Cell transformation Definition Characteristics of transformed cells

1. OXIDATIVE STRESS AND IN VITRO CELL TRANSFORMATIONMAIRE Marie-Aline2nd McKim Workshop on Reducing Data Redundancy in Cancer assessmentBaltimore, 8-10 may 2012

2. Cell transformation Definition Characteristics of transformed cells Mechanisms of cell transformation Oxidative stress in carcinogenesis Definition Sources of ROS Effect of ROS on macromolecules, modulation of gene expression Oxidative stress and cell transformation Acrylonitrile Chromium Fibres and particles (e.g. silica)

3. Cell transformation • multistage process that results in the conversion of normal cells into fully malignant cells after exposure to carcinogen substances Cell transformation assays : • ECVAM validation (2005-2009) • OECD recommendation (2007) • OECD TG (april 2012) • primary cells (Syrian hamster embryo), cell lines (Balb3T3, C3H101/2) • similar to that observed in vivo for both genotoxic and non-genotoxic carcinogens • surrogate in vitro model for carcinogenesis in vivo • mechanisms of carcinogenesis SHE cells Balb3T3

4. Mechanisms of cell transformation • Genotoxic and non-genotoxic mechanisms • disturbance in signal transduction • imbalance of cell proliferation/apoptosis • modulation of gene expression (cell cycle control, proliferation and differentiation) • alteration of DNA repair • oxidative stress • histone desacetylation, DNA methylation • inflammation • changes in intercellular communication • immunosuppression • Several studies have involved oxidative stress in cell transformation

5. Oxidative stress • Oxidative stress • physiological, endogenous and exogenous processes that directly / indirectly affect the oxidant and antioxidant balance • occurs in cells or tissues when the concentration of reactive oxygen species (ROS) generated exceeds the antioxidant capacity of the cells • cellular oxidative damage • Involved in aging process & and in the pathogenesis of a number of diseases : • cardiovascular, metabolic, inflammatory and neurodegenerative diseases, cancers • Cancers • oxidative damage can participate in all stages of carcinogenesis process • prooxidant state observed in human cancers / tumor cells

6. Free oxygen radicals : superoxide (O2•-) hydroxyl radical (•OH) nitrite oxide (NO•) organic radical (R•) peroxyl radical (ROO•) alkoxyl radical (RO•)…. Non radical ROS : hydrogen peroxide (H2O2) singlet oxygen (1O2), ozone (O3) organic hydroperoxides (ROOH) hypochloride (HOCl) peroxinitrite (ONO-) dinitrogen dioxide (N2O2)… highly reactive lipid- or carbohydrate-derived carbonyl compounds ROS : reactive oxygen species • Key players in oxidative stress • radicals, ions, molecules with a single unpaired electron in their outermost shell of electron • highly reactive • H2O2, O2•-, •OH = most studied ROS in carcinogenesis

7. Sources of ROS Endogenous sources peroxisomes, mitochondria, inflammatory cells, P450 metabolism, NADPH oxidase, Lipoxygenase… • e.g. Peroxisomes (organelle, consumption of O2) • ROS production via peroxisomal oxidase (acyl coA and xanthine oxidase) • Peroxisome proliferator increase the number and size of peroxisomes • Ex : hypolipidemic drugs, phthalates, halogenated coumpounds… • Link between peroxisome proliferation and hepatocarcinogenesis • e.g. Inflammatory cells (neutrophils, eosinophils, macrophages) • Activation (endo / exogenous stimuli) leads to a respiratory burst : • increase in oxygen uptake • generation of ROS through NADPH oxidase • Phagocyte-derived ROS may be involved in the development of cancers

8. Exogenous sources Ionizing radiations, UV Environmental toxicants Chemotherapeutic agents … The majority of environmental, occupational and industrial chemicals are able to generate free radical species primarily or through their metabolic activation Sources of ROS Endogenous sources peroxisomes, mitochondria, inflammatory cells, P450 metabolism, NADPH oxidase, Lipoxygenase…

