EVALUATION OF THE ANTIGENOTOXIC POTENTIAL OF GARLIC SULFUR COMPOUNDS IN HEPG2 CELLS

1. EVALUATION OF THE ANTIGENOTOXIC POTENTIAL OF GARLIC SULFUR COMPOUNDS IN HEPG2 CELLS Sulfur compound Genotoxic compound S S C. Belloir, M.H. Siess, C. Daurat and A.M. Le Bon INRA, UMR Toxicologie Alimentaire, 17 rue Sully, 21065 Dijon cedex, France Introduction O Numerous epidemiological studies have reported that consumption of garlic reduces the risk of cancers in humans1. Organosulfur compounds present in high amounts in garlic would account for its anticarcinogenic activity. However only few reports are available on the antigenotoxic properties of garlic constituents in human cells. The aim of the present study was to assess the antigenotoxic potential of a major constituent of garlic, diallyl sulfide (DADS) in a human cell line, HepG2 cells. The protective activities of two metabolites of DADS, allicin2 (DADSO)and allyl mercaptan3 (AM), were also determined. S S Diallyl disulfide (DADS) SH Allicin (DADSO) Methods Allyl mercaptan (AM) In order to study the mechanisms of action (modulation of drug metabolizing enzymes, scavenging of ultimate genotoxic compounds), two experimental protocols were performed : i) a pre-treatment protocol in which HepG2 cells were first incubated with the sulfur compound for 20 hr then were treated with the indirect-acting genotoxic compound (aflatoxin B1 (AFB1) or benzo(a)pyren (BaP) or nitrosodimethylamine (DMN)) for 20 hr; ii) a co-treatment protocol in which HepG2 cells were simultaneously incubated with the sulfur compound and the direct-acting genotoxic compound (hydrogen peroxyde (H2O2) or 4-nitrosoquinoline oxide (4-NQO) or methylmethane sulfonate (MMS)) for 4 hr. After the treatments, cells were harvested. DNA damage was immediately evaluated using the comet assay4. The tail moment as defined by Olive et al5 was recorded as parameter for DNA damage. Seeding 0 24 48 72 hours Experimental protocol Pre-treatment study Co-treatment study Comet assay Results PRE-TREATMENT STUDY CO-TREATMENT STUDY AFB1 DMN H2O2 MMS BaP 4-NQO 25 * 16 * * 6 20 * * 4 6 * 20 * * * * 12 * 15 3 * 4 * * * * 15 DADS 4 * Olive tail moment 8 2 10 10 2 2 1 4 5 5 0 0 0 0 0 0 C G 5 25 50 100 µM C G 5 25 50 100 µM C G 5 25 50 100 µM C G 5 25 50 100 µM C G 5 25 50 100 µM C G 5 25 50 100 µM * * 5 * 20 6 * 4 * * * 20 * * * * 6 * 4 * 15 3 * 15 4 DADSO 3 Olive tail moment 4 2 10 10 2 2 2 1 1 5 5 0 0 0 0 0 0 C G 5 25 50 100 µM C G 5 25 50 100 µM C G 5 25 50 100 µM C G 5 25 50 100 µM C G 5 25 50 100 µM C G 5 25 50 100 µM * 14 * 6 * * * 10 5 * 8 * * * * 12 20 * * * * 8 4 * * 10 6 4 15 AM 3 6 8 * Olive tail moment 4 6 10 2 4 2 4 2 1 5 2 2 0 0 0 0 0 0 C G 5 25 50 100 µM C G 5 25 50 100 µM C G 5 25 50 100 µM C G 5 25 50 100 µM C G 5 25 50 100 µM C G 5 25 50 100 µM C = control cells G = cells + genotoxic compound = cells + genotoxic compound + sulfur compound (5-100 µM) * : significantly different to values for samples treated with genotoxic compound alone (P 0.05, Mann-Whitney-U-test) Pre-treatment of HepG2 cells with DADS or DADSO inhibited the genotoxicity of aflatoxin B1. DADS and AM significantly reduced DNA damage induced by benzo(a)pyrene. AM afforded protection against dimethylnitrosamine genotoxicity in a concentration-dependent manner. In co-treatment studies,AM and, to a lower extent, DADSO and DADS were shown to inhibit hydrogen peroxide genotoxicity. DNA damage provoked by methylmethane sulfonate was significantly prevented when this genotoxic compound was incubated together with DADS or DADSO. AM and DADSO strongly reduced 4-nitrosoquinoline oxide genotoxicity. Conclusion References The present study showed that DADS and two metabolites, DADSO and AM, afford protection against DNA damage induced by direct- and indirect-acting genotoxic compounds. These sulfur compounds could act through different mechanisms of action, i.e. modulation of enzymes involved in activation/detoxification of genotoxic compounds and scavenging of ultimate species. These findings support the assumption that sulfur compounds could be responsible for the chemopreventive properties of garlic in humans. 1. Fleischauer,A.T. and Arab,L., 2001. J; Nutr., 131, 1032-1040. 2. Teyssier C. et al., 1999. Drug. Metab. Disp., 27, 835-841. 3. Egen-Schwind C. et al., 1992. Planta Medica, 58, 301-305. 4. Singh N.P. et al., 1988. Exp. Cell. Res., 175, 184-191. 5. Olive P.L. et al., 1990. Radiat. Res., 122, 86-94. This project was partly financed by the European Quality of Life and Management of Living Resources