Dinitropyrene metabolism, DNA adduct formation, and DNA amplification in an SV40-transformed Chinese hamster embryo...

The environmental pollutants 1,6-dinitropyrene (1,6-DNP) and 1,8-dinitropyrene (1,8-DNP) are strongly carcinogenic in a number of animal models. These DNPs are metabolized by nitroreduction to N-hydroxy arylamine derivatives that either directly or after acetylation bind to cellular DNA. In the experiments reported here, we examined whether DNA adduct formation by 1,6-DNP and 1,8-DNP was associated with amplification of specific DNA sequences, a process that may be causally related to tumorigenesis. CO60 cells, an SV40-transformed Chinese hamster embryo cell line, were incubated with 2.5 or 50 ng/mL [4,5,9,10(-3)H]1,6-DNP for 5 h. High-pressure liquid chromatographic analysis of organic extracts of the medium indicated the presence of 1-acetylamino-6-nitropyrene, suggesting that these cells are capable of nitroreduction and acetylation. 32P-Postlabeling analysis of DNA isolated from cells exposed to 1.0 or 2.5 ng/mL 1,6-DNP revealed dose-related formation of N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene. A similar adduct, presumably N-(deoxyguanosin-8-yl)-1-amino-8-nitropyrene, was detected after incubations with 1,8-DNP. DNA isolated from analogous experiments was slot-blotted onto nylon membranes and hybridized with 32P-labeled SV40, c-fos, or beta-actin DNA probes. beta-Actin was not amplified and c-fos was amplified only a small amount; however, there was dose-related amplification of SV40 sequences, whose levels were in some instances approximately 20 times that observed in solvent-treated controls. These data indicate that DNA adduct formation by 1,6-DNP and 1,8-DNP is associated with the amplification of certain DNA sequences, a response that may be related to the tumorigenic potential of these compounds.