Carcinogenic effects of airborne...

Carcinogenic effects of airborne particlesCarcinogenic effects of airborne particles

RoelRoel SchinsSchins

Particle Research GroupParticle Research Group

InstitutInstitut fürfür umweltmedizinischeumweltmedizinische ForschungForschung (IUF)(IUF)HeinrichHeinrich--HeineHeine University Düsseldorf,University Düsseldorf,

Germany.Germany.

CHILDREN with LEUKAEMIA Scientific Conference 6CHILDREN with LEUKAEMIA Scientific Conference 6--10 September 200410 September 2004

Health implications of particles

AMBIENT EXPOSURE (PM10 / PM2.5)

Acute: asthma attacks, exacerbations of chronic obstructive pulmonary disease (COPD) and cardiovascular disease

Chronic: immunological effects, cancer

Typical exposures: µg/m3Typical exposures: mg/m3

OCCUPATIONAL EXPOSURE

‘Classic’ occupational diseases, e. g. pneumoconiosis, emphysema, chronic bronchitis, pneumonitis, lung cancer (quartz).

Quartz and Quartz and lunglung cancercancer

•• Alveolar type II epithelial cells are target for quartz carcinogAlveolar type II epithelial cells are target for quartz carcinogenesisenesis(Johnson et al., 1987) (Johnson et al., 1987)

•• Epidemiological studies (reviewed by IARC 1997)Epidemiological studies (reviewed by IARC 1997)

GroupGroup TreatmentTreatment Tumour (%)Tumour (%)ControlControl Room airRoom air 33QuartzQuartz 1mg/m1mg/m33 DQ12 6h/day 5days/week 24monthsDQ12 6h/day 5days/week 24months 1919

MuhleMuhle et al. 1989 Am J et al. 1989 Am J IndInd HygHyg

•• Toxicological studies in ratsToxicological studies in rats

Tumour formation in rat inhalation studies is associated with duTumour formation in rat inhalation studies is associated with durability of rability of the material (the material (biopersistencebiopersistence) ) Poorly soluble particles (PSP)Poorly soluble particles (PSP)

Particle Use/Exposure Durability Tumours

Asbestos Insulation, mining, shipyard workers Insoluble +Crystalline silica Quarrying, construction Insoluble +Carbon black Pigments, toner, tires Insoluble +Titanium dioxide (TiO2) Pigments, cosmetics, Sunscreen agents Insoluble +NiO, Ni-subsulfide Exhaust Insoluble +Graphite Aluminium production Insoluble +/-Iron oxides (FexOy) Pigments, paramagnetic diagnostics Insoluble +/-Diesel exhaust Engines, cars Partly soluble +Coal mine dust Mining Partly soluble +Talc Cosmetics, mining Partly soluble +Amorphous silica Cleaning, paints, adsorbents, drugs Readily soluble -Cement (CaCO3) Construction, Building Soluble -

Carcinogenicity studies in rat

Bloodcapillary

Neutrophils

Macrophages

Lung epithelium

Exposure Exposure →→ inflammation inflammation →→ fibrosis fibrosis →→ cancer ?cancer ?

Pathogenesis

Accumulation of inflammatory cellsNeutrophils, macrophages (chronic inflammation)

Increased permeability of epithelium, cell damage

Epithelial hyperthrophy and hyperplasia, fibrosis

Expression of cytokines, chemokines and adhesion molecules e.g. Tumor Necrosis Factor-alpha (TNF), interleukin-1 (IL-1)

Macrophage Inflammatory Protein-2 (MIP-2), IL-8,Intercellular Adhesion Molecule-1 (ICAM-1)

Particles and inflammation

p50 p65 IκBαp

uupIκBαp

uup

p50 p65

p65 p50 Nucleus

Cytosol

Inflammatory genes:IκBα

TNFTNFααILIL--1, IL1, IL--22ILIL--6, IL6, IL--88iNOS, COXiNOS, COX--IIIIGMGM--CSF, GCSF, G--CSFCSFICAMICAM--11MIPMIP--11α, α, MIPMIP--22RANTES, MCPRANTES, MCP--11

Particles

“ Oxidative stess ”

Nuclear factor kappa B (NFκB)A crucial transcription factor in particle-induced inflammation ?

