Duluth, 19-21 May 2010

1. Duluth, 19-21 May 2010 IDENTIFYING GENOTOXIC AND NON GENOTOXIC CARCINOGENS WITH CELL TRANSFORMATION ASSAYS P. VASSEUR, M.A. MAIRE, C. RAST, S. ALEXANDRE, H. BESSI. University of Metz, CNRS , France

2. Duluth, 19-21 May 2010 Context, Definitions, History of CTA • SHE cells • Protocol • Mechanisms • The Balb 3T3, C3H10T1/2 cell lines • Performances of cell transformation assays (CTA) • Analyses SHE results • Conclusion

3. Duluth, 19-21 May 2010 Context Long term rodent carcinogenicity assay : expensive, time consuming → not required for the evaluation of chemicals (EU), except the genotoxic ones produced at high tonnage Short term in vitro and in vivo genotoxicity assays have been used as surrogates to predict carcinogenicity. Yet, a number of chemical carcinogens to humans and mammals are negative in genotoxicity assays, but positive in cell transformation assays (CTA) In 2007, OECD has recommended the development of guidelines for cell transformation assays for in vitro detection of chemical carcinogens

4. Duluth, 19-21 May 2010 OECD Environment, Health and Safety publications Series on Testing and Assessment N°31, 2006. Detailed Review Paper (DRP 31) on Cell Transformation Assays for Detection of Chemical Carcinogens. Environment Directorate, November 28, 2006, 170 p. http://www.olis.oecd.org/olis/2007doc.nsf/LinkTo/NT00002F0A/$FILE/JT03230941.PDF http:www.oecd.org/document/12/0,2340,en_2649_34377_1898188_1_1_1_1,00.html

5. Duluth, 19-21 May 2010 Cell Transformation Cell transformation is the induction of phenotypic alterations in cultured cells that are characteristics of tumorigenic cells. These phenotypic alterations can be induced by exposing mammalian cells to carcinogens. Transformed cells that have acquired the characteristics of malignant cells have the ability to induce tumors in susceptible animals (Berwald and Sachs, 1963, 1965).

6. MULTISTAGE TRANSFORMATION OF SYRIAN HAMSTER EMBRYO (SHE) CELLS BY CHEMICAL CARCINOGENS Subcutaneous Injection into newborn hamster Primary or secondary SHE cells + BaP or 3MC then subcultured Foci of rapidly dividing cells Tumours Earle (1943) :Morphological changes in cell culture were associated with the oncogenicity of these cells in vivo Berwald and Sachs, 1963, 1965

7. Further development and validation of SHE assay DiPaolo et al., 1969 Pienta et al., 1977 Barrett et al., 1979 Newbold et al., 1982 Chouroulinkov & Lasne, 1976 ,… demonstrated the ability of chemical carcinogens from different chemical classes to induce morphological transformation (MT) in vitro

8. Morphological transformation (MT) = Changes exhibited by transformed cells related to neoplasia and associated with behaviour and growth control modifications : . alteration of cell morphology . disorganized pattern of colony growth . acquisition of anchorage-independent growth (Combes et al., 1999) Later on, transformed cells become able to : . grow in semi-solid agar . produce autocrine growth factors . evolve to tumorigenicity when injected into appropriate hosts . divide indefinitely (immortalized), which is associated with other alterations like aneuploïd karyotype and altered genetic stability.

9. Phenotypic changes / SHE CTA • Changes in • - cytoskeleton • - morphology of cells • & colonies • Characteristic • phenotype • of transformed cells : normal transformed - a random growth pattern of spindle shaped cells, - a piling up of cells in a criss-cross pattern (a loss of growth inhibition and of cell-cell orientation at confluency)

10. Normal SHE colony

11. Morphologically transformed SHE colony

12. Morphologically transformed SHE colony

13. Chemical carcinogens classified in two groups . Genotoxic carcinogens able to initiate cells to carcinogenesis through direct interaction with DNA, resulting in DNA damages and/or structural/numerical chromosomal aberrations which can be detected by genotoxicity tests. . Non-genotoxic carcinogens carcinogenic agents devoid of direct interaction with DNA. The indirect modifications to DNA structure, amount or function may induce altered gene expression and/or signal transduction. Generally, non-genotoxic carcinogens refer to carcinogens negative in genotoxicity assays performed to measure endpoints such as gene mutations and chromosomal damages (chromosomal aberrations, micronuclei).

