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WP 6 Cancer : Milestones 2003

WP 6 Cancer : Milestones 2003. P12 : INRA, Dijon.  Investigation of the anti-initiating effects of garlic powders using the medium- term-hepatocarcinogenesis model in the rat.  Evaluation of the antigenotoxic properties of garlic powders in tissues of rat.

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WP 6 Cancer : Milestones 2003

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  1. WP 6 Cancer : Milestones 2003 P12 : INRA, Dijon Investigation of the anti-initiating effects of garlic powders using the medium- term-hepatocarcinogenesis model in the rat  Evaluation of the antigenotoxic properties of garlic powders in tissues of rat.

  2.  Previous results on the effects of garlic powder on carcinogen metabolizing enzymes : : Reduction of CYPs involved in the activation of carcinogens  Increase of enzyme involved in the detoxication of carcinogens  Correlation between the level of fertilisation and the increase of some enzyme activities  Hypothesis : This could have a consequence on the toxicity of chemicals which are metabolized by these enzymes Two approaches: genotoxicity study (DNA damage in rat tissues) carcinogenesis study (preneoplasic foci in rat liver)

  3. Antigenotoxic properties of garlic extracts in rat Anne-Marie Le Bon and Christine Belloir Objectives : To assess if garlic ingestion prevent DNA alterations induced by genotoxic compounds in rat liver and colon To determine if the effect is correlated with the level of sulfur compounds in the powder Experimental groups Alliin content (nmoles/mg) Studied material : Garlic powder «  0 kg/ha SO4 58 S0 Garlic powder «  100 kg/ha SO4  » 75 S100 Garlic powder «  200 kg/ha SO4  » 90 S200

  4. 5 6 7 4 5 %garlic powder PROTOCOL : liver intraperitoneal injection of AFB1(aflatoxin B1) DMN (nitrosodimethylamine) (4 hours before sacrifice) 60 Wistar male rats weeks Groups C(control diet) S0 S100 S200 Sacrifice DNA damage evaluation (comet test) LIVER 5 rats / group Genotoxic compound Broken DNA = comet Undamaged DNA

  5. AFQ1 CYP 2B GST A5 CYP 2B AFB1 AFB-epoxide AFB-glutathione CYP 3A CYP 1A DNA AFM1 GST = glutathione-S-transferase Metabolic pathways of Aflatoxin B1 (AFB1)

  6. Metabolic pathways of nitrosodimethylamine (DMN) CYP 2E1 spontaneous DMN CH3+ OH-DMN DNA

  7. 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 RESULTS Effects of garlic ingestion on DNA damage in rat liver Control AFB1 DMN a a b b * * b c b DNA damage (OTM) * * * c * C S0 S100 S200 AFB1 S0 S100 S200 NDMA S0 S100 S200 *: significantly different from the group treated by the genotoxic compound alone (Dunnett’s test, p < 0.05) Means having different letters are significantly different (Newman Keuls ’s test, p < 0.05)

  8. 5 6 7 4 5 %garlic powder Groups C(control diet) S0 S100 S200 (5 rats / group) PROTOCOL : colon administration of DMH (dimethylhydrazine) MNU (methylnitrosourea) (24 or 3 hours before sacrifice) 60 Wistar male rats weeks Sacrifice COLON DNA damage evaluation (comet test)

  9. spontaneous MNU CH3+ DNA Metabolic pathways of dimethylhydrazine (DMH) CYP 2E1 spontaneous OH-CH2-NO-N-CH3 DMH CH3+ methylazoxymethanol DNA Mechanism of action of methylnitrosourea (MNU)

  10. RESULTS Effects of garlic ingestion on DNA damage in rat colon Control DMH MNU 7.00 a a a 6.00 a 5.00 a * 4.00 DNA damage (OTM) b b * 3.00 * b * 2.00 1.00 0.00 C S0 S100 S200 DMH S0 S100 S200 MNU S0 S100 S200 *: significantly different from the group treated by the genotoxic compound alone (Dunnett ’s test, p < 0.05) Means having different letters are significantly different (Newman Keuls ’s test, p < 0.05)

