Detection of DNA damage in fish Oreochromis mossambicus induced by co-exposure to phenanthrene and nitrite by...

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. In the marine environment, PAHs are one of the classic anthropogenic organic pollutants, while nitrite (NO(2)(-)) is produced naturally via various biological processes like imbalance in nitrification/denitrification or eutrophication and subsequent oxygen depletion from an oversupply of nutrients. In this paper, we report the formation of PAH-DNA adducts in fish contaminated with PAHs and exposed to NO(2)(-) in the ambient water. Electrospray ionization tandem mass spectrometric (ESI-MS/MS) analysis of the bile of the euryhaline fish Oreochromis mossambicus exposed simultaneously to field relevant sublethal concentrations of phenanthrene and NO(2)(-) and collision-induced dissociation of selected ions revealed the presence of DNA-PAH adducts. The present study indicates that, although several high sensitivity techniques have been developed for the analysis of PAH derived DNA adducts, MS/MS has emerged as a powerful tool in the detection and structure elucidation of DNA adducts.

Juvenile O. mossambicus from a local estuarine fish farm were used with increasing frequency for carcinogenicity testing and comparative cancer research. The fish were exposed to the alkylating agent phenanthrene in the presence of NO(2)(-). Composite untreated bile samples after dilution with methanol: water (1:1; v/v) were analyzed by ESI-MS.

Several adducts could be evidenced in the bile by MS/MS. Deoxyadenosine/deoxyguanosine having a mass in the range of 450-650 amu is detected. In addition, a segment of modified dinucleotide with a mass that corresponds to a dimer consisting of a modified guanosine and a normal guanosine has also been identified in the bile.

The formation of certain types of DNA adducts is a crucial step in the induction of cancer and a primary stage in mutagenesis. Phenanthrene injected by i.p. route led to the transformation of phenanthrene to N-formyl amino phenanthrene-N(6)-deoxyadenosine adduct, whereas the fish co-exposed to phenanthrene and ambient nitrite metabolizes PAH to mono-, di- as well as trinitro derivatives, which then react with DNA leading to the formation of mainly modified guanosine and adenosine adducts. In the present investigation, dinitrophenanthrene diol epoxide (DNPDE) adduct with guanosine (m/z 587) seems to be the dominant adduct in the mixture, and its presence is shown first as a comparatively less stable adduct, which decomposes to give a more stable N(2) adduct (m/z 567).

MS/MS has proved to be useful in the rapid determination and discrimination of structurally different phenanthrene/derivatives DNA adducts in a complex mixture of fish bile co-exposed to phenanthrene and nitrite. However, the nature of metabolites formed is likely determined by the route of PAH administration, and there is a need to further define the early biochemical events of carcinogenesis in these species.

DNA adduct analysis in fish bile offers a promising approach to study the risk of potentiation of anthropogenic chemicals into genotoxic compounds in the presence of nitrite in the marine environment. We believe this is the first report on the formation of DNA-phenanthrene adducts on co-exposure of the fish to PAH and nitrite.