A comparison of the carcinogen-DNA adducts formed in rat liver in vivo after administration of single or multiple doses of N-methyl-4-aminoazobenzene

Abstract

N-Methyl-4-aminoazobenzene (MAB) is believed to be metabolized in the liver to an electrophilic N-sulfonyloxy ester which binds covalently to cellular macromolecules, resulting in the induction of hepatic neoplasia. Previous in vivo studies in the rat detected only two hepatic MAB-DNA adducts, 3-(deoxyguanosin-N2-yl)-MAB(N2-dG) and N-(deoxyguanosin-8-yl)-MAB(C8-dG), which respectively accounted for 25% and 70% of the total MAB bound to DNA at 8 h after a single dose of the carcinogen. Subsequently, the C8-dG adduct was shown to be rapidly lost from the DNA while the N2-dG adduct was a persistent lesion. Since a single dose of MAB is not sufficient for complete carcinogenic activity, we sought to identify the MAB-DNA adducts present in rat liver after multiple oral doses of [3H]MAB. The MAB was administered by intubation at a level of 0.2 mmol/kg for 1, 3 or 4 doses and animals were sacrificed at 8 h after the last dose. Hepatic DNA was isolated by extraction and hydroxylapatite chromatography and was enzymatically hydrolyzed to MAB-mononucleoside adducts, which were quantitated by high pressure liquid chromatography (HPLC). After 3 doses, N2-dG, C8-dG, and an unknown adduct were detected. By 4 doses, these accounted for 51%, 25% and 23% of the total adducts. This data is consistent with rapid removal of the C8-dG derivative and the relative persistence of the N2-dG and the unknown adduct. The latter was shown to exhibit chromatographic and pH-dependent solvent partitioning properties that were identical to a product also present in DNA treated with the synthetic ultimate carcinogen, N-benzoyloxy-MAB. Analysis of this adduct by field desorption mass spectrometry (M+ = 460) and, after perdeuteromethylation, by electron impact mass spectrometry (M+ = 528; M-N(CH3)(CD3) = 481) indicated the structure to be a deoxyadenosin-N6-yl derivative substituted through an aromatic ring of MAB. Further analysis by 270 MHz 1H-NMR spectroscopy allowed complete assignment of the MAB and adenyl resonances and was uniquely consistent with a 3-(deoxyadenosin-N6-yl)-MAB structure. Since this persistent adduct is potentially mutagenic due to possible tautomeric equilibria between the N6-amino and N6-imino structures, it may represent an initiating lesion in MAB hepatocarcinogenesis.