mixed populations of E. coli K12 and S. enterica serovar Typhimurium during the prolonged period of carbon starvation due to the evaluation of the GASP phenomenon. The mixture of one-day-old culture of S. enterica and sixteen-day-old cells of E. coli as minority did not show the GASP phenomenon. The same results were obtained when the mixture consisted of S. enterica as a minority cells. When we introduced isogenic mutants resistant to streptomycin or nalidixic acid the GASP phenomenon was completed. Presumably, this phenomenon is caused by additional stress, such as resistance to a growth inhibitor, or/and by increased range of the mutator cells. P156 Monitoring paraquat and redox cycling processes in S9 liver extract using screen-printed electrochemical sensors. S. Pariagh1, J. P. Hart1*, R. Hurst2 and M. R. O'Donovan3 1 Centre for Research in Analytical, Materials and Sensors Science and the 2 Centre for Research in Biomedicine, Faculty of Applied Sciences, University of the West of England, Bristol, Coldharbour Lane, Bristol BS16 1QY, UK. 3 Safety Assessment, AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK. *Corresponding author. At present, the complex redox cycling processes that occur when a post-mitochondrial rat liver extract (S9) is challenged with potentially toxic substances, e.g. paraquat (PQ2+), are poorly understood mainly due to an inability to monitor closely electron flux in the S9 system. In the present study, electrochemical techniques were used as a novel strategy to understand better these processes. Cyclic voltammetric studies were performed on 0.8 mM PQ2+ and 2 mM NADPH, in PBS pH 7.2, to simultaneously monitor and characterise their electrochemical behaviour at screen-printed carbon sensors [1]. These voltammograms showed that suitable current responses could be obtained for use in studies involving the S9 fraction. The addition of

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isocitrate and NADP+ to S9 was found to stimulate a constant production of cytosolic NADPH over 25 mins. When S9 was exposed to PQ2+, a decline in NADPH oxidation current was observed after 22 mins and was accompanied by a dramatic decrease in the magnitude of the first cathodic peak of PQ2+. This cathodic peak was found to be oxygen sensitive and is considered to result from the catalytic conversion of PQ+• radical back to PQ2+ [2]. It seems plausible that the decline in PQ2+ signal may be due to elevated oxygen consumption by S9, which is consistent with other reports [3, 4]. Simultaneous real time monitoring of these reduction and oxidation processes, and negation of turbidity issues, highlight the advantages of electrochemical sensors over established spectrophotometric methods. Although the mechanisms involved in paraquat toxicity are relatively well understood, electrochemical sensors may be very useful in investigating the role of redox cycling in the toxicity of other compounds. References 1] Hart, J.P., & Wring, S. A., Recent developments in the design and application of screen-printed electrochemical sensors for biomedical, environmental, and industrial analysis. (1997). Trends in Analytical Chemistry 16, 89-103. 2] Farrington J.A., Ebert M, Land E.J. and Fletcher K. (1973). Bipyridylium quaternary salts and related compounds. V. Pulse radiolysis studies of the reaction of paraquat radical with oxygen. Biochimica et Biophysica Acta (BBA)- Bioenergetics 314 (3), 372-381. 3] Mason R.P. and Holtzman J.L. (1975). The role of catalytic superoxide formation in the oxygen inhibition nitroreductase Biochem. Biophys. Res. Comm. 67 (4), 1267-1274. 4] Rossouw D.J., Chase C.C. and Engelbrecht F.M. (1984). The effect of paraquat on the in vitro activity of cytosol, mitochondrial and microsomal enzyme systems. S. Afr. Med. J. 65 (14), 555-563. P157 Frameshift mutations induced by nitroacridines, acridine mustards, and simple intercalators in the lacZ reversion assay in Escherichia coli E. Terry, C. C. Yin, and G. R. Hoffmann Department of Biology, Holy Cross College, Worcester, MA 01610, USA Nitroacridines and acridine mustards form covalent adducts in DNA and thus differ from acridines that are nonreactive intercalating agents. We compared the frameshift mutagenicity of five nitroacridines, two acridine mustards, and three simple intercalators in the lacZ reversion assay in Escherichia coli. The simple intercalators quinacrine, 9-aminoacridine (9AA), and 9-(3’-dimethylaminopropylamino)-acridine (des-nitracrine) induce primarily -G and +G frameshifts in runs of repetitive guanine residues. They weakly induce -2 frameshifts in an alternating CG sequence and -A frameshifts in a run of adenines. The distinctive feature of the mutagenicity of acridine mustards is a predominance of +G frameshifts. The nitroacridine Entozon differs from the mustards and simple intercalators in being a weak inducer of ±1 frameshifts and a potent inducer of -2 frameshifts. To determine whether this pattern extends to other nitroacridines, we compared the induction of +G, -G, and -CG frameshifts by the cancer chemotherapy drug nitracrine (ledakrin; 1-nitro-9-(3’-dimethylaminopropylamino)-acridine), its 2-, 3-, and 4-nitro isomers, and its des-nitro counterpart. The 1-nitro compound is more toxic than the other acridines, thereby complicating direct comparisons of mutagenic potency. Nevertheless, the data show that 1-nitracrine and 3-nitracrine induce -2 frameshifts more strongly than does des-nitracrine, a result consistent with our observations for Entozon. The most potent -2 frameshift mutagen is 1-nitracrine if judged on the basis of the lowest effective concentration, but it is Entozon if calculated as a maximum yield of revertants. The pattern for 2-nitracrine and 4-nitracrine is dominated by -G frameshifts and resembles that of nonreactive acridines, though with some reduction in potency. The mutagenicity of all these compounds can be explained by a slipped mispairing model of frameshift mutagenesis. Variations on the model can encompass a prevalence of -G, +G, or -CG frameshifts. P158 Stress proteins and tumor suppressor p53 in breast cancer Milićević Z1, Bajić V2., Petrović M3., Bogojević D3 1Institute of Nuclear Sciences ‘’Vinča’’, Lab. Mol. Biol Endocrinology 090 Belgrade 2ICN-Galenika , Pasterova 2, Belgrade 3Molecular Biology Laboratory, Institute for Biological Research ‘’Siniša Stanković’’, Serbia and Montenegro The biochemical activity of p53 most closely associated with tumor suppression is its function as a sequence-specific DNA- binding protein and transcription factor that controls the expression of a large panel of gene products implicated in normal growth control, DNA repair, cell cycle arrest, engagement of apoptosis, angiogenesis, redox regulation, metastasis, nitric oxide production and protein degradation. Exposure of cells to conditions of environmental stress including oxidative stress or pathological conditions, such as tissue damage and mutant proteins associated with genetic diseases results in the inducible expression of heat shock proteins. In this study, we characterized p53 and HSP70 complexes and their subcellular localization in the transformed breast cancer cells which contain aberrant p53 conformers. The levels of HSC73 and inducible member HSP72 in cytosalic and nuclear fractions of breast cancer

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tissues were estimated by Western blot assay and quantified by densitometric scanning using a novel anti-HSP 70 monoclonal antibody (clone, B B70). Anti-p53 antibody DO-7 and CM-1 recognize an exposed site present in aberrant conformations of p53 protein. We also used both immunohistochemical techniques (SABC and APAAP), on fresh frozen sections. Under these conditions p53 resolves as a doublet which may represent different charged isoforms of full-length p53. These immunological analyses of p53 revealed the additional presence of a broad 70kDa band, and a 90 kDa band. The interactions of aberrant p53 protein with cellular proteins are likely to be important in understanding the biology of the transformed phenotype. Over expression of HSP70 is associated with abnormal p53 expression. HSP72 was detected immunohistochemically in p53-positive cases. In these cases, high incidence of lymphnodal or distant metastasis was observed, which suggests that expression of both p53 and HSP72 may indicate biological malignancy of the breast cancer. The possible relationship of HSP complexation to p53 as a cellular stress response to abnormal protein is discussed. The association of mutant forms of p53 that transform cells with both cytosolic and nuclear localized chaperones