  11. CONCLUSION  In liver : - Garlic powder ingestion reduced DNA damage induced by AFB1 and by DMN. - A significant correlation was found between allin content and the level of DMN genotoxicity.  Mechanism of action : induction of CYP 1A and/or GST (case of AFB1). inhibition of CYP 2E1 (case of DMN).  In colon : - Garlic powder ingestion reduced DNA damage induced by DMH. - No protection against genotoxicity induced by the direct-acting compound MNU.  Mechanism of action : inhibition of CYP 2E1 in the liver and/or in the colon. No scavenging activity of garlic powders in the colon.

  12. Anticarcinogenic properties of garlic extracts in rats Raymond Bergès and Marie-Hélène Siess et al. Objectives :  To assess the anticarcinogenic effect of garlic powder on the appearence of liver preneoplasic foci initiated by diethylnitrosamine or aflatoxin B1 in the rat To determine if the effect is correlated with the level of sulfur compounds in the powder Experimental groups Alliin content (nmoles/mg) Studied material : Garlic powder «  0 kg/ha SO4 58 S0 Garlic powder «  200 kg/ha SO4  » 90 S200 Garlic powder «  400 kg/ha SO4  » 330 S400

  13. PH 0 1 2 3 4 5 6 7 8 9 weeks Basal diet Garlic powder (5% in the diet) 2-Acetylaminofluorene S PHpartial hepatectomy Ssacrifice Carcinogens: Aflatoxin B1 (AFB1) 10 x 0,025 mg/kg Diethylnitrosamine (DEN) (10 x 2,5 mg/kg) PROTOCOL Carcinogen administration Wistar rats Initiation Promotion Measurement of enzyme activities Detection of preneoplasic foci

  14. RESULTS Food intake 45 40 35 30 25 daily intake (g/day) C 20 S0 15 S200 10 S400 5 0 0 1 2 3 4 5 6 7 8 weeks Experimental diets Partial hepatectomy

  15. AFB1 DEN a 300 b a b ab 300 c b 250 c 250 200 200 weight (g) 150 weight (g) 150 100 100 50 50 0 0 Control S0 S200 S400 Control S0 S200 S400 RESULTS Effects of garlic powders on rat body weights at the end of the experimental diet period Means and SEM (n=10). Means having different letters are significantly different (P 0.05 Newman Keuls’ test).

  16. DEN AFB1 500 500 400 400 300 300 weight (g) weight (g) 200 200 100 100 0 0 Control S0 S200 S400 Control S0 S200 S400 RESULTS Rat body weights at the end of the experiment Means and SEM (n=10). Means having different letters are significantly different (P 0.05 Newman Keuls’ test).

  17. RESULTS Effects of garlic powders on the appearance of GST- P positive preneoplastic foci induced by AFB1 in the liver AFB1 AFB1 + Garlic powder S400 GST- P : glutathione S-tranferase Pi

  18. a a a b RESULTS Effects of garlic powders on the number of GST- P positive preneoplastic foci induced by AFB1 in the liver r2 = 0.91 Means and SEM (n=10). Means having different letters are significantly different (P 0.05, Newman Keuls’ test).

  19. a a a b RESULTS Effects of garlic powders on the area of GST- P positive preneoplastic foci induced by AFB1 in the liver r² = 0.95 Means and SEM (n=10). Means having different letters are significantly different (P 0.05 Newman Keuls’ test).

