1. WP 6 UMR de Toxicologie Alimentaire INRA WP6 : Effects of garlic on carcinogen metabolizing enzymes 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) Marie-Hélène Siess, Marie-France Vernevaut, Patrick Tassin

2. WP 6 UMR de Toxicologie Alimentaire INRA 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 ?

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

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

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

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

7. WP 6 UMR de Toxicologie Alimentaire INRA WP6 : Effects of garlic on carcinogen metabolizing enzymes CYP 2B: Activation of aflatoxin B 1 marker : Pentoxyresorufin dealkylation Effects of garlic powders and DADS on CYP 2B activity

8. WP 6 UMR de Toxicologie Alimentaire INRA 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

9. WP 6 UMR de Toxicologie Alimentaire INRA WP6 : Effects of garlic on carcinogen metabolizing enzymes CYP 3A: Activation of aflatoxin B 1 marker : nifedipine oxidation Effects of garlic powders and DADS on CYP 3A activity

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

11. WP 6 UMR de Toxicologie Alimentaire INRA WP6 : Effects of garlic on carcinogen metabolizing enzymes Linear regression between sulfur content of garlic and UGT activity Total sulfur (ppm) 3000400050006000 10 15 20 25 30 Activity (nmoles/min/mg protein) Coefficients: y= ax+b b=10.8 a=0.00236 r²=0.311

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

13. WP 6 UMR de Toxicologie Alimentaire INRA WP6 : Effects of garlic on carcinogen metabolizing enzymes T S0S1S2 S3 D Effects of garlic powders and DADS on the induction of GST A

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

15. WP 6 UMR de Toxicologie Alimentaire INRA WP6 : Effects of garlic on carcinogen metabolizing enzymes CONCLUSIONS  Reduction of CYP 2 E1  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.

16. WP 6 UMR de Toxicologie Alimentaire INRA 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)

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

18. WP 6 UMR de Toxicologie Alimentaire INRA 0 µM Results : inactivation of human CYP 2A6 by 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 50 µM 400 µM 100 µM 200 µM Without DADSO DADSO DADSO + GSH

19. WP 6 UMR de Toxicologie Alimentaire INRA 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