HSP70/HSC70 affords intriguing insights on a possible mechanism in which genetic mutations in p53 that affect its conformation utilize the chaperone machinery to facilitate and stabilize these altered conformational states. P159 Development of methods for the analysis of p53 gene mutations in formalin-fixed liver tumours induced in tamoxifen treated rats Helen L. Pearson, Peter B. Farmer, Karen Brown. Cancer Biomarkers and Prevention Group, The Biocentre, University of Leicester, Leicester. The antioestrogen tamoxifen, which is widely and effectively used in the treatment of breast cancer, causes hepatocarcinomas in rats through a genotoxic mechanism involving the formation of numerous liver DNA adducts. Analysis of mutation spectra in the p53 gene of tumours can reveal characteristic signatures, enabling links to be made with exposure to particular environmental carcinogens, by virtue of correlations between sites of specific DNA damage and mutation hotspots. Characteristic p53 mutations have been found in hepatocarcinomas formed in rats administered tamoxifen for 12 months (Vancutsem et al., Cancer Res. (1994) 3864-3867). These mutations were clustered at two sites in exon 6-7 and exon 8, however, the specific types of mutations detected, A→G and C→T transitions, differ from those observed in transgenic animals and caused by the major tamoxifen DNA adduct in mammalian cells in vitro, which are typically G→T transversions. Consequently, there has been some question as to whether amplification of pseudogenes may be responsible for the high frequency of p53 mutations reported (Yadollahi-Farsani et al. Cancer Res. (2002) 1947-1952). This issue warrants further investigation and clarification in order to elucidate the precise mechanisms involved in tamoxifen carcinogenesis. The aim of this work is therefore to develop methods to examine whether specific patterns of p53 mutations are present in liver tumours induced in rats administered dietary tamoxifen, alone or with phenobarbital promotion, using PCR and single-strand conformation polymorphism analysis. Formalin-fixed liver tumour samples from female Wistar (Han) rats (46) were obtained from a previous study conducted between 1995 and 1998. Since it is often difficult to obtain enough genomic DNA templates of sufficient quality from archived fixed tissues, the protocols for molecular p53 gene analysis on such samples have to be rigorously optimized. We have therefore evaluated DNA extraction methods using a variety of techniques, including the QIAamp DNA mini kit (Qiagen), phenol/chloroform extraction and a DNAce forensic kit (Bioline). Formalin was removed most effectively from the tissue by extensive washing in PBS buffer for 7 days at room temperature. The use of an additional solvent wash with either ethanol or octanol was also investigated but in each case resulted in genomic DNA of a poorer quality. Following tissue digestion with Proteinase K (16h, 65°C) and extraction by one of the three methods, samples were checked for the presence of genomic DNA on a 1% agarose gel. For all samples exons 7 and 8 were then amplified using pairs of intron-based primers to avoid possible co-amplification of pseudogenes, since these lack intronic sequences. PCR was performed using optimized conditions in buffer containing 1.5mM MgCl2, 200 µM each dNTP and 500µM of each primer. Amplification was carried out for 30 cycles with denaturation at 94°C for 1 min, annealing at 58°C for 1 min and elongation at 72°C for 1 min, then the products were assessed on a 4% Metaphor agarose gel. The results demonstrated that DNA of sufficient quality for PCR amplification was obtained most consistently using the Qiagen kit, as evidenced by detection of the appropriately sized PCR product. These samples are currently being analysed by SSCP to identify the presence of mutations and PCR conditions are being optimized for amplification of additional regions of the p53 gene. P160 Metabolic activation of 3-aminobenzanthrone by human liver microsomes and by human recombinant cytochromes P450 Volker M. Arlt1, Marie Stiborova2, Heinz H. Schmeiser3, David H. Phillips1 1 Section of Molecular Carcinogenesis, Institute of Cancer Research, Sutton, UK, 2 Charles University, Prague, The Czech Republic, 3 German Cancer Research Center, Heidelberg, Germany.

