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WP 6 CANCER

WP 6 CANCER. Marie-Hélène SIESS et al. Participants : P12 : UMR de Toxicologie Alimentaire, INRA, Dijon P5 : IRBI, University F.Rabelais, Tours P13 : University of Muenchen, Department of Pharmacy, Center of Drug Research, Muenchen.

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WP 6 CANCER

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  1. WP 6 CANCER Marie-Hélène SIESS et al. Participants : P12 : UMR de Toxicologie Alimentaire, INRA, Dijon P5 : IRBI, University F.Rabelais, Tours P13 : University of Muenchen, Department of Pharmacy, Center of Drug Research, Muenchen

  2.  Cancer is preventable by plant food (eating fruits and vegetables  The project is focused on the mechanisms of action of garlic on critical cellular and molecular events in the process of carcinogenesis RATIONALE  Cancer is one of the first causes of death and morbidity within EU

  3. ORIGINAL OBJECTIVES  to investigate the role of garlic and its sulfur compounds on the process of carcinogenesis through cellular, animal and clinical studies  in vivo studies (rat) with garlic powders  in vitro studies (cells) with isolated sulfur compounds  to clarify the metabolism of sulfur compounds of garlic which could be effective on carcinogenesis

  4. Bioavailability and metabolism of sulfur compounds from garlic

  5. APPROACHES In vivo : administration of pure DADS or garlic in rats and measurements of the concentrations of metabolites in several tissues Ex vivo : perfusion of a rat liver by pure DADS and identification of metabolites specifically formed in the liver In vitro : metabolism of DADS by rat and human subcellular fractions to validate the hypothetical pathways

  6. Allylglutathione sulfide S glutathione Allylmercaptan Allylmethylsulfide Allylmethylsulfoxide Allylmethylsulfone SH Proposed scheme for the metabolism of DADS in the rat Metabolites detected in vitro Metabolites detected in vivo

  7. CONCLUSION  The metabolism of DADS is partly elucidated in rat and man • DADS is rapidly absorbed and extensively metabolized. • AMSO and AMSO2 are probably excretion compounds • Similar metabolites profiles are observed after garlic administration

  8. Role of garlic on the process of carcinogenesis : In vivo studies with rat

  9. OBJECTIVES  To assess the anticarcinogenic effect of garlic consumption on the appearence of liver preneoplasic foci initiated by chemical carcinogens  To elucidate the mechanisms of the anticarcinogenic action To determine if the anticarcinogenic effect is correlated with the level of sulfur compounds in garlic

  10. STUDIED MATERIALS Variety and location Variety Printanor FR Variety Printanor FR Variety Printanor FR Variety Printanor SP Fertilisation SO4 kg /ha 0 100 200 400 Alliin content nmoles/mg 58 75 90 330 Experimental groups SO S100 S200 S400

  11. Activating and protecting mechanisms of carcinogenesis Activating mechanisms Protecting mechanims Activation of chemical pro-carcinogens Activation of pro-oncogenes Promoting effects Angiogenesis Detoxication of carcinogens Protection of DNA Tumor supressing genes Antipromoting effects Stimulation of immunity

  12. Initiation Promotion Sacrifice 0 1 2 3 4 5 6 7 8 9 weeks Garlic diet Carcinogen administration Aflatoxin B1 (AFB1) : 10 x 0.025mg/kg Diethylnitrosamine (DEN) : 10 X 2.5 mg/kg Detection of preneoplastic foci PROTOCOL OF HEPATOCARCINOGENESIS

  13. AFB1 AFB1 + Garlic powder S400 RESULTS Effects of garlic on the appearance of preneoplastic foci induced by AFB1 in the liver

  14. a a a b RESULTS Effects of garlic on the number of preneoplastic foci induced by AFB1 in the liver Correlation of the nb foci with S content of garlic r = - 0.53 P = 2.93 E-04 Means and SEM (n=10). Means having different letters are significantly different (P 0.05, Newman Keuls’ test).

  15. a a a b RESULTS Effects of garlic on the area of preneoplastic foci induced by AFB1 in the liver Correlation of area foci with S content of garlic r = - 0.40 P = 0.006 Means and SEM (n=10). Means having different letters are significantly different (P 0.05 Newman Keuls’ test).

