Oxidative DNA damage by an N-hydroxy metabolite of the mutagenic compound formed from norharman and aniline

Norharman (9H-pyrido[3,4-b]indole), which is a heterocyclic amine included in cigarette smoke or cooked foodstuffs, is not mutagenic itself. However, norharman reacts with non-mutagenic aniline to form mutagenic aminophenylno rharman (APNH, of which DNA adducts formation and hepatocarcinogenic potent ial are pointed out. We investigated whether N-OH-APNH, an N-hydroxy metabo lite of APNH, can cause oxidative DNA damage or not, using P-32-labeled DNA fragments. N-OH-APNH caused Cu(II)-mediated DNA damage. When an endogenous reductant, beta -nicotinamide adenine dinucleotide (NADH) was added, the D NA damage was greatly enhanced. Catalase and a Cu(I)-specific chelator inhi bited DNA damage, suggesting the involvement of H2O2 and Cu(I). Typical . O H scavenger did not inhibit DNA damage. These results suggest that the main reactive species are probably copper-hydroperoxo complexes with DNA. We al so measured 8-oxo-7,8-dihydro-2 ' -deoxyguanosine (8-oxodG) formation by N- OH-APNH in the presence of Cu(II), using an electrochemical detector couple d to a high-pressure liquid chromatograph. Addition of NADH greatly enhance d 8-oxodG formation. UV-VIS spectra and mass spectra suggested that N-OH-AP NH was autoxidized to nitrosophenylnorharman (NO-PNH). We speculated that N O-PNH was reduced by NADH. Cu(II) facilitated the redox cycle. In the prese nce of NADH and Cu(II), very low concentrations of N-OH-APNH could induce D NA damage via redox reactions. We conclude that oxidative DNA damage, in ad dition to DNA adduct formation, may play an important role in the expressio n of genotoxicity of APNH. (C) 2001 Elsevier Science B.V. All rights reserv ed.