WP 6 UMR de Toxicologie Alimentaire INRA Dijon WP 6 Cancer : General objectives  inhibition of bio-activation of carcinogens  stimulation of detoxification of carcinogens  prevention of genotoxicity  inhibition of the initiation of liver carcinogenesis  influence on apoptosis in tumour and normal cells  To elucidate the mechanisms of action of garlic and sulphur compounds on different biological events involved in carcinogenesis:  To know the transformation and the fate of garlic sulphur compounds ingested up to their targets

WP 6 UMR de Toxicologie Alimentaire INRA Dijon WP 6 Cancer : Milestones 2002  P12 : Determination of the bioavailability of garlic powder in rat by measuring the concentrations of the metabolites in blood and in main organs in progress  P12 : Evaluation of the effects of subcellular fractions from rats treated with garlic extracts on the mutagenicity of carcinogens using the Ames test in progress  P13 Determination of the specificity of garlic compouds/preparation toward apoptosis in specific types of human tumor cell lines  P12 : In vitro effects of garlic compounds on human CYP isoenzymes

WP 6 UMR de Toxicologie Alimentaire INRA Dijon In vivo metabolism of garlic (in progress)  to investigate the in vivo fate of garlic compounds and their metabolites after a single oral administration OBJECTIVES

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Proposed scheme for the metabolism of DADS in rat SH Allylmercaptan AM Allylmethylsulfide AMS Allylmethylsulfoxide AMSO Allylmethylsulfone AMSO 2 S glutathione Allylglutathione sulfide AGS

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Garlic powder i.g. 1.5 g / rat Plasma Liver Urine homogenization 4protein precipitation (TCA, 30%) 4Extraction with CH 2 CL 2 (3 times) Sample analysis (in progress) Detection and Identification: GC-MS, LC-MS EXPERIMENTAL ( in progress)

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Evaluation of the effects of hepatic fractions from rats fed garlic extracts on the mutagenicity of carcinogens in the Ames test Objectives : to assess if the modulation of drug metabolizing enzyme activities induced by garlic ingestion modifies activation/detoxication of mutagens. Anne-Marie Le Bon et al.

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Glucuronosyl transferases (UGT) Glutathione transferases (GST) Phase 1 Phase 2 ADN Mutagenesis Quinone reductase (QR) Cytochromes P450 1A (EROD) 2B (PROD) 2E1 (PNPH) (CYP) RH ROH O R RO+ ROR’ 3A (NO)

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Diet containing garlic powder (5%, 15 days) EXPERIMENTAL DESIGN Cytosol Microsomes livermale Wistar rat homogenization centrifugation Ames test Enzyme activities G 0 (0 kg/ha SO4) G 200 (200 kg/ha SO4)

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Effects of garlic ingestion on liver enzyme activities ERODPRODPNPHNOUGTGSTQR % of control * * ** * * * ** * * C G 0 G 200 Phase 1 enzymes (CYP) Phase 2 enzymes (transferases) (1A)(2B)(2E1)(3A)

WP 6 UMR de Toxicologie Alimentaire INRA Dijon AMES TEST plating on minimal glucose agar plate incubation 48 heures at 37 °C His + revertant colonies counting hepatic fraction bacteria Pro-mutagen or mutagen Salmonella typhimurium Salmonella typhimurium His -His+ Mutagen

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Effects of hepatic microsomes on PhIP mutagenicity Revertants His+ / plate PhIP (ng / plate ) Control G 0 G 200 *

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Effects of hepatic microsomes on aflatoxin B1 (AFB1) mutagenicity

WP 6 UMR de Toxicologie Alimentaire INRA Dijon G 0 G 200 Mutagens (Enzyme involved in metabolism) PhIP (CYP1A1) CP (2B) AFB1 (2B, 3A) SO (GST) 4-NQO (QR) : increased effect : no effect compared to control : slight effect : decreased effect n.d. : not determined NDMA (2E1) n.d. Phase 1 substratesPhase 2 substrates PhIP : heterocyclic amine CP : cyclophosphamide AFB1 : aflatoxin B1 SO : styrene oxide 4-NQO : 4-nitrosoquinoline oxide NDMA : N-nitrosodimethylamine