14. The multistage process of carcinogenesis in vivo Normal Foci Benign lesion Malignant lesion Initiation Promotion Progression

15. 1. Genotoxicity 2. Epigenetic events …… Factors of Cell growth, division Transcription Mutagens Viruses Radiations Initiation Promotion Progression Invasion

16. 1. Genotoxicity 2. Epigenetic events …… Factors of Cell growth, division Transcription Mutagens Viruses Radiations Efficient controls at every steps, Inhibition of DNA repair Activation of protooncogenes Inactivation of tumor-suppressor genes of antimetastasis genes

17. 1. Genotoxicity 2. Epigenetic events …… Factors of Cell growth, division Transcription Mutagens Viruses Radiations Efficient controls at every steps, BUT, IF INACTIVATED Inhibition of DNA repair Activation of proto-oncogenes Inactivation of tumor-suppressor genes of antimetastasis genes

18. 1. Epigenetic events 2. Genotoxicity …… Histone desacetylation Hypo/hypermethylation Inhibition of DNA repair Activation of proto-oncogenes Inactivation of - tumor-suppressor genes of - antimetastasis genes

19. Acetylation Histone acetyl transferase (HAT) open chromatin transcription, gene activation Desacetylation Histone desacetylase (HDAC) + Methylation Histone methyl transferase + HMT Methyl Binding Proteins (MBP) compactedchromatin blocage transcription gene inactivation tumor suppressors genes p53, p16

20. Desacetylation Acetylation Hypoacetylation H4 observed in early steps of carcinogenesis Silencing of tumor suppressor genes Hypermethylation H3 H4, OverexpressionHMT in a variety of neoplasia DNA hypermethylation of promoter sequences → transcriptional silencing Histone acetylases Histone desacetylase Histone methyl transferases active, open chromatin normal state Histones H3, H4 H3, H4 SAM Methylation methionine DNA methyl transferases DNA Hypomethylation also tumorigenic Iacobuzio-Donahue, Ann Rev Pathol. Mech. Dis.2009.

21. Desacetylation Nickel Acetylation Hypoacetylation H4 observed in early steps of carcinogenesis Silencing of tumor suppressor genes Hypermethylation H3 H4, OverexpressionHMT in a variety of neoplasia DNA hypermethylation of promoter sequences → transcriptional silencing (Sutherland et al. Ann NY Acad Sc, 2003) Histone acetylases Histone desacetylase Histone methyl transferases active, open chromatin normal state Histones H3, H4 H3, H4 SAM Methylation methionine DNA methyl transferases DNA Hypomethylation also tumorigenic Arsenic, Alcohol Preferential binding to methylated CpG sites PAH (tobacco smoke) AFB1 Cadmium Effect dose dependent (Herceg, Mutagenesis, 2007)

22. The MT phenotype of colonies expresses changes in the expression of genes involved in cell cycle control, proliferation and differentiation. resulting from genotoxicity and non-genotoxic mechanisms leading to : - alteration of DNA repair - disturbance in signal transduction - histone desacetylation, DNA hypermethylation & hypomethylation - modulation of gene expression→disturbance of cell cycle control, proliferation and differentiation (Alexandre et al., 2003) Histone desacetylation, DNA hypermethylation & hypomethylation - oxidative stress (Jiung et al., 1999, Zhang et al. 2000) inflammation - imbalance of cell proliferation/apoptosis - changes in intercellular communication (Cruciani et al. 1997) - telomerase activation …. - immunosuppression

