Letter

S14 Abstracts / Toxicology

Regulatory in vitro genotoxicity tests can generate high rates offalse positives. Without the perspective supplied by in vivo test-ing, banned under the EU Cosmetic Directive from 2009, therewill be high rates of attrition of potential products. This hasheightened interest in new in vitro models to predict geno-toxicity and carcinogenicity. The application of transcriptomics,proteomics and metabonomics, could lead to models with moreaccurate prediction. A study, co-ordinated by the Health and Envi-ronmental Sciences Institute, of the transcriptomic response ofmouse lymphoma L5178Y cells to genotoxins shows that geneexpression changes can discriminate between thresholded andnon-thresholded mechanisms, a vital distinction in hazard assess-ments. A study using P53 proficient TK6 cells has shown theimportant role of P53 dependent pathways in response to geno-toxins, whilst no such response was obtained for non-carcinogeniccompounds. Proteomics, such as the SILAC technique (Stable Iso-tope Labelling with Amino acids in Cell culture), may provideobjective markers to detect malignant transformants. Cancer cellsproduce a distinct pattern of metabolites involved in intracel-lular signalling pathways and networks. Studies of intracellularmetabolite changes in yeast strains following exposure to carcino-gens and non-carcinogens are casting light on this process. Otherrelated technologies have potential, such as array comparativegenome hybridisation of single cells to detect gene amplificationsand deletions following exposure to genotoxins. The measure-ment of compound induced epigenetic changes resulting in genesilencing may be of future interest for carcinogen detection. Newapproaches such as these will not deliver new models in theshort-term.

doi:10.1016/j.toxlet.2008.06.812

28Predictive toxicogenomics approaches to non-genotoxic car-cinogens

Michael McMillian ∗, Alex Nie, Nandini Raghavan, DhammikaAmaratunga, Peter Lord

Johnson & Johnson Pharmaceutical Research and Development,Raritan, New Jersey, United States

Although, there are many assays available to detect genotoxic

carcinogens, the non-genotoxic carcinogens (NGTCs), commonlyobserved in long-term rodent studies, have proven difficult topredict. Using toxicogenomics (gene expression responses to tox-icants) and enough appropriate samples, relatively subtle geneexpression response signatures can accurately predict multipletoxic endpoints. A large gene expression database built using cDNAmicroarrays and liver samples treated with over one hundredparadigm compounds was mined to determine gene expressionsignatures for NGTCs. Data were obtained from three male ratsper group treated for 24 h. Training/testing sets of 24 NGTCs and28 noncarcinogens were used to select genes. Samples from per-oxisome proliferator- and macrophage activator-treated rats wereexcluded as these easily identifiable compounds produced massivegene response effects (and are known NGTCs). Six genes were suffi-cient to identify NGTCs on the cDNA microarrays with 88% accuracy,and these genes migrated faithfully to a commercial CodeLink oligomicroarray (84% accuracy). As proof of principle, toxicogenomicsapproaches to predicting NGTCs have been derived and have per-formed well for several groups. We have obtained signatures thatperform much better on Codelink microarrays than our original sixgenes. Future challenges are to understand the biological relevances

s 180S (2008) S6–S27

of the predictive gene changes, which should increase acceptanceof toxicogenomic approaches to screening for NGTCs.

doi:10.1016/j.toxlet.2008.06.813

W7: The Toxicological Challenges of Chemical RespiratoryAllergy

29Respiratory allergy: What are the issues?