Schins and Donaldson (2000). Schins and Donaldson (2000). InhalInhal ToxicolcToxicolc

NF-κB

-ve +ve Saline DQ12 CC NC

NF-κB

-ve +ve Saline DQ12 CC NC(t=6h)Neutrophil influx by quartz is preceded by

Nuclear Factor kappaB (NFκB) activation in bronchoalveolar lavage macrophages

Quartz treatment causes NFκB activation in macrophages as well as in lung epithelial cells(t=72h)

DQ12PBS

DuffinDuffin et al. (2001) et al. (2001) ToxicolToxicol ApplAppl PharmacolPharmacol;;Albrecht et al. (2004) Am J Albrecht et al. (2004) Am J RespirRespir Cell Mol Cell Mol BiolBiol

Nuclear factor kappa B (NFκB)A crucial transcription factor in particle-induced inflammation ?

Alveolar space

Epithelium

particles

PMNPulmonarycapillary

EndotheliumChemokines Chemokines

CytokinesparticlesAM

Adhesionmolecules

Courtesy: Ad Courtesy: Ad KnaapenKnaapen

Proposed mechanisms for carcinogenicity of poorly soluble particles (PSP)

PSP exposure

Chronic inflammation and release of ROS/RNS

Genotoxicity Cell proliferation

Tumour formation

Schins (2002) Schins (2002) InhalInhal. . ToxicolToxicol..

Percoll gradient

epithelial cellssurfactant

PurificationHarvesting & AnalysisEPI: DNA damage

(comet assay)

Trypsin

DigestionLavage, isolation

BAL: Inflammatory cell counts, markers of inflammation and toxicity

Single Single intratrachealintratracheal instillation (2 mg quartz/rat, 5 animals per group)instillation (2 mg quartz/rat, 5 animals per group)

Determination of inflammation and DNA damage at 3 days after inDetermination of inflammation and DNA damage at 3 days after instillationstillation

In vivoIn vivo genotoxicity of particles (quartz) genotoxicity of particles (quartz)

DQ12DQ12PBSPBS

DQ12 QuartzDQ12 Quartz

2 mg/rat i.t.2 mg/rat i.t.

t = 72ht = 72h

KnaapenKnaapen et al. (2002) Carcinogenesis; Albrecht et al. (2002) Ann. et al. (2002) Carcinogenesis; Albrecht et al. (2002) Ann. OccupOccup. . HygHyg. .

p<0.05

0

10

20

30

40

50

PBS DQ12D

NA

str

and

bre

akag

e (%

cel

ls)

DNA damage in lung epitheliumDNA damage in lung epithelium(comet assay following isolation)(comet assay following isolation)

Recruitment and activation of neutrophilsRecruitment and activation of neutrophils

BAL fluidBAL fluid

Neutrophils (%)Neutrophils (%)

Myeloperoxidase (Myeloperoxidase (mUmU/ml)/ml)

AlkalineAlkaline PhosphatasePhosphatase (U/ml)(U/ml)

ControlControl

3.3 (4.9)3.3 (4.9)

0.027 (0.02)0.027 (0.02)

9.4 (2.5)9.4 (2.5)

DQ12DQ12

47.6 (8.7) **47.6 (8.7) **

99.3 (100.6) *99.3 (100.6) *

12.3 (2.7)12.3 (2.7)

**p<0.001, *p>0.01 vs. PBS**p<0.001, *p>0.01 vs. PBS







ControlControl

3.3 (4.9)3.3 (4.9)

0.027 (0.02)0.027 (0.02)

9.4 (2.5)9.4 (2.5)

DQ12DQ12

47.6 (8.7) **47.6 (8.7) **

99.3 (100.6) *99.3 (100.6) *

12.3 (2.7)12.3 (2.7)

**p<0.001, *p>0.01 vs. PBS**p<0.001, *p>0.01 vs. PBS

*

0

20

40

60

80

100

ctlr 1:1

Str

and

bre

aks

(com

et s

core

)

*

0

20

40

60

80

100

ctlr 1:1

Str

and

bre

aks

(com

et s

core

)

*

0

2

4

6

8

10

12

14

ctrl 1:1

8-O

HdG

per/

106

dG

*

0

2

4

6

8

10

12

14

ctrl 1:1

8-O

HdG

per/

106

dG

Inhibition of DNAInhibition of DNAdamage by antioxidantsdamage by antioxidants(catalase, SOD)(catalase, SOD)

In vitroIn vitro coco--incubationincubation studies: activated neutrophilsstudies: activated neutrophilscause damage to the DNA of alveolar epithelial cellscause damage to the DNA of alveolar epithelial cells

KnaapenKnaapen et al. 1999 Free et al. 1999 Free RadicRadic. Biol. Med.. Biol. Med.KnaapenKnaapen et al. 2002 Mol. et al. 2002 Mol. CelCel. . BiochemBiochem..