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3-Nitrobenzanthrone (3-NBA) is a potent human carcinogen found in diesel exhaust and ambient air pollution. A major metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), was recently demonstrated in urine samples of salt mining workers occupationally exposed to diesel emissions. Determining the capability of humans to metabolise 3-NBA and 3-ABA and understanding which human cytochrome P450 (CYP) enzymes are involved in their activation are important in the assessment of individual susceptibility to these environmental contaminants. We compared the ability of eight human hepatic microsomal samples to catalyse DNA adduct formation by 3-NBA and 3-ABA. Using the 32P-postlabelling method we found that all hepatic microsomes were competent to activate 3-NBA and 3-ABA. Qualitatively similar DNA adduct patterns with multiple adducts were observed, as found recently in vivo in rats. All major DNA adducts were derived from reductive metabolites bound to purine bases; we found no indication for ring oxidation. The role of specific CYPs and NADPH:CYP reductase in the human hepatic microsomal samples in 3-NBA activation was investigated by correlating the P450-linked catalytic activities in each microsomal sample with the level of DNA adducts formed by the same microsomes. On the basis of this analysis, we attributed most of the hepatic microsomal activation of 3-NBA to NADPH:CYP reductase. Microsomal activation of 3-ABA was correlated with CYP1A1 and CYP1A2 enzyme activity. Using recombinant human NADPH:CYP reductase and human recombinant CYP1A1 and CYP1A2 expressed in Chinese hamster V79 cells, we confirmed the participation of these enzymes in the formation of DNA adducts by 3-NBA and 3-ABA, respectively. Moreover, the role of nine individual recombinant human CYPs (1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2D6, 2E1, 3A4) expressed in microsomes of baculovirus transfected insect cells (Supersomes) in the metabolic activation of 3-ABA, catalysing DNA adduct formation, was also examined. Recombinant human CYP1A1 > CYP2A6 > CYP1B1 >> CYP1A2 = CYP2B6 were capable of activating 3-ABA, forming DNA adducts essentially similar to those found with human hepatic microsomes. These results strongly suggest a genotoxic potential of 3-NBA for humans. Because of its ubiquitous presence in diesel exhaust and ambient air pollution, exposure to 3-NBA may represent a health hazard for large sections of the population. Arlt et al. (2001) Int. J. Cancer, 93, 450-454. Arlt et al. (2002) Carcinogenesis, 23, 1937-45. Arlt et al. (2003) Biochem. Biophys. Res. Commun., 300, 107-14. Arlt et al. (2003) Int. J. Cancer, 105, 583-592. Arlt et al. (2003) Cancer Res., 63, in press. P161 Investigation into the biological relevance of high toxicity genotoxins. A molecular analysis Paul Fowler1, James Parry1, Elizabeth Parry1 and Alison Wolfreys2

1Centre for Molecular Genetics and Toxicology, University of Wales Swansea 2Safety and Environmental Assurance Centre, Unilever Plc The measurement of genotoxicity and mutagenicity of new chemicals and compounds relies on the use of a range of in vitro test systems, which includes an assessment of clastogenic activity across a range of concentrations. Where tests give positive clastogenicity results at high levels of cell survival it is reasonable to assume that the test chemical may have a similar effect in vivo, and may contribute to cancer induction, depending on additional factors including cellular metabolism and bioavailability. In cases where the test chemical fails to give a positive result for in vitro clastogenicity at high levels of cell survival, (low toxicity) but does however give a positive result at low levels of cell survival (high toxicity), predictions of in vivo clastogenicity become much more difficult due to compounding factors such as apoptosis and other forms of cell death, which may or may not eliminate mutant cells. It is also difficult to determine whether the cells that die due to apoptosis or other mechanisms, may contribute to the apparent clastogenicity of the chemical. For these reasons it is difficult to assess the relevance to human safety of compounds where the clastogenic markers are not seen until highly toxic levels are reached, this point also occurs mainly at levels that are unlikely to exist in-vivo. Using cDNA arrays, patterns of gene expression have been compared in a cultured cell line that has been exposed to pairs of model mutagenic chemicals with the same mechanism of DNA interaction but where each of the pair manifest their effects at different levels of toxicity. Using this method it is possible to differentiate between gene expression changes that are due to direct genetic damage or those that are a consequence of toxicity, thus aiding the interpretation of the in vitro data. Studies using cDNA arrays have shown that significant changes in expression of a variety of heat shock protein genes are indicative of the chemicals toxicity profile. Chemicals that induce chromosomal damage at both low and high levels of toxicity tend to have several heat shock protein genes down-regulated over a 24-hour time span. Conversely chemicals that damage chromosomes only at high levels of toxicity tend to have up-regulated gene expression in the heat shock protein genes over a similar time course. This project is supported by a BBSRC CASE studentship with Unilever.

