Cell specificity for the pulmonary metabolism of tobacco-specific nitrosamines in the Fischer rat

Abstract

The activity and distribution of the metabolic pathways of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and the structurally related nitrosamine, N'-nitrosonornicotine (NNN) were examined in pulmonary cells from F344 rats in order to investigate the mechanisms by which NNK and NNAL, but not NNN, cause lung tumors. The tritium labeled nitrosamines were incubated with Clara cells, alveolar macrophages, alveolar type II cells, or small cells and metabolites were analyzed by HPLC. O6-Methyl-guanine (O6MG) formation was also quantified in the cells incubated with NNK. Clara cells metabolized all compounds more extensively than the other cell types. Total alpha-hydroxylation, carbonyl reduction to NNAL, and pyridine N-oxidation in cells incubated with NNK, as well as concentrations of O6MG in DNA were higher in Clara cells than in other cell types. Carbonyl reduction of NNK predominated over the other metabolic pathways in all cell types. The high activity for alpha-hydroxylation of NNK in Clara cells is consistent with previous studies which proposed that the cell specificity for O6MG formation and the accumulation of this adduct during low-dose exposure to NNK may stem from the presence of a high affinity pathway in Clara cells for NNK activation. Metabolism of NNAL by alpha-hydroxylation, and by reconversion to NNK followed by alpha-hydroxylation were observed. Total alpha-hydroxylation of NNAL was less extensive than alpha-hydroxylation of NNK. NNN was metabolized by both the 2'- and 5'-alpha-hydroxylation pathways. 2'-Hydroxylation of NNN produces the same DNA pyridyloxobutylating agent as does methyl hydroxylation of NNK. However, NNN is not a methylating agent and does not induce lung tumors in rats. Metabolism of NNN by 2'-hydroxylation was, depending on cell type, 41-85% as extensive as total alpha-hydroxylation of NNK, indicating that the rates of formation of the DNA pyridyloxobutylating agent were similar from NNN and NNK. The results of this study demonstrate that Clara cells have a high capacity to metabolically activate NNK, NNAL and NNN and provide further support for the hypothesis that DNA methylation of pulmonary cells is important in NNK carcinogenesis.

Comparative Levels of O6-Methylguanine, Pyridyloxobutyl-, and Pyridylhydroxybutyl-DNA Adducts in Lung and Liver of Rats Treated Chronically with the Tobacco-Specific Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

“…Multiple factors influence levels of O 6 -methyl-G in the lungs of NNK-treated rats because the different cell types have contrasting activities for NNK metabolic activation, DNA adduct formation, and AGT activity (Belinsky et al, 1988(Belinsky et al, , 1989b(Belinsky et al, , 1990Staretz et al, 1997). Thus, the results reported here for whole lung are difficult to dissect with respect to these individual components.…”

Formation of genotoxic products from N-nitrosoheptamethyleneimine (NHMI), 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone (NNK) and N?-Nitrosonornicotine (NNN) by isolated rabbit lung cells

“…The same pyridyloxobutylating agent is also formed by hydroxylation of NNN (Belinsky et al, 1989). DNA damage can lead to cytotoxicity and/or DNA repair, and we found significantly increased levels of DNA excision repair, as measured by unscheduled DNA synthesis, in isolated Clara cells following NNK but not NNN administration in vitro.…”

“…The formation of oxobutyldiazohydroxide from NNK and NNN thus seems to be less important in the lung, which can be further supported by the fact that NNK is a lung carcinogen whereas NNN is not (Hecht et al, 1980;Hoffmann et al, 1984). The rates of DNA repair appeared greatest in Clara cells, and this cell type also metabolized NNK more extensively than any other lung cell in vitro and had the highest level of NNK-induced DNA adducts in the rat (Belinsky et al, 1987(Belinsky et al, , 1989. Clara cells are rich in P450 enzymes (Serbjit-Singh et al, 1979 which are involved in the metabolism of NNK (Hecht et al, 1986) and also of NNN (Hecht and Hoffmann, 1988).…”

“…DNA damage can lead to cytotoxicity and/or DNA repair, and we found significantly increased levels of DNA excision repair, as measured by unscheduled DNA synthesis, in isolated Clara cells following NNK but not NNN administration in vitro. The DNA repair is most evidently a result of the increased formation of 06-methylguanine observed in cells incubated with NNK (Belinsky et al, 1989) indicative of the C4-hydroxylation to methyldiazohydroxide (Castonguay et al, 1988) as the genotoxic pathway of NNK in the lung. The formation of oxobutyldiazohydroxide from NNK and NNN thus seems to be less important in the lung, which can be further supported by the fact that NNK is a lung carcinogen whereas NNN is not (Hecht et al, 1980;Hoffmann et al, 1984).…”

“…There is a two-fold difference between the cell types in P450 isozyme and NADPH-P450-reductase activities (Domin et al, 1986) and nitrosamines like NNK and NNN are more extensively metabolized in Clara cells than other cells of lung (Belinsky et al, 1989). Further, it has also been shown that nitrosamines such as diethylnitrosamine and NNK are taken up by Clara cells via /%adrenergic receptors (Schuller and Cole, 1989) which are not present in type II cells.…”