Alveolar epithelial cellAlveolar epithelial cellNeutrophilNeutrophil

Proposed mechanisms for carcinogenicity of PSP

PSP exposure

Chronic inflammation and release of ROS/RNS

Genotoxicity Cell proliferation

Tumour formation

?

secondary

primary

Schins (2002) Schins (2002) InhalInhal. . ToxicolToxicol..

Quartz particles elicit oxidative DNA damage inQuartz particles elicit oxidative DNA damage inRLE rat lung epithelial cells RLE rat lung epithelial cells in vitroin vitro

Schins et al. (2002) Schins et al. (2002) MutatMutat. . ResRes

Tail lengthTail length

QuartzQuartz 24.2 24.2 ±± 2.82.8

Quartz + Quartz + MannitolMannitol 19.8 19.8 ±± 1.7 *1.7 *

Quartz + Quartz + DMSODMSO 20.2 20.2 ±± 2.4 *2.4 *

ControlControl 19.0 19.0 ±± 0.90.9

MannitolMannitol 19.1 19.1 ±± 1.31.3

DMSODMSO 19.5 19.5 ±± 2.12.1


QuartzQuartz 24.2 24.2 ±± 2.82.8





DMSODMSO 19.5 19.5 ±± 2.12.1


QuartzQuartz 24.2 24.2 ±± 2.82.8





DMSODMSO 19.5 19.5 ±± 2.12.1


QuartzQuartz 24.2 24.2 ±± 2.82.8





DMSODMSO 19.5 19.5 ±± 2.12.1

DNA strand breaks (comet)DNA strand breaks (comet)

ControlControl

QuartzQuartz

88--OHdGOHdG

Ambient particulate matter (PM)

8 % increase with a 10 µg/m3 change in PM 2.5

EPIDEMIOLOGICAL OBSERVATIONS

Exacerbations of airways disease in COPD and asthma (Pope and Dockery, 1999)

Cardiovascular deaths/hospital admissions (Schwartz and Morris, 1995)

Cancer (Beeson et al., 1998: Pope et al., 2002)

Susceptible groups Elderly, diseased individuals,...CHILDREN (asthma, allergies, cancer ?)

Carcinogenesis of PM ?Carcinogenesis of PM ?

PM causes inflammation

Human volunteer studies (CAPS inhalation, intratracheal instillation of PM)Role for transition metals, ultrafine particles and/or endotoxin(e.g. Ghio and Devlin, AJRCCM 2002; Schaumann et al. AJRCCM 2004)

Role for transition metals, ultrafine particles and/or endotoxin

In vitro studiesActivation of NFκB pathway, enhanced expression of proinflammatory cytokines (e.g. TNF, MIP-2, IL-6, IL-8)(e.g. Becker et al. Toxicol Appl Pharmacol 1996; Frampton et al., Am J Physiol 1999)

Inhalation / intratracheal instillation studies in ratsEnhanced cytokine/chemokine expression, neutrophil influx(e.g. Shukla et al., Am J Respir Cell Mol Biol 2000; Schins et al., Toxicol Appl Pharmacol 2004)

Ambient exposure to PM and cancer?

Exposure to ambient PM

CarcinogenicityInflammation

Toxicological evidence(animal experiments& volunteer studies)

Epidemiology observations

Experimental observations with PSP

?

Important research questions:

Investigation of chronic effects of PM (inflammation, tumorigenesis?)

Experiments with model PM to determine the role of constituents and/or characteristics of PM that drive inflammation and genotoxicity

Secondary particles (atmospheric chemistry: SO2, NOx, ammonia, sunlight)

Ammonium sulphates, nitrates, chlorides

Biologically-derived material

Endotoxin

Fungal spores, plant parts, bacteria, etc.

Crustal minerals (e.g. wind blown)

mineral rich: aluminium silicate clays, soil particles

Primary particles (predominantly traffic/combustion)

ultrafine particles i.e. <100nm (poorly soluble)

organics e.g. polycyclic aromatic hydrocarbons (PAH)

transition metals (Fe, Ni, Cu, V, Zn, Cr, ..) 200 nm

Primary genotoxicity?PAH, nitro-PAH,

metals, ketones,

carboxylic acids,

aldehydes,

peroxides....

PM and DNA damagePM and DNA damage

Oxidative DNA damage by PMOxidative DNA damage by PM

--rr--ll

--ss

PMPM PMPMsupsup PM PM supsup+ + MannitolMannitol

ControlControl

KnaapenKnaapen et al. (2000) et al. (2000) InhalInhal. . ToxicolToxicol; ; KnaapenKnaapen et al. (2002) Mol. et al. (2002) Mol. CelCel. . BiochemBiochem..