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P162 Analysis of mutation induction in cultured cells by the model genotoxin methyl methane sulphonate (MMS) George E. Johnson, Emma L. Quick, James M. Parry and Elizabeth M. Parry Centre for Molecular Toxicology, University of Wales Swansea, Singleton Park, Swansea, SA2 8PP DNA reactive genotoxins are predicted to exhibit a linear relationship between exposure dose and mutagenic response (Henderson et al., 2000). However, only limited data is available on the mechanisms of mutation in the low dose region. Current risk assessment assumes that there is no safe level of DNA damage, however low its abundance with the DNA. However, living organisms can be predicted to tolerate low levels of DNA damage, due to the activity of protective mechanisms such as DNA repair. Other interactions leading to threshold effects in genotoxicity tests include: (a) effects concerning redundant targets; and (b) physiological protective mechanisms such as compound inactivation (Speit et al., 2000). We have been investigating the low dose region of compound exposure using the model genotoxin methyl methane sulphonate (MMS) and the hormone 17-β-oestradiol using the metabolically active cell lines, AHH-1 and MCL-5. In these studies gene mutation activity has been monitored by the measurement of TK and HPRT mutations and clastogenic and aneugenic activity using the micronucleus assay. Current data suggests a possible threshold for genotoxic activity at 1µg/ml for MMS, and estradiol showing mutagenic activity at concentrations of 0.27µg/ml. Henderson, L., Alberini, S., and Aardema, M (2000) Thresholds in genotoxicity responses. Mut. Res. 464: 123-128 Speit, G., Autrup, H., Crebelli, R., Henderson, L., Kirsch-Volders, M., Madle, S., Parry, J.M., Sarrif, A.M. and Virijhof, H (2000) Thresholds in genetic toxicology-concluding remarks. Mut. Res. 464: 149-153 P163 Chemical mixtures tested in vitro and in vivo using the SupF gene B. Meneshian, and J.M Parry. Department of Biological Sciences, University of Wales Swansea, Singleton Park, SA2 8PP, Swansea, UK. Polycyclic aromatic hydrocarbons (PAHs) and Heterocyclic amines (HCAs) are members of a large group of organic compounds widely distributed in the atmosphere, water, diet and cigarettes. Studies of occupational exposure to PAHs have shown an increased incidence of tumours of the lung, skin, bladder and other sites. Other studies have reported that HCA, formed when meat containing food is cooked, induces cancer of the colon, prostate and mammary gland in rats, tumours which are common in the Western world and strongly related to diet. However, air, water, diet and cigarettes are a complex mixture of chemicals. Thus, while most studies have investigated the effect of a single compound, this study evaluates the role of PAH mix and HCA mix on carcinogenesis. Shuttle vector plasmid pSP189, carrying the SupF target gene was treated with the chemical mixtures and replicated in human embryonic adenovirus-transformed Kidney cells (Ad293) and DNA-repair deficient Xeroderma Pigmentosum neoplasma cells (XP14BRneo). After replication in human cells and amplification in E.coli MBM7070 modified strain, the tRNA gene insert contained in the plasmid is sequenced. The mutagenic profiles of these mixtures will be presented.

