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longated spindle-like morphology, as well as apoptotic and earlypoptotic cells were seen. EnnB reduced cellular ability to capturend internalize receptors as illustrated by the lipid marker ganglio-ide GM1. Furthermore, transmission electron microscopic (TEM)ictures revealed accumulation of lipids inside of the lysosomesorming lamellar structures/myelin bodies, suggesting inducedysosomal damage. Enhanced levels of activated caspase-1 werebserved after EnnB exposure, and pre-treatment with the caspase-specific inhibitor, ZYVAD-FMK reduced EnnB-induced apoptosis.nnB increased the release of interleukin-1beta (IL-1�) in cellsrimed with lipopolysaccharide (LPS). ZYVAD-FMK, as well as theathepsin B inhibitor CA-074Me reduced the IL-� release.

In conclusion, we suggest that EnnB may activate the inflam-asome through lysosomal damage, leakage of cathepsin B and

leavage of caspase-1, resulting in apoptosis as well as processingnd release of IL-1�.

oi:10.1016/j.toxlet.2012.03.489

20-09dentification of novel biomarkers of mercury-inducedmmune dysregulation

ennifer Nyland 1, Jonathan Motts 1, Renee Gardner 2, Ellenilbergeld 3

University of South Carolina, United States, 2 Karolinska Institute,weden, 3 Johns Hopkins University, United States

Purpose: Mercury (Hg) is an ubiquitous environmental con-aminant, causing both neurotoxicity and immunotoxicity. Theoal of this project was to identify novel serum biomarkers ofg-induced immune dysregulation in humans. Methods: An anal-sis of serum samples from an epidemiological study on minersorking in Amazonian Brazil was completed on stratified sam-les (based on Hg level and antinuclear autoantibody titer) usingprotein microarray to probe for the induction of elevated auto-

ntibodies. These auto-antibodies were then analyzed in termsf immune system pathways. Pathways examined include thosenvolved in antigen presentation, oxidative stress, and macrophageignaling/activation. Results and conclusions: We found statisti-ally significant correlations between high levels of Hg exposurend positivity for certain auto-antibodies. Auto-antibodies iden-ified as potential novel biomarkers include antibodies to theollowing proteins: interferon induced transmembrane proteinIFITM-1), heat shock transcription factor (HSF-1), colony stim-lating factor (CSF-1), ghrelin/obestatin prepropeptide (GHRL),etallopeptidase inhibitor (TIMP-1), peroxiredoxin (PRDX-2), glu-

athione S-transferase alpha (GSTA-1), and chaperonin (HSPD-1).hese proteins play a wide variety of roles, including as antioxi-ants, in the regulation of pro- and anti-inflammatory cytokines, asell as danger and oxidative stress signaling. Dysregulation of theseroteins is believed to play a role in autoimmune diseases such asheumatoid arthritis, Sjögren’s syndrome, and multiple sclerosis.

ur next step is to validate these findings by probing the entire

ample cohort for titers of these auto-antibodies.

oi:10.1016/j.toxlet.2012.03.490

211S (2012) S43–S216

P20-10Increased liver somatic index and plasma Apo A-I levels in ratsexposed to Bisphenol A

Helen Karlsson 1, Monika Rönn 2, Stefan Ljunggren 3, Lars Lind 4,P.M. Lind 2

1 County Council Ostergotland, Sweden, 2 Occupational andEnvironmental Medicine, Sweden, 3 Center of Occupational andEnvironmental, Sweden, 4 Department of Medical Sciences, Uppsala,Sweden

Increased knowledge about harmful effects of environmentalcontaminants such as endocrine disruptors and elucidation of theirmechanisms of action in biological systems are of significant impor-tance for optimal preventive guidance regarding human health.

In this study female rats were fed with addition of fructose(5%) and different doses of Bisphenol A (fructose 5% + 0.025, 0.25and 2.5 mg/L respectively) for 10 weeks in purpose to investigate,among others, effects on plasma lipids, liver somatic index, insulin,adiponektin, leptin and blood glucose levels. Insulin, adiponektin,leptin and blood glucose levels were measured by Elisa. Plasmaapo A-I was separated by SDS PAGE and protein intensities wereevaluated after Western Blot.