9. Antioxidant defenses ROS Antioxidants • Enzymatic e.g. superoxide dismutase (SOD), catalase, glutathione peroxidase (GP), peroxiredoxin, … • Non-enzymatic e.g. Glutathione (GSH), flavonoids (EGCG), carotinoids, vitamins (E, C), pyruvate, urate, plant-derived antioxidants, metallothioneins, …

10. Antioxidants Enzymatic (SOS, CAT, GSH perox…) Non-enzymatic (VitE, GSH, VitC…) Effect of ROS on macromolecules ROS Oxidative damage Alteredgene expression Lipids Proteins ADN, ARN Signalingpathways DNA methylation …

11. Lipid peroxidation DNA damage single or double strand breakage, base modifications, deoxyribose and phosphate modifications, DNA cross-link, DNA adducts (e.g. 8OHdG) Lipid peroxyl radical Lipid hydroxyperoxide Modification of membrane structure & function Reactive aldehydes formation (MDA, 4-HNE…) cell death, mutation, induction of transcription, induction of signaling pathways, genomic instability Loss of cell homeostasis Effect of ROS on macromolecules PROTEINS LIPIDS DNA Formation of carbonyl derivatives Direct oxidation of amino acid chains Oxidation-induced peptide cleavage Modification of structure and function of the membrane Changes to receptor proteins and gap junction proteins Stimulation or inhibition of enzymes activity

12. ROS Antioxidants Enzymatic (SOS, CAT, GSHperox…) Non-enzymatic (VitE, GSH, VitC…) Oxidative damage Altered gene expression Alteration of gene expression by ROS Lipids Signaling pathways DNA methylation … Proteins ADN, ARN

13. Effect of ROS on signaling pathways ROS are integrated in signaling pathways Homeostasis Response to oxidative stress High level of ROS Low or transient level of ROS Normal growth and metabolism Apoptosis and necrosis Regulation of cellular migration, proliferation, survival, death response • alteration of expression of proto-oncogenes / growth factors • activation of transcription factors (Nfr2, AP-1, Nf-kb, HIF-1, p53, Foxo…) • activation of protein kinase pathways (MAPKs, Akt, PKC…) dysregulation of these pathways = potential mechanisms of ROS-induced carcinogenesis

14. Signaling pathways : e.g. Nrf2 and NFkB • Nfr2 • protective enzymes : xenobiotic detoxification, antioxidative response, proteome maintenance • low level / loss of activity :  ROS production, DNA damage, predisposition to tumorigenesis

15. Hydroxyl radical OH• base modifications, deletions, strand breakage, chromosomal rearrangement … 8-OHdG, 8-hydroxyguanine, O6- methylguanine… DNA damage modified DNA structure DNA methyltransferases (DNMTs) reduction or inhibition of the methyl-accepting ability HYPOMETHYLATION • transcriptional level, aberrant genes expression, activation of proto-oncogenes, genomic instability Effects of ROS on DNA methylation

16. ROS Antioxidants Enzymatic (SOS, CAT, GSHperox…) Non-enzymatic (VitE, GSH, VitC…) Oxidative damage Lipids, ADN, ARN, Proteins Altered gene expression Signaling pathways, DNA methylation… CARCINOGENESIS Oxidative stress and carcinogenesis

17. Oxidative stress and carcinogenesis • most of these compounds induce cell transformation (Klaunig et al., 2011)

18. Oxidative stress and cell transformation 1. Acrylonitrile 2. Chromium 3. Fibres and particles (e.g. Silica)

19. Example : Acrylonitrile • Acrylonitrile (ACN) : • intermediate used in manufacture of acrylic fibres, plastics, synthetic rubbers and resins • exposition : manufacturing process, end-product usage, cigarette smoke, drinking water… • IARC 2B • genotoxicity equivocal in vitro and in vivo (Whysner et al., 1998) • absence of ACN-DNA-adducts in brain (major target organ) : mechanisms of carcinogenesis others than ACN-DNA reactivity (epigenetic mechanisms) • mechanisms of carcinogenesis associated with oxidative stress(Jiang et al., 1998 ; Kamendulis et al., 1999)

