Biotransformation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in freshly isolated human lung cells

Abstract

Metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was characterized in human lung cells isolated from peripheral lung specimens obtained from 12 subjects during clinically indicated lobectomy. NNK biotransformation was assessed in preparations of isolated unseparated cells (cell digest), as well as in preparations enriched in alveolar type II cells, and alveolar macrophages. Metabolite formation was expressed as a percentage of the total recovered radioactivity from [5-(3)H]NNK and its metabolites per 10(6) cells per 24 h. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was the major metabolite formed in all lung cell preparations examined, and its formation ranged from 0.50 to 13%/10(6) cells/24 h. Formation of alpha-carbon hydroxylation end-point metabolites (bioactivation) and pyridine N-oxidation metabolites (detoxification), ranged from non-detectable to 0.60% and from non-detectable to 1.5%/10(6) cells/24 h, respectively, reflecting a large degree of intercellular and inter-individual variability in NNK metabolism. Formation of the alpha-hydroxylation end-point metabolite 4-hydroxy-1-(3-pyridyl)-1-butanol (diol) was consistently higher in alveolar type II cells than in cell digest or alveolar macrophages (0.0146 +/- 0.0152, 0.0027 +/- 0.0037 and 0.0047 +/- 0.0063%/10(6) cells/24 h, respectively; n = 12; P < 0.05). SKF-525A was used to examine cytochrome P450 contributions to the biotransformation of NNK. SKF-525A inhibited keto reduction of NNK to NNAL by 85, 86 and 74% in cell digest, type II cells, and macrophages, respectively (means of 11 subjects, P < 0.05). Type II cell incubates treated with SKF-525A formed significantly lower amounts of total alpha-hydroxylation metabolites compared with type II cells without SKF-525A (0.0776 +/- 0.0841 versus 0.1694 +/- 0. 2148%/10(6) cells/24 h, respectively; n = 11; P < 0.05). The results of this first study examining NNK biotransformation in freshly isolated human lung cells indicate that NNK metabolism is subject to a large degree of inter-individual and intercellular variability, and suggest a role for P450s in human lung cell NNK metabolism. Both alveolar type II cells and alveolar macrophages may be potential target cells for NNK toxicity based on their alpha-carbon hydroxylation capabilities. In addition, carbonyl reduction of NNK to NNAL is SKF-525A sensitive in human lung cells.

CYP2A13: Variable Expression and Role in Human Lung Microsomal Metabolic Activation of the Tobacco-Specific Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

“…By the same token, the rates of NNK ␣-hydroxylation were, on average, only ϳ40% higher in microsomes containing 3 to 20 fmol/mg CYP2A13 than in microsomes containing Ͻ2 fmol/mg CYP2A13; the rate difference would be much greater if the rates were normalized by total protein in airway epithelial cells, rather than by proteins of all lung cells. In support of this idea, a previous study had reported increased formation of NNK ␣-hydroxylation products in an enriched human alveolar type II cell preparation, compared with the amounts formed by a mixed population of lung cells or an enriched alveolar macrophage preparation (Smith et al, 1999). Even in individuals with Ͻ2 fmol CYP2A13/mg microsomal protein, CYP2A13 may still play an important role in NNK metabolic activation in airway epithelial cells, a notion supported by the 30% reduction in activity rendered by the addition of 8-MOP to CYP2A13-Low samples.…”

Analysis of CYP2A Contributions to Metabolism of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone in Human Peripheral Lung Microsomes

“…Before use, the purity of H]NNK was assessed, since high purity (Ͼ98%) was needed to ensure that impurities would not interfere with quantification of metabolites. If purity was Ͻ98%, H]NNK was purified by high-performance liquid chromatography as described previously (Smith et al, 1999). Incubation mixtures were prepared as described previously (Smith et al, 2003), using 4.2 M [5-3 H]NNK and 1.0 mg of microsomal protein in a total volume of 1.0 ml.…”

“…These endpoint metabolites are used in assessing the degree of NNK bioactivation, since their formation is indicative of the formation of the DNA-reactive metabolites. The detoxification of NNK and NNAL occurs mainly through pyridine N-oxidation, which results in the formation of excretable N-oxides (Hecht, 1998).Cytochromes P450 (P450s), specifically CYP2A6/2A13, CYP2B6, CYP3A4/3A5, and CYP2E1 (Smith et al, 1992(Smith et al, , 1995(Smith et al, , 1999(Smith et al, , 2003Hecht, 1998), and prostaglandin H synthase (Smith et al, 1995(Smith et al, , 1999 but not lipoxygenases (Bedard et al, 2002) have been implicated in human pulmonary NNK metabolism. Of particular interest are the CYP2A isozymes CYP2A13 and CYP2A6.…”

“…Cytochromes P450 (P450s), specifically CYP2A6/2A13, CYP2B6, CYP3A4/3A5, and CYP2E1 (Smith et al, 1992(Smith et al, , 1995(Smith et al, , 1999(Smith et al, , 2003Hecht, 1998), and prostaglandin H synthase (Smith et al, 1995(Smith et al, , 1999 but not lipoxygenases (Bedard et al, 2002) have been implicated in human pulmonary NNK metabolism. Of particular interest are the CYP2A isozymes CYP2A13 and CYP2A6.…”

Serum levels of CC16, SP-A and SP-B reflect tobacco-smoke exposure in asymptomatic subjects

“…Cytochrome P450 may also biotransform the tobacco specific carcinogens to 4-(methylnitrosamine)-1-(-3-pyridine)-1-butadone (NNK). If so, both alveolar type II cells and macrophages may be target cells for NNK toxicity [39].…”