WP 6 UMR de Toxicologie Alimentaire INRA Dijon CONCLUSIONS  Garlic diet : weak effect on enzyme activities  no consequence on the reduction of mutagenesis in the Ames test  Different results between this ex-vivo study and the in vivo study (protection)  Induction of GST by garlic = mechanism of protection? (demonstrated for DADS, see Guyonnet et al, Carcinogenesis, 2002)

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Working hypothesis: Anticarcinogenic properties of garlic compouds could be explained by the inhibition of several CYPs which are involved in the activation of carcinogens Objectives : To assess the inhibitory effects of garlic compounds and compounds issued from the metabolism of DADS, on human CYP activities In vitro effects of garlic compounds on human CYP enzyme activities Marie-Hélène Siess et al.

WP 6 UMR de Toxicologie Alimentaire INRA Dijon In vitro effects of garlic compounds on human CYP enzyme activities Compounds selected: Garlic compounds : S-allylcysteine, allicine, diallyldisulfide Metabolites of DADS : Allylmercaptan, allylmethyl sulfide, allylmethylsulfone allylmethylsulfoxide, allylglutathione sulfide Doses : 50 µM and 500 µM

WP 6 UMR de Toxicologie Alimentaire INRA Dijon In vitro effects of garlic compounds on human CYPs enzyme activities CYP 1A2 CYP 2A6 CYP 2E1 CYP 3A4 Activation of carcinogens Heterocyclic amines Tobacco nitrosamines Nitrosamines, solvents... Aflatoxine B1, polycyclic aromatic hydrocarbons Enzyme activities Ethoxyresorufine deethylase Coumarine hydroxylase Chlorzoxazone hydroxylase Nifedipine oxidase Enzyme activities measured with microsomes isolated from human livers

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Effects of garlic compounds on human CYP 1A2 N = 4 different human samples

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Effects of garlic compounds on human CYP 2A6

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Effects of garlic compounds on human CYP 2E1

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Effects of metabolites of DADS on human CYP 2E1

WP 6 UMR de Toxicologie Alimentaire INRA Dijon In vitro effects of garlic compounds on human CYP enzyme activities CONCLUSIONS  Most garlic compounds fail to inhibit CYP enzyme activities, at 50 µM  Allicine is the most active inhibitor of CYP 1A2  CYP 2E1 is inhibited (40-60 %), by allicine, DADS, allyl glutathione sulfide and allyl mercaptan (500 µM)

WP 6 UMR de Toxicologie Alimentaire INRA Dijon WP 6 Cancer : General conclusions Are animal and human studies succesful in showing that garlic can prevent cancer ? YESNO  Increase of detoxication enzymes (rat)  Decrease of CYPs (animal, human samples)  In vivo decrease of the mutagenicity of some carcinogens (animal)  Increase of CYP enzymes (rat)  Decrease of CYPs for high concentrations (human samples)  Ex vivo no decrease of the mutagenicity of some carcinogens (animal)

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Bottelnecks: There will be some delay in the experiments because :  Caroline left the lab on february A postdoc will be involved in the program in  Anne-Marie will be absent from the lab (November March 2003) for a maternity leave. No replacement is possible.

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Next steps :2002 and 2003  In vivo metabolism of garlic in rat (in progress)  Hepatocarcinogenesis protocol will be performed to confirm the antigenotoxic effects of garlic powders  Participation to the human intervention study

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Stocks of garlic powders available at September 2002 in my freezer Varieties : Printanor (Morasol, Messidrome are not in my freezer) Field experiments : 2000 and 2001 Countries ; France and Spain Analysis:  2000 trials : analyses from Jacques and Thomas are different  2001 trials : analyses were done by Jacques on individual or pooled levels of fertilisation

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Overview of the garlic powders Printanor FR 2000 ~ 8000 g Printanor SP 2000 ~ g Printanor FR 2001 ~ g Printanor SP 2001 ~ g Levels of fertilisation mixed before making the powders In red color : samples burnt and not available

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Pre-treatment studies DADSDADSOSACAM NDMA AFB1  ()() BaP     In vitro evaluation of antigenotoxic properties of garlic compounds in HepG2 cells (comet test) Garlic compoundPro-mutagen  : inhibition of the genotoxicity (  ) : slight inhibition : no effect