Nitroreduction of 4-nitropyrene is primarily responsible for DNA adduct formation in the mammary gland of female CD rats

We determined whether DNA adducts derived from 4-nitropyrene (4-NP) are for med via nitroreduction or ring oxidation. DNA adduct markers derived from b oth pathways were prepared and, consequently, were compared with those obta ined in vivo in rats treated with 4-NP. Following in vitro reaction of 9,10 -epoxy-9,10-dihydro-4-nitropyrene (4-NP-9,10-epoxide), an intermediate meta bolite derived from ring oxidation of 4-NP, with calf thymus DNA (average l evel of binding in two determinations was 8.5 nmol/mg of DNA), DNA was enzy matically hydrolyzed to deoxyribonucleosides and the DNA hydrolysates were analyzed by HPLC. Electrospray mass and H-1 NMR spectra of the major produc ts indicated that these adducts are deoxyguanosine (dG) derivatives that re sulted from N-2-dG substitution at the 9- or 10-position of the pyrene nucl eus. However, these adducts were not detected in vivo in the rat mammary gl and and liver following the administration of 4-NP. Nitroreduction of 4-NP catalyzed by xanthine oxidase in the presence of DNA resulted in three majo r putative DNA adducts (level of binding of 12.0 +/- 1.1 nmol/mg of DNA, n = 4) designated as peak 1 (46%), peak 2 (25%), and peak 3 (17%). Although p eak 1 was further resolved into peaks la and Ib, both were unstable and gra dually decomposed to peak 2, and the latter was unequivocally identified as pyrene-4,5-dione. On the basis of electrospray mass spectral analysis, pea k 3 was tentatively identified as a deoxyinosine-derived 4-aminopyrene addu ct. None of the adducts derived from nitroreduction of 4-NP catalyzed by xa nthine oxidase coeluted with the synthetic standard N-(deoxyguanosin-8-yl)- 4-aminopyrene prepared by reacting dG with N-acctoxy-4-aminopyrene. Neverth eless, HPLC analysis of the hydrolysates of liver and mammary DNA obtained from rats treated with [H-3]-4-NP yielded four radioactive peaks, all of wh ich coeluted with the markers derived from the nitroreduction pathway. Thes e results indicate that nitroreduction is primarily responsible for DNA add uct formation in the liver and, especially, in the mammary gland which is t he organ susceptible to carcinogenesis by this environmental agent.