DINITROPYRENE METABOLISM, DNA ADDUCT FORMATION, AND DNA AMPLIFICATIONIN AN SV40-TRANSFORMED CHINESE-HAMSTER EMBRYO CELL-LINE

The environmental pollutants 1,6-dinitropyrene (1,6-DNP) and 1,8-dinit ropyrene (1,8-DNP) are strongly carcinogenic in a number of animal mod els. These DNPs are metabolized by nitroreduction to N-hydroxy arylami ne derivatives that either directly or after acetylation bind to cellu lar DNA. In the experiments reported here, we examined whether DNA add uct 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-H-3]1,6-DNP f or 5 h. High-pressure liquid chromatographic analysis of organic extra cts of the medium indicated the presence of 1-acetylamino-6-nitropyren e, suggesting that these cells are capable of nitroreduction and acety lation. P-32-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-(deox yguanosin-8-yl)-1-amino-6-nitropyrene. A similar adduct, presumably N- (deoxyguanosin-8-yl)-1-amino-8-nitropyrene, was detected after incubat ions with 1,8-DNP. DNA isolated from analogous experiments was slot-bl otted onto nylon membranes and hybridized with P-32 labeled SV40, c-fo s, or beta-actin DNA probes. beta-Actin was not amplified and c-fos wa s amplified only a small amount; however, there was dose-related ampli fication of SV40 sequences, whose levels were in some instances approx imately 20 times that observed in solvent-treated controls. These data indicate that DNA adduct formation by 1,6-DNP and 1,8-DNP is associat ed with the amplification of certain DNA sequences, a response that ma y be related to the tumorigenic potential of these compounds. (C) 1993 Wiley-Liss, Inc.