  16. AFQ1 CYP 2B Phase II enzymes CYP 2B AFB1-epoxide AFB1-glutathione CYP 3A CYP 1A DNA AFM1 Initiationof carcinogenesis Metabolic pathways of Aflatoxin B1 (AFB1) AFB1

  17. Treatments S0 S200 S400 CYP 1A -   CYP 3A - - - GST     UGT     CYP 2B - - - RESULTS Effects of garlic on carcinogen metabolizing enzymes CYP : cytochrome P450 GST : glutathione transferase UGT : UDP-glucuronosyltransferase

  18. Undamaged DNA a b genotoxic b b Broken DNA = comet Means (n=5) having different letters are significantly different (P 0.05 Newman Keuls ’s test) RESULTS Effects of garlic on DNA damage induced by AFB1 in rat liver

  19. phase 1 (CYP) Procarcinogen Non toxic metabolites phase 2 (transferases) Ultimate metabolite DNA Initiation of carcinogenesis

  20. RESULTS Effects of garlic on the apparition of preneoplastic foci induced by DEN in the liver Means and SEM (n=10).

  21. Metabolic pathways of nitrosamine CYP 2E1 spontaneous DEN CH3+ OH-DEN DNA Initiationof carcinogenesis

  22. RESULTS Effects of garlic on CYP 2E1 activity a b b b Means and SEM (n=5). Means having different letters are significantly different (P 0.05 Newman Keuls ’s test)

  23. a b c c RESULTS Effects of garlic on DNA damage induced by nitrosamine in rat liver Means (n=5) having different letters are significantly different (P 0.05 Newman Keuls ’s test)

  24. CONCLUSION  Garlic powder ingestion reduced preneoplasic foci induced by AFB1. Mechanisms of action :  induction of CYP 1A and/or GST and UGT  no effect on CYP 2B and 3A  A significant correlation was found between allin content and the anticarcinogenic efficacy  No protection against the carcinogenicity induced by DEN even if there is an inhibition of the genotoxicity of nitrosamine  Mechanisms of action unknown

  25. Antigenotoxic effects of sulfur compounds in human cells

  26. The studied molecules were : DADS (diallyl disulfide) DADSO (allicin) SAC (S-allyl cysteine) AM (allyl mercaptan) Range : 5 to 100 µM DNA alterations were measured using the comet test. Genotoxic compound Broken DNA = comet Undamaged DNA OBJECTIVES To assess if garlic compounds prevent DNA alterations induced by mutagenic compounds in a human cell line, HepG2 cells.

  27.  Pre-treatment studies : cells are treated firstwith the garlic compound and then with the pro-mutagen Garlic compound Pro-mutagen Hypothesis : inhibition or induction of xenobiotic metabolizing enzymes Mutagens : benzopyrene (BaP) aflatoxin B1 (AFB1) nitrosodimethylamine (NDMA) CYP 1A CYP 1A, 3A CYP 2E1  Co-treatment studies : cells are treated with both the garlic compound and the direct-acting mutagen at the same time Garlic compound +mutagen Hypothesis : scavenging of mutagenic compounds Mutagens : hydrogen peroxide (H2O2) methyl methane sulfonate (MMS) 4-nitrosoquinoline oxide (4-NQO) PROTOCOLS

  28. RESULTS Pre-treatment studies DADS DADSO AM SAC   BaP    () AFB1 ()  NDMA   : inhibition of the genotoxicity () : slight inhibition - : no effect

  29. SAC DADS DADSO AM  ()   H2O2    MMS ()   () 4-NQO RESULTS Co-treatment studies  : inhibition of the genotoxicity () : slight inhibition - : no effect

  30. CONCLUSIONS  Sulfur compounds from garlic prevent the genotoxicity of carcinogens in a human cell line They act through different mechanisms of action : modulation of enzymes involved in activation or detoxication scavenging of ultimate species

  31. WP6 : GENERAL CONCLUSION  The metabolism of sulfur compounds such diallyl disulfide is partly elucidated in rat and man. New metabolites and pathways are identified in the rat.  Garlic consumption prevents the initiation step of chemical hepatocarcinogenesis in the rat. Some mechanism of action are elucidated. The efficacy is correlated with the S-content of garlic In human cells, garlic sulfur compounds inhibit the genotoxicity induced by different chemicals.

  32. Expected impacts of the WP 6 outputs  To contribute to improve the knowledge for the prevention of cancer (scientific community)  To contribute to policy design on the prevention of cancer (health public agencies)  To contribute to fully optimize the health effects of garlic (breeders, farmers, agrofood industries)

  33. Deliverables WP6 Po Po Po Po = poster

  34. Participants of my lab Scientists : Anne-Marie, Caroline, Engineer : Raymond Technicians : Christine, Joëlle, Lucien, Marie-France, Marie-France Post-docs : Emmanuelle and Varsha Students : Caroline, Néjia, Sophie, Marlène, Anne, Céline

  35. phase 1 (CYP) pro-carcinogen Activity/expresion of XME phase 1 (CYP) non toxic metabolites Bio- activation phase 2 (transferases) ultimate metabolite Alteration of DNA(Comet test Ames test) DNA Preneoplastic foci (Immunohistology) initiation of carcinogenesis APPROACHES

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