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WP 6 Cancer : General objectives

WP 6 Cancer : General objectives.  To elucidate the mechanisms of action of garlic and sulphur compounds on different biological events involved in carcinogenesis:.  inhibition of bio-activation of carcinogens  stimulation of detoxification of carcinogens  prevention of genotoxicity

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WP 6 Cancer : General objectives

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  1. WP 6 Cancer : General objectives To elucidate the mechanisms of action of garlic and sulphur compounds on different biological events involved in carcinogenesis: inhibition of bio-activation of carcinogens stimulation of detoxification of carcinogens prevention of genotoxicity inhibition of the initiation of liver carcinogenesis influence on apoptosis in tumour and normal cells To know the transformation and the fate of garlic sulphur compounds ingested up to their targets WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  2. WP 6 Cancer : Milestones 2002 P12 : Evaluation of the effects of subcellular fractions from rats treated with garlic extracts on the mutagenicity of carcinogens using the Ames test in progress P12 : Determination of the bioavailability of garlic powder in rat by measuring the concentrations of the metabolites in blood and in main organs in progress P12 : In vitro effects of garlic compounds on human CYP isoenzymes P13 Determination of the specificity of garlic compouds/preparation toward apoptosis in specific types of human tumor cell lines WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  3. Invivo metabolism of garlic (in progress) OBJECTIVES • to investigate the in vivo fate of garlic compounds and their metabolites after a single oral administration WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  4. SH S glutathione Proposed scheme for the metabolism of DADS in rat Allylglutathione sulfide AGS Allylmercaptan AM Allylmethylsulfide AMS Allylmethylsulfoxide AMSO Allylmethylsulfone AMSO2 WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  5. EXPERIMENTAL (in progress) Sampleanalysis (in progress) Garlic powder i.g. 1.5 g / rat • homogenization • protein precipitation (TCA, 30%) • Extraction with CH2CL2 (3 times) Liver Urine Detection and Identification: GC-MS, LC-MS Plasma WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  6. Evaluation of the effects of hepatic fractions from rats fed garlic extracts on the mutagenicity of carcinogens in the Ames test Anne-Marie Le Bon et al. Objectives : to assess if the modulation of drug metabolizing enzyme activities induced by garlic ingestion modifies activation/detoxication of mutagens. WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  7. ADN Mutagenesis O R R O R’ RH Phase 2 R O + R O H Phase 1 Cytochromes P450 1A (EROD) Glucuronosyl transferases (UGT) Glutathione transferases (GST) (CYP) 2B (PROD) 2E1 (PNPH) Quinone reductase (QR) 3A (NO) WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  8. homogenization Cytosol centrifugation Microsomes G 0 (0 kg/ha SO4) G 200 (200 kg/ha SO4) male Wistar rat liver Enzyme activities Ames test EXPERIMENTAL DESIGN Diet containing garlic powder (5%, 15 days) WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  9. 400 C G 0 300 G 200 * * 200 * % of control * * * * * * 100 * * 0 EROD PROD PNPH NO UGT GST QR (1A) (2B) (2E1) (3A) Phase 1 enzymes (CYP) Phase 2 enzymes (transferases) Effects of garlic ingestion on liver enzyme activities WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  10. Mutagen Salmonella typhimurium His - His+ hepatic fraction plating on minimal glucose agar plate bacteria His+ revertant colonies counting Pro-mutagen or mutagen . . Salmonella typhimurium . . . . . incubation 48 heuresat 37 °C AMES TEST WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  11. 300 G 200 250 Control 200 G 0 Revertants His+ / plate 150 * 100 50 0 0 500 1000 2000 3000 PhIP (ng / plate ) Effects of hepatic microsomes on PhIP mutagenicity WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  12. 