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Exhibition Stands 1. Covance Covance is one of the world’s largest and most comprehensive drug development services companies. The breadth of our service spans late stage discovery support and nonclinical safety assessment through clinical trials and commercialization. As the world’s largest provider of genetic and molecular toxicology testing services we provide a complete line of assays to support the discovery, development and registration of pharmaceutical, food, medical device, agrochemical, and industrial products. Employing over 100 genetic and molecular toxicology staff and Study Directors averaging nearly 20 years of experience, Covance has conducted over 4000 studies since 1998. Our wide range of standard screening and regulatory studies include assays to evaluate gene mutation, chromosomal aberration, cell transformation, primary DNA damage, DNA binding, cytotoxicity and cell proliferation. We also provide customized programs and regulatory consultancy. TEL: +44 (0)1423 500888 EMAIL: info@covance.com WEBSITE: www.covance.com 2. Gentronix Ltd Gentronix Ltd is an innovative biotechnology company helping to accelerate the pace of drug development in the pharmaceutical industry. Our new product, GreenScreen GC, is a combined genotoxicity / cytotoxicity assay targeted at the pre-clinical drug discovery market. GreenScreen GC is a cell-based eukaryotic assay for simple and rapid screening of lead compounds, designed to precede the standard test battery for genotoxicity. It provides a cost-effective preview of these regulatory tests (e.g. AMES, MLA, MNT). The patented assay system uses a microplate format compatible with standard laboratory automation or a manual set-up mode and allows a higher throughput than existing genotoxicity tests. Consequently, GreenScreen GC helps ease the ADME-Tox bottleneck in drug development by targeting resources more effectively on those NCE’s most likely to make it to the clinic. For further information, please visit our website at http://www.gentronix.co.uk, or contact: Dr Gordon Barker Gentronix Ltd The Fairbairn Building PO Box88 Manchester, M60 1QD UK t: +44 (0)161 200 3127 e: gordon.barker@gentronix.co.uk 3. Safepharm Laboratories SafePharm Laboratories is a world leader in the safety evaluation of chemicals, providing a comprehensive service to more than 1200 companies worldwide, helping them meet the requirements of increasingly complex regulations relating to the safe use of these products. Our broad client base covers all sectors of the chemical industry, including pharmaceuticals, agrochemicals, intermediates, dyestuffs, specialty and performance chemicals. All testing is conducted according to GLP and SafePharm’s reports have been successfully filed with all the leading regulatory agencies around the world. We provide a “turn-key” service for the testing and notification of new and existing substances, as well as bespoke packages for programs such as HPV, ICCA, REACH, POPS and Endocrine Disrupter screening programs, as well as the Biocidal Products Directive. SafePharm is committed to increasing the use of alternative toxicity methods and has recently opened dedicated laboratory facilities for the development and validation of such new techniques. Contact: Chris A. Newcombe (Mr) Marketing Executive, UK SafePharm Laboratories Limited PO Box 45 Derby DE1 2BT UK Tel: +44 (0)1332 792896 Fax: +44 (0)1332 799018

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4. Xenometrix Ames II is a new liquid format version of the traditional Ames Assay and is performed in microplates with two strains, TAMix, Ta98. The traditional Ames assay requires too much chemical and labor to serve as a screening tool for the increasing number of chemicals in drug development, and the increasing demand for early indications of potential carcinogenesis. The new Ames II assay offers a higher speed format (can be fully automated), colorimetry detection and requires less amount of compound. The assay shows good correlation with the traditional AMES test. The Fastube Blood Sampling System is for storage and further purification of RNA. Samples may be stored for 36 hours at RT or for 1 year at minus 20°C before RNA extraction. We offer other systems for high throughput cell harvesting (without centrifugation), plasmid isolation, protein expression analysis, isolation of recombinant proteins systems for endotoxin removal, 5-channel-flowchamber for in vitro microscopy. Contact: Dr.Nicole Weiland Xenometrix by Endotell Gewerbestrasse 25 CH-4123 Allschwil Tel ++41 61 482 14 34 Fax ++41 61 482 20 72 email info@xenometrix.ch 5. Syngenta CTL Central Toxicology Laboratory (CTL) has an established international reputation for its skills and scientific excellence in toxicology. This reputation is built upon wide ranging regulatory and leading edge research studies. CTL offers cost effective and comprehensive toxicology services to the Pharmaceutical, Agrochemical and Industrial Chemical industries based on our scientific expertise, providing our customers with a key competitive advantage. For further information please contact our Business Development Group on the following numbers:- Tel No: +44 (0)1625 514534/514149 Fax No: +44 (0)1625 517314 6. RCC Ltd RCC is: a contract product development and registration company located at the heart