Formation of genotoxic nitro-PAH compounds in fish exposed to ambient nitrite and PAH

Mutagenic nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) have been known to arise in the environment through direct emissions from combustion sources and nitration of PAHs, primarily in the atmosphere. Here, we report the formation of nitro-PAH compounds in fish contaminated with PAH and exposed to nitrite (NO2-) in the ambient water. Electrospray ionization mass spectrometric analysis of the bile of the euryhaline fish Oreochromis mossambicus exposed simultaneously to field-relevant, sublethal concentrations of phenanthrene (1 microg/g) and NO2- (1 microM) and collision-induced dissociation of selected ions revealed the presence of two strongly genotoxic nitro-PAH metabolites, namely phenanthrene-6-nitro-1,2-dihydrodiol-3,4-epoxide (mass/charge [m/z] 273) and dihydrodihydroxy acetylamino nitrophenanthrene (m/z 359). These two metabolite peaks present only in the bile of fish exposed simultaneously to phenanthrene and NO2- constituted, respectively, about 3.1 and 2.7% of the highest peak among the putative unconjugated phenanthrene metabolites in the mass spectrum. The presence of the oxidized phenanthrene metabolite dihydroxyphenanthrene (m/z 233) in fish exposed to phenanthrene alone as well as phenanthrene plus NO2- suggested that oxidation of phenanthrene precedes nitration in the sequence of reactions leading to the formation of the observed nitrophenanthrene metabolites. However, the route of PAH administration seems to determine the nature of metabolites formed. Nearly 92% of the hepatic cells of the fish exposed to phenanthrene in the presence of NO2- were found to have suffered extensive DNA fragmentation on comet assay.