Damage to ‘naked’ DNA(8-HOdG immunodotblot)

--

--

TSP

d.f.

H2O2H2O2

--

++

++

--

++

++PM

Damage to ‘naked’ DNA(plasmid unwinding)

Fenton reaction:Fe2+ + H2O2 + H+ Fe3+ + H2O + •OH

OHOH..HH22OO22

DMPODMPO DMPO-OHDMPO-OH5, 5-5, 5- dimethyldimethyl-1--1- pyrrolinepyrroline-N-oxide-N-oxide

PM

H2O2(125mM)

H2O2(25mM)

H2O2(5mM)

without H2O2

15 G

H2O2(125mM)

H2O2(25mM)

H2O2(5mM)

without H2O2

15 GShi et al. (2003) J. Exp. Monitor.Shi et al. (2003) J. Exp. Monitor.

Reactive oxygen species (ROS) generation by PMReactive oxygen species (ROS) generation by PM

#######

**

0

1

2

3

control PM PM DMPO

PM DMSO

DMPO DMSOta

il m

omen

t .

DNA strand breakage in epithelial cells(A549, 3h, 100µg/cm2)

Oxidative DNA damage by PMOxidative DNA damage by PM

Uptake of PM by epithelial cells

cells treated with PM2.5cells treated with PM2.5

88--OHdG OHdG immunocytochemistryimmunocytochemistry

020406080

100120

0 50 100 150 200 250

Deferoxamine (µM)

DM

PO-O

H si

gnal

(% c

ontr

ol)

PM

PM + Def

Hydroxyl-radical generationby PM (EPR)

0

100

200

300

DFO PM PM+ DFO

% o

f con

trol

*

#

8-OHdG

KnaapenKnaapen et al. (2000) et al. (2000) InhalInhal. . ToxicolToxicol.; Shi et al. (2002) .; Shi et al. (2002) OccupOccup. . EnivironEniviron. Med.. Med.

untreated cellsuntreated cells

Role of PAH in genotoxicity of PM ?Role of PAH in genotoxicity of PM ?

Mutagenicity of airborne PAH has been clearly established

Effects of PM filter extracts (organic solvents) in Ames test, SCE test, etc

Extracts cause bulky DNA adducts in vitro and in vivo

Particles are carriers of PAH into the lungs

Availability of PAH from PM, in vitro, in vivo ?

N

O

N

N

HN

OH

O

H

OH

OH

OH

NH

OH

N

O

N

N

HN

OH

O

H

OH

OH

OH

NH

OH

OH

OH

NH

OH

Bioavailability of PAH from carbon black particles- Adduct formation in A549 human lung epithelial cells -

0 10 20 30 40 50 60 70 80

CB (extracted) - coatedwith EPA-mix

EPA mixture

Extract of CB

CB - extracted

CB -original

Blank (DMSO)

Control

adducts / 10e8 n.t.

spotDRZ

PM sampling using high volume cascade impactor (HVCI) in Amsterdam, Athens, Barcelona, Duisburg, Helsinki & Prague

Coarse PM, fine PM, ultrafine particles.

Chemical characterisation e.g. metals, PAH, endotoxin

Inflammation & genotoxicity: in vitro and in vivo

- in vitro (macrophages, lung epithelial cells)

- in vivo (SHR rats, whole lung, isolated epithelial cells)

PAMCHAR SIX CITIES STUDYPAMCHAR SIX CITIES STUDY

PAHPAH--DNA adduct formation by PM inDNA adduct formation by PM inlung epithelial cells lung epithelial cells in vitroin vitro

((3232PP--postlabelling)postlabelling)

Total DNA adduct level

1

10

100

Prague Duisburg Helsinki Amsterdam Barcelona Athens

Add

ucts

/108

nuc

leot

ides

Fine PM

Coarse PM

DuffinDuffin et al. in preparationet al. in preparation

DNA damage in lung epithelial cellsDNA damage in lung epithelial cellsisolated from rats exposed to PMisolated from rats exposed to PM

*

0

1

2

3

4

5

water Duisburg(fine)

Prague (fine) Prague(coarse)

BaP

tail

mom

ent

Particles also induced inflammation: Secondary genotoxicity?