23. Signal transduction kinase cascade Disturbance in signal transduction from cell environment nucleus Nuclear receptors Lipophilic hormones Syntheses, replication,mitosis Nuclear transcription factors Non lipophilicMembrane growth + receptor factor cascade phosphorylation / dephosphorylation transient activation of a number of intermediates

24. Disturbance in signal transduction from cell environment nucleus Syntheses, replication, mitosis Growth + Receptor factor A signal transduction pathway may be disrupted, activated or blocked, by analogs that substitute or interfere with some intermediates or the receptor itself. An activation may be permanent, instead of transient, leading to a sustained response ( ex : cell cycle dysregulation, increased rate of mitosis)

25. The tumor promoter TPA 12-O-Tetradecanoylphorbol-13-acetate substitutes to diacylglycerol (DG) and activates the PKC pathway DG Phorbol ester TPA Signal C OH Phorbol ester Kinase C Inactive protein Phosphorylated protein active cell response Ca++

26. Oxidative stress is involved in acrylonitrile (ACN)-induced morphological transformation in SHE cells 8-oxodGuo in SHE cells Vit. E Vit. E Con ACN % MT colonies ACN 0 25 50 75 0 25 50 75 µM -tocopherol 5 5 5 5 µM Zhang et al., 2000. Carcinogenesis 21, 727-733.

27. DEHP, Di-(2-ethylhexyl)phthalate, a non genotoxic carcinogen induces SHE cell transformation at doses inhibiting apoptosis (50 µM)in serum-deprived cells CONTROLAPOPTOTIC Consequence : survival of abnormal cells Mechanisms : Surexpression of the antiapoptotic gene bcl-2 Repression of the protooncogene c-myc DEHP 10 µM DEHP 50 µM Maire et al., 2005. Toxicol Lett, 158, 237-245

28. DEHP inhibits apoptosis via surexpression of bcl-2 (antiapoptotic) → change in bax/bcl-2 ratio bcl-2 (500 pb) Bcl-2 (26 kDa) and represses the protooncogene c-myc expression (Maire et al., 2005, Toxicol Lett,158, 237-245)

29. Protocol of the SHE assay Obtention of SHE cells (feeder and target cells) Hamster 5000 rads embryos primary feeder cells 13 days cultures Gestation nontarget cells differentiated cells storage - 196°C 7 days of exposure SHE cells tested at clonal density 150 cells / dish 25-45 colonies / dish Fixation, coloration scoring Cloning : 1 cell → 1 colony of hundred(s) cells ) Cloning efficiency (> 20%) : 150 cells → 25-45 colonies

30. Transformed colony Normal colony Scoring of coded plates, under stereomicroscope

31. Experimental design(continued…) • *Preliminary experiment for dose-range finding • *Definitive test • - 5 dose levels, vehicle control and positive control (BaP) • - Cytotoxicity evaluated by clonal efficiency • - Nb target cells adjusted in order to obtain 20-45 colonies/dish • - 40 dishes per concentration (or 10/conc. x 4 experiments) • Transformation frequency and cloning efficiency established • from 1000 scored colonies per concentration • Statistical analyses • Criteria of acceptance fulfilled

32. Experimental design (continued…) • statistical analysis for comparison between vehicle control • and concentration level (Fisher’s exact test or 2) and • positive dose-response trend (Cochran-Armitage test) • positive response is declared when : • . 2 positive (successive) concentrations, at least • . or one positive concentration plus positive trend • criteria for acceptance fulfilled • . 20% cloning efficiency in controls • . Nb transformed colonies in the range 25-45/dish

33. Experimental design (continued…) Test medium : DMEM (without phenol red) with fetal calf serum (12-15%), 10% CO2 pH / exposure physiological pH : 7.0 - 7.35 exposure 7 days or LeBoeuf’s modification (1986) pH: 6.7 exposure to the tested chemical 24 h or 7 days

