Protection of garlic against carcinogen genotoxicity Anne-Marie Le Bon, Christine Belloir, Varsha Singh Milestones :  In vitro evaluation of the antigenotoxic properties of garlic compounds in HepG2 cells  Evaluation of the antigenotoxic properties of garlic extracts in tissues of rat

The studied molecules were : Range : 5 to 100 µM DADS (diallyl sulfide) DADSO (allicin) SAC (S-allyl cystein) AM (allyl mercaptan) In vitro evaluation of antigenotoxic properties of garlic compounds in HepG2 cells 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

 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 Garlic compoundPro-mutagen  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 : benzopyrene (BaP) aflatoxin B1 (AFB1) nitrosodimethylamine (NDMA) Mutagens : hydrogen peroxide (H2O2) methyl methane sulfonate (MMS) 4-nitrosoquinoline oxide (4-NQO) PROTOCOLS P450 1A P450 1A, 3A P450 2E1

RESULTS Pre-treatment studies DADSDADSOSACAM NDMA n.d. AFB1  ()() BaP    n.d.  : inhibition of the genotoxicity (  ) : slight inhibition - : no effect n.d. : not determined

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

% of DNA alterations H202H202 5 µM25 µM50 µM100µM Effects of S-allylcystein on H 2 O 2 genotoxicity in HepG2 cells RESULTS

CONCLUSIONS  Garlic sulfur compounds prevent genotoxicity induced by direct- and indirect-acting compounds  DADS and AM can act throught both mechanisms : modulation of EMX and scavenging DADS is more efficient than AM  DADS and SAC act rather by scavenging genotoxic compounds  The metabolites of DADS, DADSO and AM, could be the effective compounds in vivo

Studied material : Garlic powder « 0 kg/ha SO4 » Garlic powder « 100 kg/ha SO4 » Garlic powder « 200 kg/ha SO4 » In vivo evaluation of antigenotoxic properties of garlic extracts in rats Objectives : to assess if garlic ingestion prevent DNA alterations induced by genotoxic compounds in rat liver

% garlic powder Sacrifice Liver DNA damage evaluation (comet test) intraperitoneal injection of a genotoxic agent : AFB1 NDMA (4 hours before sacrifice) Group C : control diet SA : Garlic 0 kg/ha SO4 SB : Garlic 100 kg/ha SC : Garlic 200 kg/ha 60 Wistar male rats PROTOCOL weeks

RESULTS CSASBSCCSASBSCCSASBSC * * * (control) + AFB1 + NDMA DNA damage (OTM) Effects of garlic ingestion on DNA damage in rat liver (5 rats / group) * : significantly different from the group C (Dunnett ’s test, p < 0.05)

CONCLUSIONS  Garlic powders are not genotoxic by themselves.  Garlic powder ingestion reduce liver DNA damage induced by AFB1. No statistical difference was observed between fertilisation levels. Mechanims of action : induction of CYP 1A and/or GST ?  A slight reduction of NDMA genotoxicity was observed but this effect was not significant. Mechanims of action : inhibition of CYP 2E1 ?  To be done shortly : colon study (preliminary studies in progress)

WP 6 Cancer : General conclusion  Metabolism of DADS almost elucidated, proposition of a scheme of the metabolic pathway.  In vivo after garlic powder administration to rats, we observed a modulation of hepatic enzymes involved in carcinogen activation or detoxication.  Sulfur compounds prevent the genotoxicity of carcinogens in a human cell- line. They act by scavenging ultimate metabolites and/or by modulating the activity of carcinogen metabolizing enzymes

WP 6 Cancer : Deliverables DH. 8: In vitro metabolism of diallyl disulfide, ajoene and allicin (P12) : done, publication in progress DH. 9: In vitro effects of sulphur compounds on human P450 iso-enzymes (P12) : done DH. 10: In vitro evaluation of the anti-genotoxic properties of garlic compounds in HepG2 cells (P12) : done DH.13: In vivo metabolism of diallyl disulfide (P12) : done, publication submitted DH. 14: Effects of garlic extracts on carcinogen-metabolising enzymes (P12) : done, publication in progress DH. 15: In vivo evaluation of the antigenotoxic properties of garlic extracts in rats (P12) : in progress