  20. RESULTS Effects of garlic powders on CYP 1A1 activity (ethoxyresorufin O-deethylation) r² = 0.84 b b a a Means and SEM (n=5). Means having different letters are significantly different (P 0.05 Newman Keuls’ test)

  21. RESULTS Effects of garlic powders on glutathione S-transferase activity c r2=0.84 Subunits levels : GST A5 :  S400 b b a Means and SEM (n=5). Means having different letters are significantly different (P 0.05 Newman Keuls’ test)

  22. RESULTS Effects of garlic powders on UDP-glucuronosyl transferase activity r² = 0.89 c b b a Means and SEM (n=5). Means having different letters are significantly different (P 0.05 Newman Keuls’ test)

  23. RESULTS Effects of garlic powders on the apparition of GST- P positive preneoplastic foci induced by DEN in the liver Means and SEM (n=10). Means having different letters are significantly different (P 0.05 Newman Keuls’ test).

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

  25. 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

  26. AFQ1 CYP 2B GST A5 CYP 2B AFB1 AFB-epoxide AFB-glutathione CYP 3A CYP 1A DNA AFM1 GST = glutathione-S-transferase Metabolic pathways of Aflatoxin B1 (AFB1)

  27. 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 CYP 2E1 in the liver  Mechanisms of action unknown

  28. WP6 : GENERAL CONCLUSION  Garlic powder prevents the initiation step of hepato carcinogenesis in the rat.

  29. phase 1 (CYP) PRO-CARCINOGEN phase 1 (CYP) Bio- activation NON TOXIC METABOLITES phase 2 (transferases) ULTIMATE METABOLITE DNA INITIATION OF CARCINOGENESIS

  30. AFB1 phase 1 (CYP)  PRO-CARCINOGEN phase 1 (CYP) NON TOXIC METABOLITES Bio- activation phase 2 (transferases)  ULTIMATE METABOLITE  DNA The efficacy of the inhibition is correlated with the amount of alliin in garlic  INITIATION OF CARCINOGENESIS

  31. DMN PRO-CARCINOGEN CYP 2E1 Bio- activation ULTIMATE METABOLITE The efficacy of the inhibition is correlated with the amount of alliin in garlic  DNA INITIATION OF CARCINOGENESIS

  32. WP6 : GENERAL CONCLUSION  Garlic powder prevents the initiation step of chemical carcinogenesis in the rat.  Garlic sulfur compounds inhibit the genotoxicity induced by different chemicals in human cells.

  33. WP6 : GENERAL CONCLUSION Main results  Garlic powder prevents the initiation step of chemical carcinogenesis in the rat.  Garlic sulfur compound inhibit the genotoxicity induced by different chemicals in human cells.  New metabolic pathways of DADS have been identified in the rat.

  34. 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

  35. Deliverables WP6 Po Po Po Po = poster

  36. Deliverables WP6 DH 8 In vitro and ex vivo metabolism of diallyldisulfide by rat liver enzymes and in vitro by human enzymes by Germain et al.  2002 Sent to Drug Metabolism and Disposition: unacceptable « The data are largely confirmatory of previous in vivo experiments and provide new little information »  2003 Sent to Xenobiotica: provisionally accepted, minor revisions

  37. Deliverables WP6 DH 9 : Inhibition of humancytochromes P450 by allicin and other sulfur compounds from garlic, by Siess et al.  Results non relevant : allicin is the only one compound which has efficacy but for non physiological concentrations (500 µM)  For other compounds (8) no observable effect  Variability observed when repeating the experiments

  38. Deliverables WP6 DH 14Effects of garlic powders with varying alliin contents on hepatic drug-metabolizing enzymes in ratsby Le Bon et al.  June 2003 Sent to Journal of Agricultural and Food Chemistry : requesting to revise the manuscript Referee 1 : « good quality, importance in the field : excellent » « Publish after minor revisions » Referee 2 : « Study is interesting but several weaknesses need to be changed » Questions : « Why is diallyl disulfide included in the study ? » « Why not include pure alliin ? » « Is hepatic enzyme activities of relevance ? »

  39. 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

  40. 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

  41. 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

  42. WP 7 Human intervention study : Milestones 2003 P12 : Participation to the human intervention study by measuring some biomarkers of carcinogenesis P12 : Identification of metabolites of sulfur compounds in urine and plasma

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