Marie-Hélène SIESS et al. WP 6 CANCER Marie-Hélène SIESS et al. 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

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 The 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

ORIGINAL 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

Bioavailability and metabolism of sulfur compounds from garlic

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

Proposed scheme for the metabolism of DADS in the rat Allylmercaptan Allylglutathione sulfide S glutathione SH Allylmethylsulfide Allylmethylsulfoxide Allylmethylsulfone Metabolites detected in vitro Metabolites detected in vivo

CONCLUSION  The metabolism of DADS is partly elucidated in rat and man DADS is rapidly absorbed and extensively metabolized. AMSO and AMSO2 are probably excretion compounds Similar metabolites profiles are observed after garlic administration

Role of garlic on the process of carcinogenesis : In vivo studies with rat

OBJECTIVES  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 action To determine if the anticarcinogenic effect is correlated with the level of sulfur compounds in garlic

STUDIED MATERIALS Variety and location Variety Printanor FR Variety Printanor SP Fertilisation SO4 kg /ha 100 200 400 Alliin content nmoles/mg 58 75 90 330 Experimental groups SO S100 S200 S400

Activating and protecting mechanisms of carcinogenesis Activating mechanisms Protecting 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 Carcinogenesis 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 process Initiation 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.

PROTOCOL OF HEPATOCARCINOGENESIS Sacrifice 0 1 2 3 4 5 6 7 8 9 weeks Garlic diet Promotion Initiation Detection of preneoplastic foci Carcinogen administration Aflatoxin B1 (AFB1) : 10 x 0.025mg/kg Diethylnitrosamine (DEN) : 10 X 2.5 mg/kg

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

RESULTS Effects of garlic on the number of preneoplastic foci induced by AFB1 in the liver a b Correlation of the nb foci with S content of garlic r = - 0.53 P = 2.93 E-04 Means and SEM (n=10). Means having different letters are significantly different (P 0.05, Newman Keuls’ test).

RESULTS Effects of garlic on the area of preneoplastic foci induced by AFB1 in the liver b a Correlation of area foci with S content of garlic r = - 0.40 P = 0.006 Means and SEM (n=10). Means having different letters are significantly different (P 0.05 Newman Keuls’ test).

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

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

RESULTS 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 DNA Broken DNA = comet genotoxic The 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

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

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

Initiation of carcinogenesis Metabolic pathways of nitrosamine CYP 2E1 spontaneous DEN OH-DEN CH3+ DNA DEN undergoes an oxidative N demethylation by CYP 2E1 resulting in the production of a methyl ion, a potent alkylator of DNA Initiation of carcinogenesis

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

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

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 the genotoxicity of nitrosamine  Mechanisms of action unknown

Antigenotoxic effects of sulfur compounds in human cells

OBJECTIVES To 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 µM DADS (diallyl disulfide) DADSO (allicin) SAC (S-allyl cysteine) AM (allyl mercaptan) DNA alterations were measured using the comet test. Undamaged DNA Broken DNA = comet Genotoxic compound

PROTOCOLS Garlic compound Pro-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  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 (H2O2) methyl methane sulfonate (MMS) 4-nitrosoquinoline oxide (4-NQO)

Pre-treatment studies RESULTS Pre-treatment studies DADS DADSO AM SAC BaP   AFB1   ()  () NDMA   OSC 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

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

CONCLUSIONS  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

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.  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 In human cells, garlic sulfur compounds inhibit the genotoxicity induced by different chemicals.

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)

Deliverables WP6 Po Po Po Po = poster

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

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