Previous studies have shown that the tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is hepatocarcinogenic and results in alkylation of hepatic DNA in F344 rats. In this study, we have characterized the metabolism of NNK in cultured rat hepatocytes and have established the relationship between various metabolic pathways and the induction of DNA single strand breaks. DNA fragmentation by NNK and two other related N-nitrosamines, N'-nitrosonornicotine and nitrosodimethylamine, were compared. Metabolism of [5-3H]NNK (4.5 microM) by carbonyl reduction, alpha-carbon hydroxylation, and pyridine N-oxidation was linear from 0 to 6 h and with 0 to 2 x 10(6) hepatocytes. Using the alkaline elution assay, we observed that NNK induces DNA single strand breaks (SSB) in a dose (1-10 mM) and time (0.5-6 h) dependent manner. SSB induced by NNK (5 mM; rate of elution between 3 and 9 h, 0.117) are rejoined partially within 2 h (rate, 0.039) and totally 12 h after exposure. NNK N-oxide (5 mM) produces a smaller number of SSB (rate, 0.017) than NNK (rate, 0.105) suggesting that pyridine N-oxidation of NNK is a deactivation pathway. Hydrolysis of carbethoxy-nitrosaminomethane and 4-(N-carbethoxy-N-nitrosamino)-1-(3-pyridyl)butanone yields methyldiazohydroxide and 4-(3-pyridyl)-4-oxobutyl-diazohydroxide, respectively. These two alkylating intermediates are generated during alpha-carbon hydroxylation of NNK. After treatment of hepatocytes with 5 microM carbethoxynitrosaminomethane and 1 mM 4-(N-carbethoxyl-N-nitrosamino)-1-(3-pyridyl)butanone, the rates of DNA elution were 0.092 and 0.120, respectively. Carbonyl reduction of NNK leads to 4-(methylnitrosamino)-1-(3-pyridyl)butan-1-ol (NNAl). Reaction of NNK with methyl magnesium iodide gives 1-MeNNAl with 82% yield, NNAl but not 1-MeNNAl can be reoxidized to NNK. Both 5 mM NNAl (rate, 0.073) and 5 mM 1-MeNNAl (rate, 0.054) induce SSB indicating that NNAl does not require reconversion to NNK to be activated to DNA damaging intermediates. alpha-Methylene hydroxylation of NNK results in an equimolar formation of methyldiazohydroxide and 4-oxo-4-(3-pyridyl)-butanal. This aldehyde, at a concentration of 1 mM, induces the same frequency of SSB (rate, 0.116) as 5 mM NNK (0.105) and could possibly play a role in the carcinogenicity of NNK.(ABSTRACT TRUNCATED AT 400 WORDS)