After 10 weeks, no significant differences regarding insulin,adiponektin, leptin and blood glucose levels were detected. On theother hand, rats fed with fructose plus BPA (low, medium and highdose) had significantly raised plasma apo A-I levels compared tocontrol rats. Interestingly, the apo A-I levels correlated positivelywith the LSI (% of total weight, p = 0.014) while the mean totalcholesterol levels did not follow the apo A-I and LSI trend in the BPAfed groups. Our results indicate a role of apo A-I in immune defence.We suggest that apo A-I may act as a scavenger by interaction withBPA followed by clearance via the liver.

doi:10.1016/j.toxlet.2012.03.491

P20-11Comparative immunotoxicity study of single-walled carbonnanotubes

Soyoung Lee, Sang-Hyun Kim

Kyungpook National University, Republic of Korea

Purpose: Studies on the single-walled carbon nanotubes (swC-NTs) are becoming popular, not only for their promising applicationin the electronics, optics, and mechanical materials, but also in bio-logical applications, such as imaging and drug delivery. However,its safety of swCNTs has not yet been fully investigated. Since swC-NTs will be more likely to be used in biomedical applications, theinvestigation and elucidation of cellular effects and the associatedmolecular mechanism are urgently needed. In the present study, weinvestigated the interrelationship between different swCNTs prop-erties (such as functionalization and dispersion) and immunotoxiceffects, and defined underlying mechanisms of action. Methods:We used in vitro cell culture system for immunotoxicity of swC-NTs using MTT assay, ROS production, SOD activity and confocalmicroscope observation in macrophages. Results and conclusions:The swCNTs samples were composed of different functionaliza-tion including swCNT, swCNT-COOH, swCNT-OH, and swCNT-NH2.

As observed in the MTT and NO assay, swCNT-COOH inducedmuch greater cytotoxicity than other functionalized swCNTs inmacrophages. This is because swCNT-COOH showed significantlyenhanced cellular uptake and thus, increased ROS production than

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thers. Furthermore, we examined comparative immunotoxicity inerms of dispersion rate of swCNT-COOH (such as COOH-1, COOH-2,nd COOH-3). Obtained result clearly demonstrated that increasedhe degree of dispersion of swCNT-COOH sample became less cyto-oxic. Although different dispersed swCNT-COOH induced similarellular uptake in macrophages, COOH-3 dramatically decreasedOS production and SOD activity. These results suggest that prop-rties of functionalization and dispersion are relevant factors onegulating immunotoxicity of swCNTs.

oi:10.1016/j.toxlet.2012.03.492

20-12redicting sensitization via in silico and in chemicossessment of protein binding

era Teubner 1, Susanne Kolle 2, Tzutzuy Ramirez 2, Brittaareing 2, Annette Mehling 3, Bennard van Ravenzwaay 2, Robert

andsiedel 2

BASF Schweiz AG, Switzerland, 2 BASF SE, Switzerland, 3 3BASFersonal Care and Nutrition GmbH, Germany

In chemico and in silico tools for protein binding were examinedo identify potentially skin sensitizing substances. 54 substancesere assessed using the direct peptide reactivity assay (DPRA) and

he OECD QSAR Toolbox Version 2.0. Of these, 32 or 36 are sensitiz-ng in humans or the LLNA, respectively; eight are pre/pro-haptens.

The OECD toolbox correctly predicted 42 or 39 substances, whenompared to human or LLNA data, respectively. False negative pre-ictions included all pre/pro-haptens, allergens with a non-proteininding mechanism, the two metal salts and 2,4,6-trinitrobenzeneulfonic acid. Interestingly, there were no false positive predictions.sing the skin metabolism simulator of the toolbox, protein bind-

ng metabolites were identified for all pre/pro-haptens. However,any of the non-sensitizing substances were also predicted to have

rotein binding metabolites.The DPRA correctly detected 47 or 42 substances, when com-

ared to human or LLNA data, respectively. False negativesncluded the pro-hapten anilin, three non-binding effectors andexylcinnamicaldehyde and there were four false positives. There/pro-haptens p-phenylenediamine, propyl gallate, isoeugenol,thylene diamine, eugenol, 4-allylanisole and cinnamic alcoholave positive results in the DPRA assay indicating that reactionsther than metabolic activation induced peptide depletion.

