Factors Regulating Activation and DNA Alkylation by 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone and Nitrosodimethylamine in Rat Lung and Isolated Lung Cells, and the Relationship to Carcinogenicity

The molecular dosimetry for O⁶-methylguanine (O⁶MG) formation in DNA from rat lung and pulmonary cells was compared following treatment for 4 days with equimolar doses of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent pulmonary carcinogen or nitrosodimethylamine (NDMA), a weak carcinogen in rat lung. The dose response for O⁶MG formation from NNK was biphasic; the O⁶MG to dose ratio, an index of alkylation efficiency, increased dramatically as the dose of carcinogen was decreased. In contrast, the dose-response curve for methylation by NDMA appeared opposite of that for NNK with alkylation efficiency increasing as a function of dose. These results suggested that high and low K_m pathways exist for the activation of NNK, whereas only high K_m pathways may be involved in NDMA activation. Furthermore, DNA methylation by NNK was cell selective with the highest levels in the Clara cell, whereas methylation by NDMA was not. DNA methylation in the Clara cell was 50-fold greater by NNK than by NDMA at equimolar doses (0.005 mmol/kg). Thus, differences in O⁶MG formation, specifically the presence of a high affinity pathway in the Clara cell for activation of NNK, may explain why following low dose exposure, NNK is a potent pulmonary carcinogen while NDMA is not. Different cytochrome P-450 isozymes also appear to be involved in the activation of NNK and NDMA. Inhibition of in vitro methylation (with calf thymus DNA and lung microsomes) by antibodies to cytochrome P-450 isozymes provided evidence that a homolog of rabbit cytochrome P-450₂ (cytochrome P-450_b) may be important in the activation of NNK in rat lung, whereas cytochrome P-450₅ may activate NDMA. A 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible cytochrome P-450 isozyme (P-450_c) may also be involved in the activation of NNK but not NDMA. Treatment with TCDD increased both NNK activation by pulmonary microsomes and the formation of O⁶MG in Clara cells and type II cells incubated in vitro with NNK. α-Naphthoflavone (α-NF), a specific inhibitor of cytochrome P-450_c, reversed the increase in methylation by TCDD-induced microsomes but did not inhibit in vitro activation of NNK using microsomes from untreated rats. However, NNK mediated O⁶MG formation in Clara cells, but not in type II cells incubated with α-NF, was decreased by 21%. These data indicate that both cytochrome P-450_b and P-450_c are probably involved in the activation of NNK in Clara cells from untreated rats. Based on the localization of DNA methylation after treatment with NNK or NDMA and experiments with antibodies to cytochrome P-450 isozymes, results from this study also support the hypothesis that cytochrome P-450_b is more concentrated in Clara cells while cytochrome P-450₅ appears to be distributed more uniformly within the rodent lung. The significant differences in dosimetry, DNA methylation patterns, and pathways for activation of NNK and NDMA strongly reinforce the importance of dose-response studies in both target tissues and cell populations before accurate low dose risk estimates can be predicted for the specific carcinogen being investigated.