of Europe in Switzerland (close to Basle) and Germany (close to Frankfurt). RCC provides: successful partnerships with large and small companies for the testing and evaluation of human and environmental safety of their pharmaceutical crop protection or industrial chemical products. RCC services: design of product development programs with clear development milestones, conduct of toxicological and analytical chemistry studies, preparation and submission of dossiers to support marketing authorisations. RCC expertise: mammalian toxicology and pharmacology, genetic toxicology and cell biology, metabolism & pharmacokinetics, ecotoxicology and environmental fate, regulatory affairs, field trials, transgenic technology and animal breeding. RCC corporate culture: getting it right first time, awareness of the latest regulatory thinking through close links with industry-specific regulatory bodies, progress through focused science. If you would like more information please visit our home page www.rcc.ch 7. Oxford University Press Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide.It is the world's largest university press and is widely recognised as one of the world's premier journal publishers, successfully combining traditional values of quality and service together with innovative use of the latest technologies. Mutagenesis is published on behalf of UKEMS by Oxford University Press. The journal is a bi-monthly, international multi-disciplinary journal that publishes high quality research and review papers. It is designed to bring together research aimed at the identification, characterization and elucidation of the mechanisms of action of physical, chemical and biological agents capable of producing genetic change in living organisms and the study of the consequences of such changes. Mutagenesis is available at substantially reduced subscription rates to members of the Environmental Mutagen Societies. Visit our stand to pick up a sample copy.

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Tel: +44 (0)1865 353384 Fax +44 (0)1865 353835 http://www.oupjournals.org 8. RTC RTC is a CRO offering an ample range of Preclinical Toxicology and Analytical Chemistry. International industrial sectors served: pharmaceuticals, chemicals, biocides, veterinary pharmaceuticals, nutraceuticals, agrochemicals. Services include: Acute, Subchronic, Chronic and Oncogenicity Studies, Reproductive Toxicology, Safety Pharmacology, Specialised Studies for Anti-Cancer and Biotech Products, Quality Control Tests, MRL Studies on Farm Animals, Toxicological Safety and Risk Assessments, Regulatory Assistance. Genetic Toxicology is one of RTC’s traditional disciplines. Our GTX group has been active since the mid 1970s in the performance of all relevant regulatory in vitro and in vivo assays, more recently also the Mouse Lymphoma Assay. GTX screening assays, the in vitro Micronucleus Test, bacterial and mammalian photomutagenicity, Comet assay and transgenic mutation models are performed to meet various research objectives. The expertise and quality of the genetic toxicology group at RTC are completely dedicated to our clients and their need for a scientifically sound, rapid and well communicated service. RTC, Research Toxicology Centre S.p.A. Via Tito Speri, 12 00040 Pomezia (Rome) Italy Contact: Dr. Marion Weber (Business Development Director) Phone: ( +39) 06 91095.216 Fax: ( +39) 06 9105737 E-mail: mkt@rtc.it Web: www.rtc.it 9. Perceptive Instruments Perceptive Instruments develops and markets products for use in genetic toxicology laboratories. These include automatic colony counting systems for the Ames test and mammalian culture assays, and image analysis systems for Unscheduled DNA synthesis and the Comet assay. We will be showing the latest version of our Ames Study Manager which was developed specifically for conducting and reporting Ames test studies according to regulatory guidelines e.g. FDA, EPA, OECD. It is also designed to be compliant with FDA 21 CFR Part 11 Final Rule on Electronic Records & Electronic Signatures. It uses an Oracle relational database either on a local PC or in a client/server environment for complete security of data and audit trails. The Ames Study Manager offers seamless integration with Microsoft Word for report generation. Also on display will be new versions of the Sorcerer image analysis/colony counting system and fully GLP compliant Comet assay III scoring system. Contact: Dr Paul Pover Perceptive Instruments Ltd Blois Meadow Business Centre Steeple Bumpstead Haverhill CB9 7BN UK Tel. +44 (0)1440 730773 Fax. +44 (0)1440 730630 www.perceptive.co.uk 10. Huntingdon Life Sciences Huntingdon Life Sciences (HLS) provides a comprehensive range of pre-clinical safety testing for the Pharmaceutical, Agrochemical, Industrial Chemical and other industries. With testing facilities in the UK and the USA, 1400 staff world-wide and half a century of experience, our expertise in safety testing is unrivalled. For the last 25 years HLS has played an integral role in shaping world-wide Genetic Toxicology regulations through it’s participation in international inter-laboratory validation trials and with its lead scientists on important advisory committees. In addition to our complete regulatory packages, we offer screening assays, mechanistic investigations, and a range of other in vitro and in vivo assays.