34. In parallel to SHE cell MT assay , Development of cell transformation assays (CTA) on mouse established cell lines * Balb 3T3, clone A31 Kakunaga, 1972 Yamasaki, 1985 * C3H10T ½ Chen and Heidelberger, 1969, Reznikoff et al., 1973

35. Cell Transformation Assays (CTA) • SHE • diploïd, normal cells • metabolically competent • secondary cultures • low level of spontaneous transformation • short term (7 days) exposure • mimics the first stages of the neoplastic transformation • Balb 3T3, C3H10T1/2 • aneuploïd cell lines • limited metabolic ability • infinite life span • high level of spontaneous transformation • long term (> 4 weeks) • mimic the late stages of the neoplastic process

36. The multistage process of carcinogenesis in vivo (a) Normal Foci Benign lesion Malignant lesion (a) Initiation Promotion Progression SHE Balb/c 3T3, C3H 10T1/2 From Combes et al., 1999. ATLA 27, 745-767.

37. Morphologically transformed and non-transformed foci of BALB/c 3T3 cells (foci induced by 1 µg/ml 3-methylcholanthrene) Non-transformed Transformed Photo Dr H Yamasaki.

38. Morphologically transformed and non-transformed foci of C3H 10T1/2 cells (treatment with 1µg/ml 3-methylcholanthrene for 24h) Type I normal Type II Type III Photo Dr J. Landolph.

39. PERFORMANCES OF THE CELL TRANSFORMATION ASSAYS - OECD - Comparison with commonly used short-term genotoxicity tests for assessing carcinogenic potential Salmonella (Ames) test (mutagenesis assay) Mouse lymphoma L5178Y cell mutagenesis assay HPRT mutagenesis assay In vitro chromosomal aberrations In vivo chromosomal aberrations In vivo micronucleus test

40. Data set Rodent Carcinogens 191 127 117 Non Carcinogens 73 59 24 Inorganic 61 21 20 Nb chemicals SHE : 264 BALB: 186 C3H : 141 Organic 203 165 121 • Data banks • IARC, NTP, GENETOX, CCRIS, CPDB/Gold and Zeiger (1997) • - Heidelberger et al. (1983), Matthews et al.(1993), Leboeuf et al. (1996) and many other published articles …

41. SHE results on 64 metals and inorganic compounds Asbestosis, ceramic fibres, cadmium, nickel, chromium compounds, … Elias et al. Carcinogenesis, 10-11, 2043-2052, 1989 ; Elias et al.,Toxicology In Vitro, 14, 409-422, 2000; Elias et al., J. Toxicol Environ Health, 65, 2007-2027, 2002; Elias et al, Ann. Occup. Hyg., 46, 53-57, 2002. …

42. Comparison with rodent carcinogenicity Definitions In vivo Concordance = % agreement with in vivo exp. (a+b)/(a+b+c+d)*100 Sensitivity = % carcinogens that are positive (a/a+c)*100 Specificity = % noncarcinogens that are negative (d/b+d)*100 Positive Pred.= % positive calls that are carcinogens (a/a+b)*100 Negative Pred.=% negative calls that are noncarcinogens (d/c+d)*100 False negative = c/ a+c False positive = b/ b+d

43. Performance of CTA relative to rodent bioassay SHE 264 86% 91% 74% 9% 26% (10%) BALB 149 68% 75% 53% 25% 47% (28%) C3H 96 73% 72% 80% 28% 20% (30%) n = Concordance Sensitivity Specificity False negative False positive (Inconclusive Not included)

44. Performance of CTA relative to rodent bioassay SHE pH 7.0 204 85% 92% 66% 8% 34% (12%) SHE pH 6.7 88 74 66 85 33 15 (2) BALB 149 68% 75% 53% 25% 47% (28%) C3H 96 73% 72% 80% 28% 20% (30%) n = Concordance Sensitivity Specificity False negative False positive (Inconclusive Not included)