20. Metabolism of ACN via P450 ROS by-products via P450 2E1 and through futile cycling Cyanide compounds (reactive epoxide cyanide ethylene oxide) Inhibition of the mitochondrial respiratory chain Inhibition of antioxidant enzymes activity (e.g. catalase) Acrylonitrile : oxidative stress Acrylonitrile Conjugaison with the cellular antioxidant GSH (major route of detoxification of ACN) Early depletion of GSH Contribution to an overall decrease in antioxidants Oxidative stress

21. Acrylonitrile : SHE cell transformation • Acrylonitrile induces cell transformation for treatment >24 h: • Decrease of ACN-cell transformation by antioxidants : • Vitamin E (a-tocopherol) • EGCG (-)-epigallocathecin-3-gallate(green tea flavonoid) (adapted from Zhang et al., 2000 ; 2002)

22. Increase of ROS and 8OHdG • Not statistically increase of 8OHdG at 24h • Consistent with the failure of 24h ACN treament to induce TF • Co-treatment with antioxydants result in a decrease in 8OHdG formation Antioxidants inhibit both TF and oxidized DNA damage Acrylonitrile : formation of 8OHdG (adapted from Zhang et al., 2000)

23. Acrylonitrile : effect on oxidant/antioxidants Effects of ACN on catalase and xanthine oxidase can be cancelled by inhibition of P450 activity. P450 metabolism required for ACN-induced oxidative stress and cell transformation ? • Early and temporal depletion of enzymatic and non enzymatic antioxidant • GSH, catalase, superoxide dismutase • Activation of oxidant enzyme • xanthine oxidase (adapted from Zhang et al., 2002)

24. Acrylonitrile : P450 metabolism & TF • P450 metabolism is required for acrylonitrile effects : • Co-treatment with 1-aminobenzotriazole (ABT) (nonspecific suicidal inhibitor of P450) (adapted from Zhang et al., 2002 ; Kamendulis et al., 2002)

25. Acrylonitrile : conclusion • Involvement of oxidative stress in cell transformation induced by ACN : • Cell transformation is correlated with the formation of ROS and the increase of 8OHdG • Cell transformation can be inhibited by antioxidants (EGCG and vitamin E) • ROS induced by ACN may regulate expression of antioxidant defenses (Cat, SOD, GSH) and oxidant enzyme (xanthine oxidase) • Production of ROS requires oxidative metabolism and/or a metabolite of ACN

26. Oxidative stress in cell transformation 1. Acrylonitrile 2. Chromium 3. Fibres and particles (e.g. Silica)

27. Chromium • Metalloid compounds : • toxic metals : As, Cd, Ni, Pb, Cr... • known to induce adverse effects on humans & promote carcinogenesis • toxicological properties partly related to generation of ROS • e.g. direct mitochondrial respiration damage, ROS production via Fenton reaction, lipid peroxidation, depletion of antioxydants… • Chromium compounds : • associated with malignant disease (e.g. lung cancer) • exposure : welding, tanneries, chromium plating, exhaust from cars, cigarette smoke... • Cr(VI) compounds : human carcinogen IARC 1 • Cr(III) : IARC 3, but key role in carcinogenesis induced by Cr(VI)

28. Chromium (VI) SHE cell transformation • Hexavalent chromium compounds : • Zn, Ca, Sr, Pb, Ba • induce dose-dependant cell transformation • in vivo validation of transformed SHE cells • MT colonies checked for colony formation in soft agar and injected back in newborn hamsters • SHE morphologically transformed colonies induced by chromium compounds acquire tumorigenic potential in time strontium chromate calcium chromate (Elias et al., 1989)

29. Chromium (VI) compounds • Potential carcinogenic mechanisms of chromium compounds Cr(III) CrSO42- Fenton-like reaction : generation of hydroxyl radicals Reduction Cr(VI) → Cr(III) Cr-DNA adducts (e.g. Cr-Asc, Cr-GSH…) Oxidative stress may be involved in carcinogenesis and cell transformation induced by Cr(VI) (adapted from Henkler et al., 2010)