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11. Elsevier Elsevier is a world-leading multiple media publisher of superior STM information products and services. Elsevier publishes a wide array of journals and books in the field of mutagenesis. Visit our stand to browse Mutation Research, DNA Repair and other titles. Publishing Editor: Hendrik van Leusen e-mail: h.leusen@elsevier.com www.elsevier.com 12. Microptic Cytogenic Services Microptic Cytogenetic Services offers a unique service in cytogenetics in genetic toxicology, covering a range of aspects - everything except for the laboratory work: Training, Slide analysis, Consultancy, Computer/microscope link with associated software Microptic has been offering slide analysis services since 1985, with currently eight analysts in total for chromosome aberrations and micronuclei. One-to-one and group training courses have been available since 1987, and the computerised system since 1990. Microptic is part of the UK Good Laboratory Practice Compliance Programme and slide analysis, whether carried out at the main premises or with an outworker, can operate as part of a GLP study. Contact: Microptic Cytogenetic Services 2 Langland Close Mumbles Swansea SA3 4LY UK Tel/Fax: +44 (0)1792 366 714 Email: natalie.danford@microptic.com for further information. Website: www.microptic.com 13. BD Biosciences BD Biosciences (www.bdbiosciences.com) is a business segment of BD (Becton, Dickinson and Company) and is built on BD's 100-year foundation of quality, reliability, and commitment to customers and business partners around the world. As one of the largest companies supporting the life sciences today, BD Biosciences offers expertise in molecular biology, cellular biology, and immunology. With four industry leading business units; Clontech (Palo Alto, CA), Discovery Labware (Bedford, MA), Immunocytometry Systems (San Jose, CA), and Pharmingen (San Diego, CA); BD Biosciences can provide integrated, high-value products and services for genomics, proteomics, drug discovery and development, and cell analysis. Its customers include academic and government institutions focused on basic research in the life sciences; biotechnology and pharmaceutical companies engaged in drug discovery and development; and hospitals, reference laboratories, and blood banks that perform patient testing and monitoring for quality control. From genes to proteins to cells, BD Biosciences provides a comprehensive portfolio of reagents, systems, and technical expertise to support the life sciences arena and accelerate the pace of discovery and diagnosis. web: www.bdbiosciences.com general e-mail : bdb@europe.bd.com general tel : 0032 53 720 202 general fax : 0032 53 720 450 14. MD Biotech Inc. MD Biotech will present the AutoComet, automated microscopy system to the attendees at EEMS. The Automated Comet Assay System, AutoComet is a fully-integrated computer-controlled microscopy system to automatically detect and quantify DNA damage in cells induced by various chemicals, drugs and environmental insults. This high-throughput system is optimized for the complete unattended scoring of the sample area by automatic location of single cells and accurate measurement of DNA damage. Automatic comet acquisition and statistical analysis is accomplished in a timely and cost-effective manner. The AutoComet consists of a state-of-the-art microscope, motorized stage, motorized focus, epifluorescence illumination system, high-sensitivity electronic video imaging system, a personal computer containing a software package and image processing algorithms designed to quantify a number of damage parameters within the comet cells. The analysis program allows for user interaction and an immediate comparison and analysis of on screen data with descriptive distribution histograms and statistical summaries. Contact: Bob Kolanko Chief Financial and Operating Officer MD Biotech, Inc. 511 Burroughs Street Morgantown, WV 26505

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USA (304) 598-1101 (304) 598-1183 FAX www.mdbiotechinc.com bobkolanko@mdbiotechinc.com

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