45. Carcinogens CTA positive GenotoxicNon genotoxic Direct alkylating agents lactones, epoxides aldehydes alkylsulfonates Indirect acting alkylating agents N-nitroso compounds Halogenated aliphatic hydrocarbons Indirect acting, DNA covalent binding, Intercalating agents, Polycyclic aromatic hydrocarbons Aromatic amines, nitroarenes mycotoxins Steroïds Phthlates & HPP (fibrates) (SHE) Polyhalogenated biphenyls Halogenated aryl (insecticides) PCDD Biotoxins, cyanotoxins Tumor promoters (TPA, okadaïc acid…)

46. False negatives in CTA SHE Aniline Anthraquinone Arochlor 1254 DDT Ethinyl estradiol Ethyl alcohol d-Limonene Metaproterenol Methylcarbamate Nitrilotriacetate NTA 5-nitro-o-toluidine Pyridine Tetrahydrofuran TEHP tris(2ethylhexyl) phosphate BALB 3T3 2-Aminoanthracene Chlorinated aliphatic hydro mono, di, tetra, hexachloroethane Clofibrate 1,2-epoxybutane Ethinyl estradiol d-Limonene Monuron 2-4-Dinitrotoluene Phthlates Butylbenzyl phthlate, DEHP Procarbazine TEHP C3H Inorganics lead acetate potassium dichromate nickel chloride sodium arsenate Organics BrdU Phenobarbital Propyleneimine Styrene Thioacetamide Divergent responses Diethylstilbestrol DEHP Hexamethy phosphoramide 5-nitro-o-toluidine

47. Many known or suspected aneugens induce CT in SHE cells In vivo MN + + -/? +/? + + ? + In vitro ABS + + +/- +/- + + + + Rodent Carcinog + + + - + + - + - + + - - SHE + + + + + + + + + + + + + Acrylamide Asbestos Benzene Benomyl Cadmium chloride Chloral hydrate Colcemid DES Econazole nitrate Griseofulvine Hydroquinone Pyrimethamine Vincristine

48. Performances of short-term genotoxicity tests on the chemicals of the data set In vitro ABS 184 64% 65% 63% 35% 37% 15% In vivo MN 158 56% 57% 52% 43% 48% 31% MLA 170SHE 74% 86% 34% 14% 66% 29% Ames 252 51% 37% 81% 63% 19% 21% n = Concordance Sensitivity Specificity False negative False positive (Inconclusive Not included)

49. Non genotoxic (S. typhi) carcinogens SHE positive Ames negative Acetamide Acrylamide Actinomycine D Amitrole Auramine Benzene BrdU Butylhydroxytoluene Butylbenzylphthlate Catechol Chlordane Chlorothalonil Cinnamyl anthranilate Clofibrate Cyclosporine Decabromodiphenyloxide Dieldrin Diethanolamine DEHP Reserpine Safrole Sulfamethoxazole TPA phorbol ester Thiourea Trichlorophenol TEHP tris(2ethylhexyl) phosphate Wyeth 14043 (HPP) Diethylstilbestrol Diethylthiourea Dimethylhydrazine Estradiol Ethionine Ethylbenzene EGBE Butyl glycol Hexachlorobutadiene Hexamethylphosphoramide Hydroquinone Methylpyrilene, HCl Methyl eugenol Methylclofenapate Mezerein Monuron N-nitroso ethylaniline Okadaïc acid Oxymetholone Procarbazine, HCl Progesterone

50. One embryo 20-50 tests One female (m ≈5-8 embryos) : 160-400 tests Renewal of target cells every year Yet, cells and kits are now available and provided by some companies Training necessary 6-8 weeks required for a confirmed result Quite performant as alternative to rodent carcinogenicity assays. CTAs, in vitro assays necessary for non genotoxic carcinogens !