1000 G 0 Control 800 * G 200 600 Revertants His+/ plate * 400 * 200 * 0 0 6 13 25 50 100 AFB1 (ng / plate ) Effects of hepatic microsomes on aflatoxin B1 (AFB1) mutagenicity WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  13. PhIP : heterocyclic amine CP : cyclophosphamide AFB1 : aflatoxin B1 NDMA : N-nitrosodimethylamine SO : styrene oxide 4-NQO : 4-nitrosoquinoline oxide Mutagens (Enzyme involved in metabolism) Phase 1 substrates Phase 2 substrates AFB1 (2B, 3A) NDMA (2E1) PhIP (CYP1A1) CP (2B) SO (GST) 4-NQO (QR) G 0 n.d. G 200 n.d. : decreased effect : increased effect compared to control : no effect : slight effect n.d. : not determined WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  14. CONCLUSIONS  Garlic diet : weak effect on enzyme activities  no consequence on the reduction of mutagenesis in the Ames test  Different results between this ex-vivo study and the in vivo study (protection)  Induction of GST by garlic = mechanism of protection? (demonstrated for DADS, see Guyonnet et al, Carcinogenesis, 2002) WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  15. In vitro effects of garlic compounds on human CYP enzyme activities Marie-Hélène Siess et al. Working hypothesis: Anticarcinogenic properties of garlic compouds could be explained by the inhibition of several CYPs which are involved in the activation of carcinogens Objectives : To assess the inhibitory effects of garlic compounds and compounds issued from the metabolism of DADS, on human CYP activities WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  16. In vitro effects of garlic compounds on human CYP enzyme activities Compounds selected: Garlic compounds : S-allylcysteine, allicine, diallyldisulfide Metabolites of DADS : Allylmercaptan, allylmethyl sulfide, allylmethylsulfone allylmethylsulfoxide, allylglutathione sulfide Doses : 50 µM and 500 µM WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  17. In vitro effects of garlic compounds on human CYPs enzyme activities Enzyme activities Ethoxyresorufine deethylase Coumarine hydroxylase Chlorzoxazone hydroxylase Nifedipine oxidase CYP 1A2 CYP 2A6 CYP 2E1 CYP 3A4 Activation of carcinogens Heterocyclic amines Tobacco nitrosamines Nitrosamines, solvents ... Aflatoxine B1, polycyclic aromatic hydrocarbons Enzyme activities measured with microsomes isolated from human livers WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  18. Effects of garlic compounds on human CYP 1A2 N = 4 different human samples WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  19. Effects of garlic compounds on human CYP 2A6 WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  20. Effects of garlic compounds on human CYP 2E1 WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  21. Effects of metabolites of DADS on human CYP 2E1 WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  22. In vitro effects of garlic compounds on human CYP enzyme activities CONCLUSIONS Most garlic compounds fail to inhibit CYP enzyme activities, at 50 µM Allicine is the most active inhibitor of CYP 1A2 CYP 2E1 is inhibited (40-60 %), by allicine, DADS, allyl glutathione sulfide and allyl mercaptan (500 µM) WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  23. WP 6 Cancer : General conclusions Are animal and human studies succesful in showing that garlic can prevent cancer ? YES NO • Increase of detoxication enzymes (rat) • Decrease of CYPs (animal, human samples) • In vivo decrease of the mutagenicity of some carcinogens (animal) Increase of CYP enzymes (rat) Decrease of CYPs for high concentrations (human samples) Ex vivo no decrease of the mutagenicity of some carcinogens (animal) WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  24. Bottelnecks: There will be some delay in the experiments because : Caroline left the lab on february 2002. A postdoc will be involved in the program in 2003. Anne-Marie will be absent from the lab (November 2002 -March 2003) for a maternity leave. No replacement is possible. WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  25. Next steps :2002 and 2003 In vivo metabolism of garlic in rat (in progress) Hepatocarcinogenesis protocol will be performed to confirm the antigenotoxic effects of garlic powders Participation to the human intervention study WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  26. Stocks of garlic powders available at September 2002 in my freezer Varieties : Printanor (Morasol, Messidrome are not in my freezer) Field experiments : 2000 and 2001 Countries ; France and Spain Analysis: 2000 trials : analyses from Jacques and Thomas are different 2001 trials : analyses were done by Jacques on individual or pooled levels of fertilisation WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  27. Overview of the garlic powders Printanor FR 2000 ~ 8000 g Levels of fertilisation mixed before making the powders Printanor SP 2000 ~ 17273 g Printanor FR 2001 ~ 20750 g In red color : samples burnt and not available Printanor SP 2001 ~ 13140 g WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  28. Garlic compound Pro-mutagen DADS DADSO AM SAC   BaP  ()  AFB1   NDMA  : inhibition of the genotoxicity () : slight inhibition : no effect In vitro evaluation of antigenotoxic properties of garlic compounds in HepG2 cells (comet test) Pre-treatment studies WP 6 UMR de Toxicologie Alimentaire INRA Dijon

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