Comparison of DNA alkali-labile sites induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 4-oxo-4-(3-pyridyl)butanal in rat hepatocytes

Abstract

4-Oxo-4-(3-pyridyl)butanal (OPB) is an aldehyde formed during the activation of the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Using the DNA alkaline elution technique, the properties of DNA alkali-labile sites induced in the isolated rat hepatocytes by NNK and OPB were compared. The DNA single-strand break (SSB) frequencies in vitro, as measured by the elution rate (ER), ranged from 0.015 to 0.479 and were proportional (r2 = 0.991) to the dose (0-2 mM) of OPB. These concentrations, however, were slightly cytotoxic. For example, the LC50 after 4 h of exposure was 2.8 mM. This suggests that OPB-induced DNA SSB result from additive effects of OPB-DNA interaction and the indirect DNA damage associated with OPB cytotoxicity. NNK induced a significant and dose-dependent increase of DNA fragmentation at concentrations ranging from 0.5 to 5.0 mM with ER values ranging from 0.012 to 0.274 (r2 = 0.951). Genotoxicity as measured by the DNA-damaging potency coefficient (DDP) was 810, 345, 131 and 75 for N-methyl-N-nitrosourea (MNU), N-nitrosodimethylamine (NDMA), OPB and NNK respectively. Both MNU- and NNK-induced DNA lesions showed increased lability with increased pH (from 12.1 to 12.5) of the eluting buffer (r2 = 0.979 and 0.967 respectively). In contrast, the number of OPB-induced labile sites were not affected by increases in the pH. These results indicate that OPB is not the metabolite contributing the majority of alkali-labile sites generated by NNK. The filter elution procedure was used to study the in vitro rejoining of SSB in DNA induced by NNK. The extent of DNA SSB rejoining after 18 h of culture of hepatocytes in NNK-free medium were dependent on the concentration of NNK (0.5, 2.0 and 5.0 mM) and ranged from 50 to 90%. Rats were injected s.c. with NNK (0.39 mmol/kg). SSB frequency in liver DNA increased rapidly and reached a maximum 12 h after injection. DNA SSB frequency declined during the next 2 weeks with biphasic kinetics. The fast phase (75% rejoining of DNA SSB between 12 h and 2 days) was followed by a slow one (25% of DNA SSB maintained during the next 5 days but not present after 2 weeks). The results of this study better define the role of OPB-induced DNA damage. The persistence of DNA SSB in the liver of NNK-treated rats reflects the inability of this tissue to repair all DNA lesions.