32P-postlabeling analysis of 1-nitropyrene-DNA adducts in female Sprague-Dawley rats

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Abstract
The 32P-postlabeling technique was used to qualitatively establish the pattern of DNA adduct formation in mammary tissue and liver following administration of 1-nitropyrene to female Sprague-Dawley rats. 1-Nitropyrene (100 mg/kg b.w.) was administered by gavage in trioctanoin and the rats were sacrificed 24 h later. DNA was isolated from mammary fat pads and liver, enzymatically hydrolyzed to deoxy ribonucleoside-3′-monophosphates and then converted to [5′-32P]3′,5′-bisphosphates. The polyethyleneimine-cellulose (PEI-cellulose) TLC 32P-fingerprints revealed the presence of multiple putative adducts in the mammary fat pads and in the livers. To investigate the role of nitroreduction in the formation of these adducts, calf thymus DNA was incubated with [3H]1-nitropyrene in vitro in the presence of xanthine oxidase. The DNA was isolated and analyzed by the 32P-postlabeling technique. A major adduct spot was detected and confirmed as N-(deoxyguanosin-8-yl)-1-amino-pyrene. This adduct cochromatographed with a minor in vivo adduct of DNA obtained from mammary fat pads and livers. However, the major adducts detected in vivo did not appear to originate from simple nitroreduction of 1-nitropyrene. The results of this study suggest that other metabolic pathways, such as ring oxidation, or ring oxidation followed by nitroreduction, may be responsible for the putative 1-nitropyrene—DNA adducts observed in mammary fat pads and livers of female Sprague-Dawley rats.