Presentation on theme: "Marie-Hélène SIESS et al."— Presentation transcript:
1Marie-Hélène SIESS et al. WP 6 CANCERMarie-Hélène SIESS et al.Participants :P12 : UMR de Toxicologie Alimentaire, INRA, DijonP5 : IRBI, University F.Rabelais, ToursP13 : University of Muenchen, Department of Pharmacy,Center of Drug Research, Muenchen
2RATIONALE 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 carcinogenesisThe probability to have a cancer in our lives is evaluated to 1 out of 2 for men and 1 out of 3 for women.One european citizen out of 4 dies from a cancer.Several means to combat cancer and several progresses have been made in therapy.Among the others prevention is a valuable (effective approach to combat .There is a conse,nsus about the role of consuming fruits & vegetables and the reduction of the risk of cancer.In this context our WP was focused on
3ORIGINAL OBJECTIVES 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 to clarify the metabolism of sulfur compounds of garlic which could be effective on carcinogenesis
4Bioavailability and metabolism of sulfur compounds from garlic
5APPROACHESIn vivo : administration of pure DADS or garlic in rats and measurements of the concentrations of metabolites in several tissuesEx vivo : perfusion of a rat liver by pure DADS and identification of metabolites specifically formed in the liverIn vitro : metabolism of DADS by rat and human subcellular fractions to validate the hypothetical pathways
6Proposed scheme for the metabolism of DADS in the rat AllylmercaptanAllylglutathione sulfideSglutathioneSHAllylmethylsulfideAllylmethylsulfoxideAllylmethylsulfoneMetabolites detected in vitroMetabolites detected in vivo
7CONCLUSION The metabolism of DADS is partly elucidated in rat and manDADS is rapidly absorbed and extensively metabolized.AMSO and AMSO2 are probably excretion compoundsSimilar metabolites profiles are observed after garlic administration
8Role of garlic on the process of carcinogenesis : In vivo studies with rat
9OBJECTIVES To assess the anticarcinogenic effect of garlic consumption on the appearence of liver preneoplasic foci initiated by chemical carcinogens To elucidate the mechanisms of the anticarcinogenic actionTo determine if the anticarcinogenic effect is correlated with the level of sulfur compounds in garlic
11Activating and protecting mechanisms of carcinogenesis Activating mechanismsProtecting mechanimsActivation of chemical pro-carcinogensActivation of pro-oncogenesPromoting effectsAngiogenesisDetoxication of carcinogensProtection of DNATumor supressing genesAntipromoting effectsStimulation of immunityCarcinogenesis is made of a series of events which transform a normal cell to a tumoral cell.Overall the formation of maligant cells results from a balance between mechanisms which promote or activate the process and mechanisms which slow down or inhibit the processInitiation is made of a series of events whereby a carcinogen induces alterations in the genetic make-up of the cell. This results in a lesion that can be inherited and which confers to the cell the potential for neoplastic growth.
12PROTOCOL OF HEPATOCARCINOGENESIS SacrificeweeksGarlic dietPromotionInitiationDetection ofpreneoplastic fociCarcinogen administrationAflatoxin B1 (AFB1) :10 x 0.025mg/kgDiethylnitrosamine (DEN) :10 X 2.5 mg/kg
13RESULTSEffects of garlic on the appearance of preneoplastic foci induced by AFB1 in the liverAFB1AFB1 + Garlic powder S400
14RESULTSEffects of garlic on the number of preneoplastic foci induced by AFB1 in the liverabCorrelation of the nb foci with S content of garlicr =P = 2.93 E-04Means and SEM (n=10).Means having different letters are significantly different (P 0.05, Newman Keuls’ test).
15RESULTSEffects of garlic on the area of preneoplastic foci induced by AFB1 in the liverbaCorrelation of area foci with S content of garlicr =P = 0.006Means and SEM (n=10).Means having different letters are significantly different (P 0.05 Newman Keuls’ test).
