The metabolic activation and DNA adducts of dinitropyrenes

Dinitropyrenes are contaminants in diesel emissions that are mutagenic in bacteria and mammalian cells, and tumorigenic in laboratory animals. In this project, we investigated the factors that contributed to the extreme genotoxicity of dinitropyrenes in bacteria and determined if these factors were important in mammalian cells. Xanthine oxidase, a mammalian nitroreductase, catalyzed the conversion of the dinitropyrenes to DNA-bound products, but the level of binding did not exceed that observed with 1-nitropyrene. This suggested that factors in addition to nitroreduction were important in the metabolic activation of dinitropyrenes. 1-Nitro-6-nitrosopyrene and 1-nitro-8-nitrosopyrene were synthesized and reacted with DNA under reducing conditions. The same C8-substituted deoxyguanosine adducts were formed that were found in the xanthine oxidase-catalyzed reactions, which confirmed that incubation with this nitroreductase generated reactive N-hydroxy arylamine intermediates. In incubations with rat and human liver microsomes and cytosol, 1-nitropyrene and 1,3-dinitropyrene were reduced to a lesser extent than 1,6- and 1,8-dinitropyrene, which was in accord with their relative mutagenicities. Each of the cytosolic incubations were similar in that oxygen decreased aminopyrene, but not nitrosopyrene, formation. The data indicated that reduced derivatives of the nitrosopyrenes were redox cycling with oxygen, which decreased cytosolic aminopyrene formation. In cytosolic incubations, oxygen inhibited the reduction of 1-nitropyrene and 1,3-dinitropyrene to a greater extent than 1,6- and 1,8-dinitropyrene. By comparison, in microsomal investigations, the nitroreduction of each nitrated pyrene was equally oxygen-sensitive. This apparently was caused by the initial nitroanion radicals reacting with oxygen to decrease nitrosopyrene formation. Although more extensive nitroreduction of each compound was detected in anaerobic incubations, aerobic reduction of these compounds did occur and may be important during in vivo exposure to nitrated pyrenes. When rat liver cytosol was incubated with the nitrated pyrenes, very low levels of DNA binding were detected. Addition of acetyl coenzyme A (AcCoA) to these incubations increased the binding of the dinitropyrenes 20- to 40-fold, while the binding of 1-nitropyrene was not affected. The extent of AcCoA- dependent binding of the dinitropyrenes reflected the amount of nitroreduction; however, the increase in binding did not occur with dog liver cytosol, which was known to be deficient in N-acetylases. These results indicated that cytosolic nitroreductases catalyzed the formation of N-hydroxy arylamine intermediates, which in the case of dinitropyrenes were converted to reactive N-acetoxy arylamines by cytosolic AcCoA-dependent acetylases.