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WP6 : Effects of garlic on carcinogen metabolizing enzymes

WP6 : Effects of garlic on carcinogen metabolizing enzymes. Marie-Hélène Siess, Marie-France Vernevaut, Patrick Tassin. Milestones :. In vitro effects of garlic compounds on human CYP isoenzymes (Year 1).

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WP6 : Effects of garlic on carcinogen metabolizing enzymes

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  1. WP6 : Effects of garlic on carcinogen metabolizing enzymes Marie-Hélène Siess, Marie-France Vernevaut, Patrick Tassin Milestones : • In vitro effects of garlic compounds on human CYP isoenzymes (Year 1) • In vivo effects of garlic extracts on several carcinogen metabolising enzymes (phase I and II) (year 2) WP 6 UMR de Toxicologie Alimentaire INRA

  2. In vivo effects of garlic extracts on several carcinogen metabolizing enzymes in the rat liver • To better understand the anticarcinogenic capacities of garlic extracts by measuring enzymes which are involved either in the activation (CYPs) or in the detoxification (Phase II) of carcinogens • Is there a relation between the sulfur content of the garlic and its effect on these enzymes ? WP 6 UMR de Toxicologie Alimentaire INRA

  3. liver Garlic extracts Printanor 2000 (0,50,100,200 SO4) In vivo effects of garlic extracts on several carcinogen metabolizing enzymes in the rat liver EXPERIMENTAL CytosolsMicrosomes Enzyme assaysCYPs 1A, 2B, 2E1, 3A Phase II: Glutathione transferase Quinone reductase UDP-glucuronyl transferase Immunodetectionof isoenzymes WP 6 UMR de Toxicologie Alimentaire INRA

  4. WP6 : Effects of garlic on carcinogen metabolizing enzymes Effects of garlic powders and DADS on food intake of rats WP 6 UMR de Toxicologie Alimentaire INRA

  5. WP6 : Effects of garlic on carcinogen metabolizing enzymes Effects of garlic powders and DADS on the weights of rats WP 6 UMR de Toxicologie Alimentaire INRA

  6. WP6 : Effects of garlic on carcinogen metabolizing enzymes CYP 1A : Activation of hydrocarbons marker : Ethoxyresorufin deethylation Effects of garlic powders and DADS on CYP1A activity WP 6 UMR de Toxicologie Alimentaire INRA

  7. WP6 : Effects of garlic on carcinogen metabolizing enzymes CYP 2B: Activation of aflatoxin B1 marker : Pentoxyresorufin dealkylation Effects of garlic powders and DADS on CYP 2B activity WP 6 UMR de Toxicologie Alimentaire INRA

  8. WP6 : Effects of garlic on carcinogen metabolizing enzymes CYP 2E1: Activation : nitrosamines, low molecular weight chemicals marker : p-nitrophenol hydroxylation Effects of garlic powders and DADS on CYP 2E1 activity WP 6 UMR de Toxicologie Alimentaire INRA

  9. WP6 : Effects of garlic on carcinogen metabolizing enzymes CYP 3A: Activation of aflatoxin B1 marker : nifedipine oxidation Effects of garlic powders and DADS on CYP 3A activity WP 6 UMR de Toxicologie Alimentaire INRA

  10. WP6 : Effects of garlic on carcinogen metabolizing enzymes UGT: Detoxication of polycyclic hydrocarbons, heterocyclic amines Effects of garlic powders and DADS on UDP glucuronosyl transferase activity WP 6 UMR de Toxicologie Alimentaire INRA

  11. 30 25 Activity (nmoles/min/mg protein) 20 15 10 3000 4000 5000 6000 Total sulfur (ppm) WP6 : Effects of garlic on carcinogen metabolizing enzymes Coefficients: y= ax+b b=10.8 a=0.00236 r²=0.311 Linear regression between sulfur content of garlic and UGT activity WP 6 UMR de Toxicologie Alimentaire INRA

  12. WP6 : Effects of garlic on carcinogen metabolizing enzymes GST: Detoxication of several carcinogens (ex aflatoxin B1) Effects of garlic powders and DADS on glutathion S-transferase activity WP 6 UMR de Toxicologie Alimentaire INRA

  13. WP6 : Effects of garlic on carcinogen metabolizing enzymes T S3 S0 S1 S2 D Effects of garlic powders and DADS on the induction of GST A WP 6 UMR de Toxicologie Alimentaire INRA

  14. WP6 : Effects of garlic on carcinogen metabolizing enzymes QR: Detoxication of quinones Effects of garlic powders and DADS on quinone reductase activity WP 6 UMR de Toxicologie Alimentaire INRA

  15. WP6 : Effects of garlic on carcinogen metabolizing enzymes CONCLUSIONS • Reduction of CYP 2E1  reduction of the activation of nitrosamines or low molecular weight carcinogens. • Increase of of CYP 1A activation of polycyclic aromatic hydrocarbons. • Induction of UGT prevention of the genotoxic action of polycyclic aromatic hydrocarbons and heterocyclic aromatic amines. • correlation between the level of fertilisation and the increase of UGT. • Increase of GST and QR increase of detoxication of carcinogens. • These hypotheses will be further checked by performing experiments, where the modulation of genotoxicity of these carcinogens will be assessed by the Ames test. WP 6 UMR de Toxicologie Alimentaire INRA

  16. In vitro effects of garlic compounds on human CYPs (2A6, 2E1 and 3A4) Anticarcinogenic properties of garlic compounds could be explained by the inhibition of the activities of CYPs which are able to activate carcinogens Objective : to determine and to compare the inactivation capacities of DADSO and DADS on these human CYPs (in vitro) WP 6 UMR de Toxicologie Alimentaire INRA

  17. Procedure for monitoring the inactivation of human CYPs DADSO, DADS NADPH Microsomes Substrate Cofactors Preincubation Enzyme assay WP 6 UMR de Toxicologie Alimentaire INRA

  18. Results : inactivation of human CYP 2A6 by DADSO Without DADSO 0 µM 50 µM DADSO + GSH 100 µM 200 µM 400 µM DADSO Criteria for mechanism-based inactivation: - time dependent loss of activity - rate of inactivation is proportional to the concentration of inhibitor Prevention of CYP inactivation by glutathione Hypothesis : DADSO reacts directly with SH of the enzyme WP 6 UMR de Toxicologie Alimentaire INRA

  19. Conclusions : Inactivation of human CYPs by DADSO and DADS DADSO and DADS inactivate CYPs 2A6, 2E1 and 3A4, involved in carcinogen activation The rate of inactivation is more important for DADSO as compared with DADS (No inactivation of CYP 2E1 by DADS) The affinity of DADSO for different CYPs is higher as compared with DADS WP 6 UMR de Toxicologie Alimentaire INRA

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