Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in human kidney epithelial cells transfected with rat CYP2B1 cDNA

In all species where it has been tested, the tobacco-specificnitrosamine 4-(methylnitrosainino)-l-(3-pyridyl)-l-butanone(NNK) has been shown to be a potent carcinogen, and NNK andother nitrosamines may play a role in human tobacco-relatedcarcinogenesis. Purified rat CYP2B1 has been shown to metabolizeNNK, and the CYP2B1 gene is expressed constitutively in ratlung. The objectives of this study were to test the capacityof CYP2B1, synthesized from a rat hepatic cDNA in Ad293 cells,to metabolize NNK, and to define the type and the proportionsof the final metabolites produced. Ad293 cells were transfectedwith a CYP2B1 expression vector (pMT2-2Bl), or with a controlvector and incubated in culture medium containing [³H]NNK, afterwhich -carbon hydroxylation and pyridine N-oxidation metaboliteswere identified by HPLC analysis and quantitated by scintillationcounting. pMT2-2Bl-transfected cells were capable of catalyzing-carbon hydroxylation and pyridine N-oxidation of NNK, althoughthe reduction product 4-(methylnitrosamino)-l-(3-pyridyIH)-butan-l-ol(NNAL)was the major metabolite formed in cells regardless of transfectiontreatment. The total amount of -carbon hydroxylation metabolitesproduced by pMT2-2Bl-transfected cells was greater than thatof pyridine N-oxidation metabolites. However, pMT2-2Bl transfectedcells produced sim; ten-fold more pyridine N-oxidation metabolitesand only two-fold more -carbon hydroxylation metabolites thancontrol cells. Furthermore, the amount of NNAL-N-oxide was muchlower than that of NNK-N-oxide in the medium of pMT2–2Bl-transfectedcells, even though the amount of available NNAL, resulting fromcarbonyl reduction of NNK, was very high; this suggests thatNNAL is poorly N-oxidized by CYP2B1 compared to NNK. These resultsshow that within living cells NNK was metabolized by CYP2B1via both the pyridine N-oxidation and -carbon hydroxylationpathways. However, CYP2B1 preferentially catalyzed pyridineN-oxidation, which is considered to be a deactivation reaction.