Rebecca Dearman

University of Manchester, Manchester, United Kingdom

Chemical respiratory allergy, characterised typically by rhinitis andasthma, is an important occupational health problem associatedwith considerable morbidity and related economic and social costs.Compared with those chemicals implicated as skin sensitisers,fewer chemicals are known to cause sensitisation of the respiratorytract. These include: diisocyanates, acid anhydrides, some platinumsalts, certain reactive dyes, plicatic acid and chloramine-T. Chemicalrespiratory allergy poses significant challenges for the toxicologist,not least of which is the fact that there are, as yet, no validated orwidely accepted methods available for the identification and char-acterisation of chemicals that have the potential to cause allergicsensitisation of the respiratory tract. This is attributable, at least inpart, to the fact that there is continuing uncertainty about the exactnature of immunological mechanisms through which respiratorysensitization to chemicals can be acquired, and pulmonary reac-tions elicited. Presently, chemical respiratory sensitisation hazardis assigned to chemicals on the basis of epidemiological evidenceor, in the case of diisocyanates, as a default classification. This pre-sentation will review chemical respiratory allergy with respectto what is known of clinical characteristics and immunologicalmechanisms, including the relevance of route of exposure. Thepresentation will also include consideration of the definitions ofrespiratory allergy, with particular reference to those chemicalsthat cause allergic respiratory hypersensitivity (by definition viaa mechanism or mechanisms that involve and require specificimmunological responsiveness) compared with those chemicalsthat have been implicated as causing or exacerbating asthma vianon-immunological mechanisms (asthmagens).

doi:10.1016/j.toxlet.2008.06.806

30Progress in the development of alternative approaches

Silvia Casati ∗, Costanza Rovida

EC Joint Research Centre, Ispra, Italy

Considerable efforts have been directed towards the developmentof alternative methods to obviate the use of laboratory animals.For complex endpoints such as allergic sensitisation, the challengeof replacing whole animal studies with in vitro assays requiresan integration of approaches that together reflect the key bio-logical mechanisms. To date standardised alternative methods forthe identification of chemicals that are able to cause respiratorysensitisation are not available. However, various approaches areunder investigation including the exploitation of methods cur-rently being developed/optimised for the identification of skinsensitising chemicals. These include: (Q)SAR models, protein- andpeptide-binding assays and cell-based assays usually incorporat-

Letter

receptor (CAR NR1I3) are nuclear receptors activated by vari-ous chemically unrelated compounds, including environmentalpollutants and drugs. To characterize the specificity of diverse com-pounds for human and mouse PXR and CAR, four stable cell linesexpressing the ligand binding domain (LBD) of human hPXR, mPXR,

Abstracts / Toxicology

ing dendritic cells (DC) or DC-like cells. Furthermore, there is inplace a significant research investment through an integrated EUFramework Programme 6 “Sens-it-iv”. This programme aims todeliver novel in vitro testing strategies for the assessment of bothskin and respiratory sensitisers. Two recent workshops convenedby the European Centre for the Validation of Alternative Meth-ods (ECVAM) have focused on reviewing current state-of-the-art.Specifically, these workshops have considered the approaches thatmay be available for the prospective identification of chemical andprotein respiratory allergens and have developed recommenda-tions on what is now needed for further progress to be made. Anoverview on the outcome of these workshops will be provided.

doi:10.1016/j.toxlet.2008.06.807

S8: Receptor-Mediated Toxicity (Epigenetic Processes in Car-cinogenicity)

31The aryl hydrocarbon receptor—Role in carcinogenesis

Dieter Schrenk

Food Chemistry and Toxicology, University of Kaiserslautern,Kaiserslautern, Germany

The aryl hydrocarbon receptor (AhR) acts as nuclear transcriptionfactor upon binding of agonists, most notably of a variety of chem-ical carcinogens/liver tumour promoters such as 2,3,7,8-TCDD. Incarcinogenic mixtures of polychlorinated biphenyls (PCBs), AhR-activating ‘dioxin-like’ PCBs seem to play a predominant role asliver carcinogens, when compared to their ‘non-dioxin like’ coun-terparts. Interestingly, both TCDD and technical PCB mixtures weresignificantly more potent in female than in male rats.

AhR-dependent changes in gene expression are considered toplay a role in carcinogenicity of TCDD. Oxidative DNA damage wasreported to occur in female but not in male or ovariectomizedfemale TCDD-treated rats. A role for metabolic activation of estra-diol by AhR-regulated cytochromes P450 has been postulated. Inour experiments in rat hepatocytes in culture, TCDD led to increasesin 8-oxo-desoxyguanosine (8-oxo-dG) in nuclear DNA, and in DNAdouble strand breaks after Fpg-treatment. Both effects were notenhanced by addition of estradiol. Furthermore, TCDD treatment ofadult female rats over 21 days did not result in increased oxidativehepatic DNA damage.