16Metabolic pathways of Aflatoxin B1 (AFB1) AFM1AFQ1CYP 1ACYP 2BPhase II enzymesAFB1-glutathioneAFB1-epoxideCYP 2BCYP 3AAFB1DNAInitiation of carcinogenesis
17Effects of garlic on carcinogen metabolizing enzymes RESULTSEffects of garlic on carcinogen metabolizing enzymesTreatmentsS0S200S400CYP 1A-CYP 2BCYP 3AGST UGTCYP : cytochrome P450GST : glutathione transferaseUGT : UDP-glucuronosyltransferase
18RESULTS Effects of garlic on DNA damage induced by AFB1 in rat liver Means (n=5) having different letters are significantly different (P 0.05 Newman Keuls ’s test)Undamaged DNABroken DNA = cometgenotoxicThe comet assay is a very simple , rapid and sensitive technique to analyse DNA damge at the individual cellular level. Under highly alcaline conditions, single-, double strand-breaks and alkali-labile lesions are detected
19Initiation of carcinogenesis Non toxicmetabolitesphase 2(transferases)phase 1(CYP)ProcarcinogenUltimate metaboliteDNAInitiation of carcinogenesis
20RESULTSEffects of garlic on the apparition of preneoplastic foci induced by DEN in the liverMeans and SEM (n=10).
21Initiation of carcinogenesis Metabolic pathways of nitrosamineCYP 2E1spontaneousDENOH-DENCH3+DNADEN undergoes an oxidative N demethylation by CYP 2E1 resulting in the production of a methyl ion, a potent alkylator of DNAInitiation of carcinogenesis
22Effects of garlic on CYP 2E1 activity RESULTSEffects of garlic on CYP 2E1 activityabbbMeans and SEM (n=5).Means having different letters are significantly different (P 0.05 Newman Keuls ’s test)
23RESULTSEffects of garlic on DNA damage induced by nitrosamine in rat liverbcaMeans (n=5) having different letters are significantly different (P 0.05 Newman Keuls ’s test)
24CONCLUSION 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 DENeven if there is an inhibition of the genotoxicity of nitrosamine Mechanisms of action unknown
25Antigenotoxic effects of sulfur compounds in human cells
26OBJECTIVESTo assess if garlic compounds prevent DNA alterations induced by mutagenic compounds in a human cell line, HepG2 cells.The studied molecules were :Range : 5 to 100 µMDADS (diallyl disulfide)DADSO (allicin)SAC (S-allyl cysteine)AM (allyl mercaptan)DNA alterations were measured using the comet test.Undamaged DNABroken DNA = cometGenotoxic compound
27PROTOCOLSGarlic compoundPro-mutagen Pre-treatment studies : cells are treated first with the garlic compoundand then with the pro-mutagenHypothesis : inhibition or induction of xenobiotic metabolizing enzymesMutagens : benzopyrene (BaP)aflatoxin B1 (AFB1)nitrosodimethylamine (NDMA)CYP 1ACYP 1A, 3ACYP 2E1 Co-treatment studies : cells are treated with both the garlic compoundand the direct-acting mutagen at the same timeHypothesis : scavenging of mutagenic compoundsGarlic compound + mutagenMutagens : hydrogen peroxide (H2O2)methyl methane sulfonate (MMS)4-nitrosoquinoline oxide (4-NQO)
28Pre-treatment studies RESULTSPre-treatment studiesDADSDADSOAMSACBaPAFB1()()NDMAOSC display anti G activity when administered before the genotoxic agent, suggesting an effect of these compounds on the metabolic pathway of different carcinogens. : inhibition of the genotoxicity() : slight inhibition- : no effect
29RESULTS Co-treatment studies () DADS DADSO SAC AM H2O2 MMS 4-NQO : inhibition of the genotoxicity() : slight inhibition- : no effect
30CONCLUSIONS Sulfur compounds from garlic prevent the genotoxicity of carcinogens in a human cell lineThey act through different mechanisms of action :modulation of enzymes involved in activation or detoxicationscavenging of ultimate species
31WP6 : 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. 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 garlicIn human cells, garlic sulfur compounds inhibit the genotoxicity induced by different chemicals.
32Expected 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)
37initiation of carcinogenesis APPROACHESnon toxicmetabolitesphase 2(transferases)phase 1(CYP)pro-carcinogenultimate metaboliteBio-activationphase 1(CYP)Activity/expresion of XMEAlteration of DNA (Comet testAmes test)Preneoplastic foci(Immunohistology)DNAinitiation of carcinogenesis