Oxidative DNA Damage Induced by Carcinogenic Dinitropyrenes in the Presence of P450 Reductase

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Abstract
Nitropyrenes are widespread in the environment due to mainly diesel engine emissions. Dinitropyrenes (DNPs), especially 1,8-dinitropyrene (1,8-DNP) and 1,6-dinitropyrene (1,6-DNP), are much more potent mutagens than other nitropyrenes. The carcinogenicity of 1,8-DNP and 1,6-DNP is stronger than 1,3-dinitropyrene (1,3-DNP). It is considered that adduct formation after metabolic activation plays an important role in the expression of carcinogenicity of nitropyrenes. However, Djuric et al. [(1993) Cancer Lett.] reported that oxidative DNA damage was also found as well as adduct formation in rats treated with 1,6-DNP. We investigated oxidative DNA damage by DNPs in the presence of NAD(P)H-cytochrome P450 reductase using 32P-5‘-end-labeled DNA. After P450 reductase treatment, DNPs induced Cu(II)-mediated DNA damage in the presence of NAD(P)H. The intensity of DNA damage by 1,8-DNP or 1,6-DNP was stronger than 1,3-DNP. We also examined synthetic 1-nitro-8-nitrosopyrene (1,8-NNOP) and 1-nitro-6-nitrosopyrene (1,6-NNOP) as one of the metabolites of 1,8-DNP and 1,6-DNP, respectively, to find that 1,8-NNOP and 1,6-NNOP induced Cu(II)-mediated DNA damage in the presence of NAD(P)H but untreated DNPs did not. In both cases of P450 reductase-treated DNPs and NNOPs, catalase and a Cu(I) specific chelator attenuated DNA damage, indicating the involvement of H2O2 and Cu(I). Using a Clarke oxygen electrode, oxygen consumption by the reaction of NNOPs with NAD(P)H and Cu(II) was measured to find that NNOP was nonenzymatically reduced by NAD(P)H and that the addition of Cu(II) promoted the redox cycle. Therefore, these results suggest that DNPs are enzymatically reduced to NNOPs via nitro radical anion and that NNOPs are further reduced nonenzymatically by NAD(P)H. Subsequently, autoxidation of nitro radical anion and the reduced form of NNOP occurs, resulting in O2- generation and DNA damage. We conclude that oxidative DNA damage in addition to DNA adduct formation may play important roles in the carcinogenesis of DNPs via their metabolites.