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Polycyclic aromatic hydrocarbons (PAH) are a well-characterized group of mutagens and carcinogens. Benzo(a)pyrene [B(a)P], the best known compound in the group, exerts its genotoxic activity following metabolic activation, when it acquires the properties of an electrophilic reagent that is capable of interacting with DNA. Reactive oxygen species (ROS) can remerge during the PAH metabolic activation. Because of their antioxidant activity, vitamins C and E are thought to act as antimutagenic agents. We designed an in vitro protocol to study the potential protective effect of vitamins C and E toward B(a)P-induced DNA damage. In this study, we examined peripheral blood lymphocytes obtained from healthy nonsmoking female volunteers, 22 to 25 years of age. The cells were exposed in vitro to 1 microM B(a)P in the presence of 40 microM or 100 microM of vitamin C or, alternatively, to 30 microM or 100 microM of vitamin E. The B(a)P-induced DNA damage and repair were estimated as the generation and removal of single-strand DNA breaks measured by the alkaline version of the single-cell gel electrophoresis (comet) assay. The protective effect of vitamins C and E was demonstrated when the vitamins were applied simultaneously with or after the B(a)P. The background level of DNA damage in the presence of vitamins C and E was lower than in the system without the vitamins. The experiments were conducted according to various protocol schemes of the vitamin treatment and the results offer additional evidence of the antigenotoxic activity of vitamins C and E. The vitamin activity does not appear to be connected with the steps in metabolic activation or DNA repair. It seems that both vitamins act as competitors of DNA molecule in reaction with the reactive oxygen species.