Benzene, a tobacco constituent, is a leukemogen in humans and a carcinogen in rodents. Several benzene metabolites generate superoxide anion (O2·−) and induce nitric oxide synthase in the bone marrow of mice. We hypothesized that the reaction of nitric oxide (·NO) with O2·− leads to the formation of peroxynitrite as an intermediate during benzene metabolism. This hypothesis was supported by demonstrating that the exposure of mice to benzene produced nitrated metabolites and enhanced the levels of protein-bound 3-nitrotyrosine in the bone marrow of mice in vivo. In the current study, we investigated the influence of nitric oxide, generated from sodium 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, on DNA strand breaks induced by each single or binary benzene metabolite at different doses and compared the levels of the DNA damage induced by each benzene metabolite in the presence of nitric oxide with the levels of DNA strand breaks induced by peroxynitrite at similar doses in vitro. We found that among benzene metabolites only 1,2,4-trihydroxybenzene (BT) can induce significant DNA damage in the absence of nitric oxide. While 1,4-dihydroxybenzene (HQ), 1,4-benzoquinone (BQ) and 1,2-dihydroxybenzene (CAT) require ·NO to induce DNA strand breaks, hydroquinone was the most potent DNA-damaging benzene metabolite in the presence of ·NO. The order of DNA breaks by benzene metabolites in the presence of ·NO is: Peroxynitrite = HQ > BT > BQ > CAT. The ·NO and O2·− scavengers inhibited DNA damage induced by [HQ+·NO]. Benzene, trans,trans-muconaldehyde, and phenol, do not induce DNA strand breaks either in the absence or presence of ·NO. However, adding phenol to [HQ+·NO] leads to greater DNA damage than [HQ+·NO] alone. Collectively, these results suggest that nitric oxide is an important factor in DNA damage induced by certain benzene metabolites, probably via the formation of the peroxynitrite intermediate. Phenol, the major benzene metabolite that does not induce DNA damage alone and is inactive in vivo, synergistically enhances DNA damage induced by potent benzene metabolite in the presence of nitric oxide.

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