Antigenotoxic Effect of Grape Seed Procyanidin Extract in Fao Cells Submitted to Oxidative Stress § NIURKA LLO Ä PIZ, † FRANCESC PUIGGRO Å S, ‡ ELA CE Ä SPEDES, † LLUI ÄS AROLA, ‡ ANNA ARDE Ä VOL, ‡ CINTA BLADE Ä , ‡ AND M. JOSEPA SALVADO Ä * ,‡ Departament de Bioquı ´mica i Biotecnologı ´a, Unitat d’Enologia del Centre de Refere `ncia en Tecnologia dels Aliments de la Generalitat de Catalunya, Universitat Rovira i Virgili, Imperial Ta `rraco 1, 43005 Tarragona, Spain The protective effects of grape seed procyanidin extract on the repair of H 2 O 2 -induced DNA lesions were tested using Fao cells. Cells were exposed to 600 μMH 2 O 2 for 3 or 21 h. A procyanidin extract from grape seed (PE) was incubated or preincubated (1 h) during the exposure to H 2 O 2 . The ability of procyanidins to protect against the genotoxicity of H 2 O 2 was compared with those of the monomeric flavanols (+)-catechin and (-)-epicatechin and the flavonol quercetin. After treatment, DNA damage was monitored using alkaline single-cell gel electrophoresis (the comet assay) (Aherne, S. A.; O’Brien, N. M. Nutr. Cancer 1999, 34, 160-166). At the end of the experiment, PE significantly decreased the damage caused by H 2 O 2 . The results also showed that quercetin was the most effective of the flavonoids tested, which is consistent with its powerful antioxidant character. The results indicate that procyanidins are more effective than the corresponding individual monomers, catechin and epicatechin, at preventing DNA lesions in hepatocytes and that this protection is higher after preincubation than after co-incubation. KEYWORDS: Procyanidins; quercetin; comet assay; DNA damage; oxidative stress; H2O2; hepatocytes INTRODUCTION Reactive oxygen species (ROS) are produced in cells by cellular metabolism and by exogenous agents. These species react with biomolecules in cells, including DNA. ROS induce numerous lesions in DNA, which cause deletions, mutations, and other lethal genetic effects. If left unrepaired, this damage may contribute to a number of degenerative processes, including cancer and aging (2, 3). Characterization of this damage to DNA has indicated that both the sugar and the bases are susceptible to oxidation. About 100 different modifications have been identified, including the appearance of AP sites, cross-linking to protein, and single- and double-strand breakage (4). This last type of breakage is highly toxic and mutagenic and can cause chromosome aberrations, whereas single-strand breaks are repaired. Living organisms have evolved several systems that recognize and repair the various forms of DNA damage induced by oxidation. These DNA repair systems are key processes in the secondary defense system, which copes with damage caused by ROS and which is not destroyed by the primary defense mechanisms. Primary defense mechanisms include antioxidant enzymes, nonenzymatic endogenous antioxidants, and exog- enous antioxidant molecules, which together are usually able to maintain ROS at nonharmful levels. Polyphenols are exog- enous antioxidants, and this is the most studied property of these compounds. Experimental and epidemiological data have re- vealed that moderate red wine consumption prevents various types of cancer. This is largely due to the phenolic compounds in wine. The flavonoids present in red wine comprise, among others, flavonols and procyanidins (derivatives of flavan-3-ols), the distinct antioxidative potentials of which are of great importance for explaining their beneficial effects. The antioxi- dative effects of flavonoids involve mechanisms such as metal- chelating, free-radical scavenging with the formation of less reactive flavonoid aroxyl radicals (5), inhibiting certain oxidative enzymes (6), and activating detoxifying/defensive proteins (7). The standard antioxidant capacity of flavonoids can be deter- mined from their reduction potentials (700-450 mV), which are lower than those of alkylperoxyl and superoxide radicals (2300-1000 mV). Therefore, flavonoids may inactivate these damaging oxyl species and prevent their deleterious conse- quences. In this study, we examined the antigenotoxic and protective effects of a procyanidin extract from grape seed, of their stable monomers the flavan-3-ols (+)-catechin and (-)-epicatechin, and of the flavonol quercetin on H 2 O 2 -induced genotoxicity in rat Fao cells. The DNA cleavage/repair was identified by alkaline single-cell electrophoresis, a very sensitive method for detecting strand breaks and measuring repair kinetics at the level of single cells. § This research was done at the Rovira i Virgili University in Tarragona, Spain. Dr. Llo ´piz and Dr. Ce ´spedes were visiting scientists from the University of Cuba and did not carry out any of the experiments reported in the paper at that institution. * Author to whom correspondence should be addressed (telephone 34- 977 55 95 67; fax 34-977 55 82 32; e-mail jsr@astor.urv.es). † Medical University of Havana, Cuba. ‡ Rovira i Virgili University. J. Agric. Food Chem. 2004, 52, 1083-1087 1083 10.1021/jf0350313 CCC: $27.50 © 2004 American Chemical Society Published on Web 02/04/2004