30. Oxidative stress in cell transformation 1. Acrylonitrile 2. Chromium 3. Fibres and particles (e.g. Silica)

31. Cell transformation induced by fibres & particles • CTA recommended as alternative methods for evaluation of carcinogenicity of solid material (Fubini et al., 1998 - ECVAM workshop 1998)

32. Diatomaceous earth Quartz & Zeolite Asbestos Fibres & particles • Factors involved in the toxicity / carcinogenicity : • chemical composition, structure, type • dimension, diameter, length (phagocytosis) • surface reactivity • ability to generate ROS • oxidative damage • Genotoxicity : • weak mutagenicity, aneugen and clastogen effects • oxidative DNA damage • e.g. Silica : • crystalline silica (quartz, cristobalite, tridymite…) : IARC 1 • amourphous silica (diatomaceous earth, calcined DE…) : IARC 3

33. ROS induced by silica Main sources of ROS generated by silica Inflammation Particle-generated free radicals and ROS Cell-generated ROS Neutrophils & Alveolar macrophages • Silica-based surface radicals • SiO•, SiO2, SiO3•, Si+ O2•- Iron site active at the surface • Cells damage & lung injury • Membrane damage through lipid peroxidation : ↑of permeability, perturbation of intracellular homeostasis • Activation of cell signaling pathways (MAPK/ERK kinase…) • Activation of transcription factors (NFkB, AP-1, Nfr2…) • Increased expression of inflammatory cytokines (TNFa, IL-1…) • Apoptosis induction : mitochondrial dysfunction • increased gene expression of death receptors & ligands (TNFa, FasL…)

34. * DE Min HF dfx Cell transformation induced by silica • Treatments surface influence cell transformation of silica: • Relationship between cell transformation, surface treatment and ROS generation : * (30.4 µg/cm²) (Fubini et al., 2001)

35. Oxidative stress in silica-induced TF • Co-treatments with antioxidants decrease cell transformation : mannitol : quencher of HO• , catalase : dismutation of H2O2, SOD : dismutation of O2-• • surface radicals react with H2O and/or H2O2 to form HO• • catalase can inhibit this reaction (not SOD) • consistent with the larger efficiency of catalase in inhibiting TF (adapted from Fubini et al., 2001)

36. Conclusion : Silica • Ability of surface to generate ROS: • relevant role in cell transformation, inhibition with antioxidant enzymes • similar results obtained with refractory ceramic fibers (Elias et al., 2002) • Role of iron present at the surface in cell transformation : • Small iron contamination present at the surface : • increase biological reactivity of particles • iron chelation but also iron coating decrease cell transformation • Large iron contamination : • decrease some adverse effects • reduce cellular uptake • decrease attachment to the cell surface and/or internalization of iron-coated particles • → decrease of cell transformation

37. Conclusion : CTA / oxidative stress • Relevance of CTA : • CTA provide evidence for several stages in neoplastic progression (long term) • accurate and comprehensive recapitulation of the in vivo neoplastic process • clarify carcinogenecity in case of ambiguous genotoxicity • may be indicative of mode of action of substances (duration treatment, sequential treatment, co-treatment…) • Mutagenicity / genotoxicity assays in case of oxidative stress : • indicative of mainly single changes • in case of reparation of oxidative DNA damage : escape to the genotoxicity screening • some events not directly related to DNA damage may occur before reparation : • activation by ROS of signaling pathways / epigenetic mechanisms • contribution to growth and neoplastic transformation

38. Thank you for your attention

40. a block in cellular differentiation visualised as morphological transformation in the SHE CTA the acquisition of immortality expressed by : unlimited lifespan, aneuploid karyotype, genetic instability the acquisition of tumourigenicity closely associated with : in vitro phenotypes of foci formation, autocrine factor production, anchorage independent growth in semi solid agar malignant growth when transformed cells are injected back into a suitable host Characteristics of transformed cells (LeBoeuf et al., 1999)