Inhibition of apoptosis of preneoplastic hepatocytes is anotherputative key event in tumour promotion by AhR agonists. In rat hep-atocytes in culture, TCDD leads to suppression of apoptosis elicitedby UV treatment. We could demonstrate that TCDD did not affectcaspase(s) activation by UV irradiation. Furthermore, no evidencefor a caspase-independent target such as AIF was found. These datasuggest that the pleiotropic response to AhR activation acts on laterstages in the way to the final execution of apoptosis.

doi:10.1016/j.toxlet.2008.06.802

32Nuclear receptor CAR-mediated signals responsible for hepato-cellular carcinoma

Masahiko Negishi

NIEHS, RTP, North Carolina, United States

CAR, a member of the nuclear hormone receptor superfamily,is activated by xenobitoics including therapeutic drugs such asPhenobarbital (PB). Chronic PB treatment is known to promote

s 180S (2008) S6–S27 S15

development of hepatocellular carcinoma (HCC) in rodents; themolecular mechanism of which remains elusive. We used wild typeand CAR KO C3H mice to find that the CAR KO mice do not developHCC, indicating that CAR is a determining factor in the PB promo-tion (Yamamoto et al., 2004). To decipher the molecular mechanismby which PB promotes HCC development via CAR, cDNA microarraywas first performed to identify the CAR-regulated genes that may beinvolved in PB promotion of HCC development. Several genes wereselected for further characterization, one of which is anti-apoptoticfactor GADD45B that is induced by CAR throughout HCC develop-ment and was further up-regulated in the tumour tissues. Usingprimary hepatocytes from Car+/+ and Car−/− mice, we confirmedthat CAR activation represses TNF�-induced cell death. In addition,CAR directly bound to the GADD45B protein, increasing GADD45Bactivity to inhibit MKK7-dependent phosphorylation of JNK1. Thus,CAR can potentiate the anti-apoptotic function of GADD45B by acti-vating the Gadd45b gene as well as by directly interacting withthe GADD45B protein. Consistent with this hypothesis, treatmentwith CAR activator resulted in repression of TNF�-induced deathof primary hepatocytes from Gadd45b+/+ but not Gadd45b−/− mice.CAR may repress cell death by inhibiting the activity of MKK7 viaGADD45B, decreasing phosphorylation of JNK1 as well as JNK1-dependent apoptosis.

Reference

Yamamoto, et al., 2004. Cancer Res. 64, 7197–7200.

doi:10.1016/j.toxlet.2008.06.803

33Pregnane-X receptor/constitutive androsterone receptor

Patrick Balaguer 1,∗, Marina Grimaldi 1, Sonia Dagnino 1, AudeMallavialle 1, Alain Chavanieu 2, Vincent Cavaillès 1

1 INSERM U896, Montpellier, France, 2 INSERM U554, Montpellier,France

Pregnane-X receptor (hPXR, NR1I2) and constitutive androstan

hCAR and mCAR fused to the yeast GAL4 DNA binding domain (DBD)were developed.

With the help of these reporter cell lines, we assessed the selec-tivity and activity of PXR and CAR environmental and syntheticligands. Furthermore these cells enabled us to detect PXR activ-ity in environmental samples. As these receptors are involved inthe regulation of drug-metabolism enzymes and therefore, playan essential role in the detoxification pathways, they are new tar-gets for endocrine disrupting compound. Due to their capacity tobind a multitude of different types of environmental pollutants, itis difficult to predict which type of molecule is responsible for theobserved activity in environmental samples. We will show that thedevelopment of a tool to purify nuclear receptor ligands in an envi-ronmental complex matrix should improve chemical detection ofPXR’s endocrine disruptors.

doi:10.1016/j.toxlet.2008.06.804