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WP 6 CANCER Participants : P12 : UMR de Toxicologie Alimentaire, INRA, Dijon P5 : IRBI, University F.Rabelais, Tours P13 : University of Muenchen, Department.

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Presentation on theme: "WP 6 CANCER Participants : P12 : UMR de Toxicologie Alimentaire, INRA, Dijon P5 : IRBI, University F.Rabelais, Tours P13 : University of Muenchen, Department."— Presentation transcript:

1 WP 6 CANCER 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 Marie-Hélène SIESS et al.

2 RATIONALE  Cancer is one of the first causes of death and morbidity within EU  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

3  to clarify the metabolism of sulfur compounds of garlic which could be effective on carcinogenesis  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 ORIGINAL OBJECTIVES

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 Proposed scheme for the metabolism of DADS in the rat AllylmethylsulfideAllylmethylsulfoxideAllylmethylsulfone Allylmercaptan Allylglutathione sulfide S glutathione SH Metabolites detected in vitroMetabolites 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 AMSO 2 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  To assess the anticarcinogenic effect of garlic consumption on the appearence of liver preneoplasic foci initiated by chemical carcinogens OBJECTIVES  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 SP Experimental groups SO S100 S200 S400 Fertilisation SO 4 kg /ha Alliin content nmoles/mg

11 Activating mechanismsProtecting 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 Activating and protecting mechanisms of carcinogenesis

12 PROTOCOL OF HEPATOCARCINOGENESIS Detection of preneoplastic foci Carcinogen administration Aflatoxin B1 (AFB1) : 10 x 0.025mg/kg Diethylnitrosamine (DEN) : 10 X 2.5 mg/kg Sacrifice weeks Garlic diet PromotionInitiation

13 RESULTS Effects of garlic on the appearance of preneoplastic foci induced by AFB1 in the liver AFB1AFB1 + Garlic powder S400

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

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

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

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

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

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

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

21 Metabolic pathways of nitrosamine DENCH3 + CYP 2E1 DNA OH-DEN spontaneous Initiation of carcinogenesis

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

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

27  Co-treatment studies : cells are treated with both the garlic compound and the direct-acting mutagen at the same time Hypothesis : scavenging of mutagenic compounds Garlic compound + mutagen Mutagens : hydrogen peroxide (H 2 O 2 ) methyl methane sulfonate (MMS) 4-nitrosoquinoline oxide (4-NQO) PROTOCOLS Garlic compoundPro-mutagen  Pre-treatment studies : cells are treated first with the garlic compound and then with the 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

28 RESULTS Pre-treatment studies DADSDADSOSACAM NDMA  AFB1   BaP  ()()   : inhibition of the genotoxicity (  ) : slight inhibition - : no effect  ()() 

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

30  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 CONCLUSIONS

31  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 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.  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 = poster Po

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

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


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