42. maintenance : inheritance of gene silencing de novo methylation : increase promoter methylation status DNMTs Me HYPERMETHYLATION : Block access to the transcriptional machinery of the promoter  Gene silencing : tumor suppressor CpG islands in promoter region (Methyl CpG binding domain MBP, Methyl CpG binding protein) HYPOMETHYLATION :  Gene activation : oncogenes Transcriptional activity regulated by the chromatin’s acetylation status : Histones acetylation/déacetylation involved in the acquisition of transformed phenotype aberrant gene expression associated with malignant transformation CARCINOGENESIS

43. Endogenous sources of ROS PEROXISOMES MITOCHONDRIA INFLAMMATORY CELLS Organelles, consomption of O2 Activation (endo / exogenous stimuli) of neutrophils, eosinophils, macrophages Mitochondrial electron transport chain Complex I : NADH-ubiquinone oxidoreductase Complex III : ubiquinol cytochrome c oxidoreductase Production of ROS involves peroxisomal oxidase (acyl coA and xanthine oxidase) • Undergo to a respiratory burst : • increase in oxygen uptake • generation of ROS through NADPH oxidase Peroxisome proliferator increase the number and size of peroxisomes Ex : hypolipidemic drugs, phthalates, ester, halogenated coumpounds… ROS-generated at complex III regulate hypoxic activation of HIFs (hypoxia inducible factors), transcription factors, involved in cell proliferation and angiogenesis Phagocyte-derived ROS may be involved in the development of cancers Link between peroxisome proliferation - induced ROS and liver tumorigenesis

44. Origin of ROS in silica cell transformation • Nature and origin of ROS implied in silica cell transformation : • Clear-cut relationship between HO• release and cell transformation • Antioxidants decrease cell transformation : • mannitol : quencher of HO• • catalase : dismutation of H2O2 • SOD : dismutation of O2-• • Iron yield of HO• radical via the Fenton reaction : • Aqueous suspensions of quartz generate : H2O2, HO•, H2O2, O2 • Fenton reaction (ferrous ions impurity at the surface) • Fe2+ + H2O2 → Fe3+ + OH- + HO• Involvement of HO•, H2O2, O2-• in cell damages

45. Origin of ROS in silica cell transformation • Iron yield of HO• radical via the Haber-Weiss cycle • In the presence of reductants (metabolites such as ascorbate, cysteine, gluthatione) • O2-• as intermediate • Fe3+ + reductant (n) → Fe2+ + reductant (n-1) • (n= redox state of the reductant molecule) • this way has already been hypothesized for asbestos fibres Any scavengers of HO•, H2O2, O2-• mannitol, catalase, SOD inhibit the overall reaction

46. Origin of ROS in silica cell transformation • Parallel free-radical mechanisms not involving iron and O2•-: • surface radicals react with H2O and/or H2O2 to form HO• Surface radicals SiO•, SiO2, SiO3•, Si+ O2•- Catalase (not SOD) inhibit this reaction • consistent with the larger efficiency of catalase in inhibiting cell transformation

48. Metal mixture Balb3T3 cell transformation • e.g. metal mixture (As, Cd, Pb) • As, Cd : IARC 1 and Pb : IARC 2B • Induced cell transformation in Balb3T3 system • Oxidative damage markers related with cell transformation of metal mixture ↑ ROS and ↓ cell viability : significantly correlated with the ↑ of cell transformation (Silva-Aguilar et al., 2011)

49. Metal mixture Balb3T3 cell transformation • Metal mixture induced cell transformation as both initiator and promoter • Co-treatment with antioxidant N-acetyl-cysteine* Decreased TF if metals are initiator-only Abolished TF if metals are promoter initiation phase promotion phase Metal mixture : 2 µM As, 2µM Cd, 5µM Pb ; TPA : 0.1 µg/mL ; MNNG : 0.5 µg/mL * Cysteine donor, promotes the reduction of glutathione (GSH) (Silva-Aguilar et al., 2011)

50. Metal mixture Balb3T3 cell transformation