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

  3. In vitro effects of garlic compounds on human CYP enzyme activities Marie-Hélène Siess et al. Working hypothesis: Anticarcinogenic properties of garlic compounds 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

  4. 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

  5. In vitro effects of garlic compounds on human CYPs enzyme activities 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

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

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

  8. In vitro effects of garlic compounds on human CYP enzyme activities CONCLUSIONS  CYP 1A2 and CYP 2A6 are inhibited by allicine. No effect of the others compounds CYP 2E1 is inhibited (40-60 %), by allicine, DADS, allyl glutathione sulfide and allyl mercaptan (500 µM) Most garlic compounds fail to inhibit CYP enzyme activities, at 50 µM WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  9. 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 and Christine Belloir Working hypothesis :Protection against the genotoxic effects of carcinogens conferred by garlic could be related to the modulation of enzymes involved in the activation or detoxication of carcinogens Objectives : to examine the ability of liver subcellular fractions from rat fed with garlic to modulate the activation or detoxication of carcinogens. WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  10. DNA 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) 2B(PROD) (CYP) 2E1(PNPH) Quinone reductase (QR) 3A(NO) WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  11. 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

  12. 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

  13. 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

  14. Effects of hepatic cytosols on nitrosoquinoline oxide mutagenicity Quinoline oxide Control G0 * G200 Histidine revertants /plate * * * Quinoline oxide (µg / plate) WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  15. Effects of hepatic cytosols on styrene oxide mutagenicity Styrene oxide Control G0 G200 Histidine revertants /plate * * * * * * Styrene oxide (µg / plate) WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  16. 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 G 200 : decreased effect : increased effect compared to control : no effect : slight effect n.d. : not determined WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  17. CONCLUSIONS  Consumption of garlic by rats, modifies the enzymes involved in the activation and the detoxication of carcinogens (weak effect) • Consequence on the reduction of mutagenesis in the Ames test • The antigenotoxic effect of garlic depends on : • the mutagen • the balance between activation and detoxication • The anticarcinogenic effects are very similar to those observed with diallyldisulfide • The increase of detoxication enzymes seems to be a relevant mechanism for the the antigenotoxic effect of garlic WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  18. INVIVO METABOLISM OF GARLIC POWDER IN THE RAT Objective : to investigate the bioavailability and the biotransformation of the sulfur constituents of garlic after a single oral administration • Analysis of several sulfur compounds in tissues of rat, at various periods following a single garlic ingestion. WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  19. EXPERIMENTAL Sampleanalysis Garlic powder i.g. 5 g / kg bw • homogenization • protein precipitation (TCA, 30%) • Extraction with CH2CL2 (3 times) Plasma Stomach Liver Intestine Kidney Urine Detection and Identification: GC-MS, WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  20. 1 : S-allyl-L-cysteine sulfoxide; 3 :  -glutamyl-S-trans-1-propenyl cysteine 2 : -glutamyl-S-allyl cysteine 4 :  -glutamyl phenylalanine Sulfur compounds identified in garlic powder (HPLC) 1 2 3 4 WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  21. Sulfur compounds detected in stomach after a single oral administration of garlic powder diallyldisulfide 2-vinyl-4H-1,3-dithiin 3-vinyl-4H-1,2-dithiin WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  22. AMS AMSO AMSO2 Sulfur compounds detected in plasma after a single oral administration of garlic powder WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  23. AMS AMSO AMSO2 Sulfur compounds detected in liver after a single oral administration of garlic powder WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  24. CONCLUSIONS • Following garlic ingestion , AMS, AMSO and AMSO2 are identified in several tissues (plasma, liver, urine) • In our analysis conditions, neither allicin nor conjugates (AGS, NASAC) are detected • The highest amounts of these metabolites are measured 2-24 hours after garlic ingestion • AMSO2 is the most abundant and the most persistent of these compounds in all analysed tissues • The metabolite profiles are very similar to those obtained when DADS is administered WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  25. GENERAL CONCLUSION Elucidation of some mechanisms of action of garlic and sulphur compounds on different biological events involved in carcinogenesis: inhibition of bio-activation of carcinogens (human cells Hep G2, human liver microsomes) stimulation of detoxification of carcinogens (garlic powder) prevention of genotoxicity (aflatoxin B1 and nitrosamine..;) • Next step : Hepatocarcinogenesis protocol will be performed to confirm the antigenotoxic effects of garlic WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  26. Human intervention study : cancer biomarkers State of the art: • Epidemiological studies indicate that garlic consumption reduces cancer risk • No studies on the effect of garlic on the genotoxic action of environmental carcinogens in man • Indications that garlic have an antigenotoxic and anticarcinogenic effects in animals Aim : to investigate the anticarcinogenic effect of garlic in man, using genetic biomarkers WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  27. Human intervention study : cancer biomarkers Study design : the same as for AS Samples : from placebo group and garlic group (simvastatin group not relevant for cancer) No necessity of duplicate samples Collect of plasma, erythrocytes, lymphocytes and urine WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  28. Human intervention study : cancer biomarkers Measurements : • Alteration of DNA in lymphocytes (comet assay) • Antioxidant status : SOD, GSPX and CAT • Antimutagenic properties of urine • Metabolites of garlic constituents in urine WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  29. Deliverables WP6 WP 6 UMR de Toxicologie Alimentaire INRA Dijon

  30. 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

  31. 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

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