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  P12 : Evaluation of the effects of subcellular fractions from rats treated with garlic extracts on the mutagenicity of carcinogens using the Ames test  P13 Determination of the specificity of garlic compounds/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 Working hypothesis: Anticarcinogenic properties of garlic compounds 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 measured with microsomes isolated from human livers

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  CYP 1A2 and CYP 2A6 are inhibited by allicine. No effect of the others compounds  CYP 2E1 is inhibited (40-60 %), by allicine, DADS, allyl glutathione sulfide and allyl mercaptan (500 µM)  Most garlic compounds fail to inhibit CYP enzyme activities, at 50 µM

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 examine the ability of liver subcellular fractions from rat fed with garlic to modulate the activation or detoxication of carcinogens. Anne-Marie Le Bon and Christine Belloir Working hypothesis :Protection against the genotoxic effects of carcinogens conferred by garlic could be related to the modulation of enzymes involved in the activation or detoxication of carcinogens

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Glucuronosyl transferases (UGT) Glutathione transferases (GST) Phase 1 Phase 2 DNA 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 Histidine revertants /plate Quinoline oxide (µg / plate) Quinoline oxide G0 Control G200 Effects of hepatic cytosols on nitrosoquinoline oxide mutagenicity * * * *

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Styrene oxide (µg / plate) Histidine revertants /plate Effects of hepatic cytosols on styrene oxide mutagenicity Styrene oxide G0 Control G200 * * **** ****

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) 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  Consumption of garlic by rats, modifies the enzymes involved in the activation and the detoxication of carcinogens (weak effect)  Consequence on the reduction of mutagenesis in the Ames test  The antigenotoxic effect of garlic depends on :  the mutagen  the balance between activation and detoxication  The increase of detoxication enzymes seems to be a relevant mechanism for the the antigenotoxic effect of garlic  The anticarcinogenic effects are very similar to those observed with diallyldisulfide

WP 6 UMR de Toxicologie Alimentaire INRA Dijon IN VIVO METABOLISM OF GARLIC POWDER IN THE RAT  Analysis of several sulfur compounds in tissues of rat, at various periods following a single garlic ingestion. Objective : to investigate the bioavailability and the biotransformation of the sulfur constituents of garlic after a single oral administration

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Garlic powder i.g. 5 g / kg bw Plasma Stomach Liver Intestine Kidney Urine homogenization 4protein precipitation (TCA, 30%) 4Extraction with CH 2 CL 2 (3 times) Sample analysis Detection and Identification: GC-MS, EXPERIMENTAL

WP 6 UMR de Toxicologie Alimentaire INRA Dijon 1 1 : S-allyl-L-cysteine sulfoxide; 2 :  -glutamyl-S-allyl cysteine 3 :  -glutamyl-S-trans-1-propenyl cysteine 4 :  -glutamyl phenylalanine Sulfur compounds identified in garlic powder (HPLC) 2 3 4

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Sulfur compounds detected in stomach after a single oral administration of garlic powder diallyldisulfide 2-vinyl-4H-1,3-dithiin 3-vinyl-4H-1,2-dithiin

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Sulfur compounds detected in plasma after a single oral administration of garlic powder AMS AMSO AMSO 2

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Sulfur compounds detected in liver after a single oral administration of garlic powder AMS AMSO AMSO 2

WP 6 UMR de Toxicologie Alimentaire INRA Dijon CONCLUSIONS  Following garlic ingestion, AMS, AMSO and AMSO 2 are identified in several tissues (plasma, liver, urine)  AMSO 2 is the most abundant and the most persistent of these compounds in all analysed tissues  The highest amounts of these metabolites are measured 2-24 hours after garlic ingestion  The metabolite profiles are very similar to those obtained when DADS is administered  In our analysis conditions, neither allicin nor conjugates (AGS, NASAC) are detected

WP 6 UMR de Toxicologie Alimentaire INRA Dijon  inhibition of bio-activation of carcinogens (human cells Hep G2, human liver microsomes)  stimulation of detoxification of carcinogens (garlic powder)  prevention of genotoxicity (aflatoxin B 1 and nitrosamine..;)  Elucidation of some mechanisms of action of garlic and sulphur compounds on different biological events involved in carcinogenesis: GENERAL CONCLUSION  Next step : Hepatocarcinogenesis protocol will be performed to confirm the antigenotoxic effects of garlic

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Human intervention study : cancer biomarkers  Epidemiological studies indicate that garlic consumption reduces cancer risk Aim : to investigate the anticarcinogenic effect of garlic in man, using genetic biomarkers State of the art:  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

WP 6 UMR de Toxicologie Alimentaire INRA Dijon 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

WP 6 UMR de Toxicologie Alimentaire INRA Dijon 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

WP 6 UMR de Toxicologie Alimentaire INRA Dijon Deliverables WP6

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