Ring opening of benzo[a]pyrene in the germ-free rat is a novel pathway forformation of potentially genotoxic metabolites

The metabolism of benzo[a]pyrene (BP) is known to lead to a large number of oxygenated compounds, some of which can bind covalently to DNA. We have st udied the integrated metabolism of BP in vivo in germ-free rats given C-14- labeled BP. Urinary metabolites were separated into groups according to aci dity using lipophilic ion exchangers. The groups were analyzed by mass spec trometry and were further fractionated by high-performance liquid chromatog raphy. The fraction of urinary metabolites previously shown to contain N-ac etylcysteine and glucuronic acid conjugates was found to contain derivative s of 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid as major components. The se compounds, which were identified by mass spectrometry and NMR, accounted for about 30% of the total metabolites in urine, demonstrating that, surpr isingly, ring opening is a major pathway for metabolism of BP in the germ-f ree rat. The dicarboxylic acid may be excreted in urine as an ester glucuro nide. By using the single cell gel electrophoresis or COMET assay, we were able to demonstrate that the anhydride of 7-oxo-benz[d]anthracene-3,4-dicar boxylic acid was an efficient inducer of DNA damage. Taken together, these results indicate that the novel ring opening metabolic pathway may provide alternative mechanisms for the toxicity of BP.