PAHPAH--DNA adduct formation DNA adduct formation in lung epithelial cellsin lung epithelial cellsisolated from rats exposed to PMisolated from rats exposed to PM

*

**

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

water Duisburg(fine)

Prague(fine)

Prague(coarse)

BaP BaP (lung)

Add

ucts

/108

nuc

leot

ides


ROS/RNS

ROS

Target cell

Neutrophil recruitment& activation

Fe++/Fe+++

BPDEB[a]P

ONOO-

Cyp/EH

Fe++

•OH

8-OHdG

BPDE-DNAModel PM

MUTAGENESIS

?

NFκB, TNF MIP-2, NO

Proposed mechanisms of genotoxicity of PMProposed mechanisms of genotoxicity of PM(studies with model PM)(studies with model PM)

PM and childhood cancersPM and childhood cancers

Carcinogenesis beyond the respiratory tract?Carcinogenesis beyond the respiratory tract?

Induction of systemic oxidative stressInduction of systemic oxidative stressand genotoxicityand genotoxicity

2. Toxicological studies (animal models):

- Particles (inhalation, instillation) elicit genotoxic/mutagenic effects outside the lungs: Endpoints include DNA strand breaks and oxidative damage, bulky DNA adducts, chromosomal aberrations, sister chromatid exchanges).

- Various tissues/organs involved such as peripheral blood/bone marrow cells, liver, colons

1. Biomarker studies:

- Enhanced oxidative DNA damage (8-OHdG) in peripheral blood leukocytes, and blood antioxidant status of individuals with (chronic) occupational dust exposure, e.g. coal miners (e.g. Schins et al., Int Arch Occup Env Hyg 1995; Schins et al., Biomarkers 1999)

- Correlation between personal PM2.5 exposure and oxidative DNA damage (e.g. comet) in peripheral blood leukocytes (e.g. Sorensen et al., 2003)

Systemic effects of ultrafine PM:Systemic effects of ultrafine PM:-- Translocation of particles Translocation of particles --

Inhaled/instilled particles translocate from the lungs into the blood implicated in the observed association between PM exposure and cardiovascular effects:

(e.g. Nemmar et al. 2001; 2003; Kreyling et al., 2002)

Carbon particles (C14, ultrafine) are found in the brain of rats following inhalatoryexposure translocation of particles along the olfactory nerves has been proposed

(Oberdorster et al., Inhal Toxicol 2004)

% of Dose

0

1

2

3

4

0 15 30 45 60 750

1

2

3

4

0 15 30 45 60 75

time (min)

Ultrafine albumine particles (80 nm) labelled with 99mTc.

0.5 mg (intratracheal) /Hamster

Nemmar et al. AJRCM 164: 1665-1668 (2001)

Germline mutation rates at expanded-simple-tandem repeat

(ESTR) DNA loci in mouse pedigrees in association with

urban/industrial air pollution (TSP = 40 – 115µg/m3; PAH = 2– 30µg/m3)

Reduction following particulate-air filtration (> 0.1µm)

Science 2004, 304:1008-1010 (14 May)

Particulate air pollution can cause genetic damage in

germ cells & transgenerational effects

Germline mutagenesisGermline mutagenesis

How may particles impact on carcinogenesisHow may particles impact on carcinogenesisin various organs other than lungs?in various organs other than lungs?

PM (ultrafine component) can translocate from the airways into the blood and thus reach various target cells/tissues. Possible implications:

Direct genotoxic effects of particles (surface reactivity, ROS production)

Induction of inflammation, apoptotic and/or (compensatory) proliferation

Carrier effect: Systemic delivery of mutagens/carcinogens (e.g. PAH)

Particles activate signalling pathways (inflammation/proliferation) within the lung

Systemic release of mediators (e.g. cytokines, growth factors, ROS) may impact on processes involved in carcinogenesis (e.g. apoptosis, proliferation)

University Maastricht,The NetherlandsRoger Godschalk

Ad KnaapenFrederik-Jan van Schooten

Edinburgh University, UKKen DonaldsonRobert Mroz

German Research Foundation (DFG) / German Collaborative Research Centre SFB503

DFG International Research Training Group "Molecular Mechanisms of Food Toxicology“ IGK738

PAMCHAR - EU (QKL4-CT-2001-00423)

IUF, DüsseldorfCatrin AlbrechtRodger Duffin

Doris HöhrHui Li

Klaus UnfriedTingming Shi

Erich JermannChristel Weishaupt

Astrid Winzer

National Institute of Public Health and the Environment

(RIVM), The NetherlandsFlemming Cassee

Miriam Gerlofs-Nijland

Hogeschool Zuyd - Heerlen, The Netherlands

Paul Borm

National Public Health Institute (KTL), Finland

Raimo Salonen

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