Among the non-animal assays to assess skin sensitization poten-ials, the in silico and in chemico tools for protein binding offer goodredictivities although a better integration of the skin metabolismould improve the accuracies.

Keywords: OECD QSAR Toolbox; Protein binding; Skin sensitiza-ion; Prediction model; In vitro

oi:10.1016/j.toxlet.2012.03.493

20-13nfluence of TNF-a and PPD on N-acetyltransferase 1 ineratinocytes and monocytes

utta Bonifas, Jutta Bonifas, Simone Scheitza, Brunhilde Blömeke

University Trier, Germany

N-acetyltransferase 1 (NAT1)-mediated N-acetylation is anmportant detoxification pathway for aromatic amines includinghe contact allergen para-phenylenediamine (PPD). Recently, we

211S (2012) S43–S216 S135

demonstrated that immune relevant cells like keratinocytes andmonocyte derived dendritic cells are able to N-acetylate PPD.However, NAT1 acetylation capacity can be modulated by variousfactors, possibly including the cellular microenvironment, whichmay be altered in the skin after allergen exposure. In this studywe aimed to evaluate the influence of tumor necrosis factor alpha(TNF-a), an important cytokine released after allergen exposure,on NAT1 activity of keratinocytes and monocytes, and studied theimpact of PPD on NAT1 mRNA splice variants in proliferating andcalcium treated keratinocytes.

Addition of TNF-a increased N-acetylation of PPD, mono-acetyl-PPD and aminofluorene by keratinocytes about 3.7 ± 0.4-fold,4.9 ± 1.4-fold and 4.2 ± 1.3-fold. Furthermore, co-treatment of THP-1 cells with PPD (concentrations that inhibit NAT1) and 10 ng/mlTNF-a reversed the PPD-induced NAT1 inhibition (about 30%).These changes were restricted to the presence of the amines sincetreatment with TNF-a alone did not influence NAT1 activity of THP-1 and keratinocytes. Analysis of mRNA splice variants of NAT1revealed that PPD enhances certain variants while treatment withcalcium led to reduction of these variants.

Overall these data demonstrate that NAT1 expression of ker-atinocytes and monocytes is variable and dependent on themicroenvironment. This may influence the detoxification capacityfor PPD and other aromatic amines.

doi:10.1016/j.toxlet.2012.03.494

P20-14Ammonium hexachloroplatinate (AHCP) exposure results in aT helper (Th) 2 profile

Rebecca Dearman, Ian Kimber

University of Manchester, United Kingdom

Prolonged (13 day) topical exposure of BALB/c mice to thecontact allergen 2,4-dinitrochlorobenzene (DNCB), or to the respi-ratory allergen trimellitic anhydride (TMA) induces selective type1 or type 2 cytokine secretion profiles, respectively. Exposure toTMA, but not to DNCB, also results in increased total serum IgE.Both TMA and DNCB are dissolved in the standard vehicle for topicalexposure; acetone:olive oil. In the current experiments responsesprovoked by the respiratory sensitising platinum salt ammoniumhexachloroplatinate (AHCP) formulated in dimethyl formamide(DMF) have been examined. Thirteen days after the initiation oftopical exposure, cytokine secretion of draining auricular lymphnode cells (LNC) and total serum IgE concentration were mea-sured. TMA-activated LNC displayed a type 2 phenotype with highlevels of interleukin (IL)-4, 5, 10 and 13 and relatively low lev-els of interferon (IFN)-gamma, whereas the converse Th1 patternwas provoked by DNCB exposure. LNC isolated from AHCP-treatedmice displayed a type 2phenotype, although cytokine expressionwas less vigorous than that provoked by TMA (3-fold lower IL-4and IL-5 production; 6-fold lower IL-13 secretion). AHCP expo-sure provoked a vigorous increase in total serum IgE comparedwith DMF- (2008 ± 280 pg/ml versus 223 ± 35 pg/ml; p < 0.001)and TMA-treated mice (1040 ± 279 pg/ml; p < 0.01). Thus, AHCPdisplays a cytokine phenotype and increases in the total serumIgE consistent with respiratory sensitising potential. The cytokineexpression pattern (a snap shot of type 2 activity at one time point)was considerably less vigorous that that provoked by TMA, how-

ever, changes in total IgE which are a cumulative measure weremore profound.

doi:10.1016/j.toxlet.2012.03.495