Evidence for an Important Role of DNA Pyridyloxobutylation in Rat Lung Carcinogenesis by 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone: Effects of Dose and Phenethyl Isothiocyanate

Abstract

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), selectively induces lung tumors in F344 rats. NNK is metabolically activated to intermediates that methylate and pyridyloxobutylate DNA. To explore the importance of pyridyloxobutyl DNA adducts in NNK-induced rat lung tumorigenesis, the first study in this report examined levels of these adducts in whole lung and pulmonary cells of F344 rats treated with different doses of NNK (0.3, 1.0, 10.0, and 50 mg/kg; 3 × weekly for 2 weeks). Pyridyloxobutyl DNA adducts were highest in Clara cells compared to alveolar Type II cells, alveolar macrophages, and small cells, suggesting that enzymes involved in the formation of the pyridyloxobutylating species are concentrated in Clara cells. When we compared lung tumor incidence at the different doses of NNK (S. A. Belinsky et al., Cancer Res., 50: 3772–3780, 1990) versus pyridyloxobutyl DNA adducts in Type II cells, we observed a significant correlation. Because NNK-induced lung tumors arise from the Type II cells, this suggests an important role for pyridyloxobutyl DNA adducts. In the second study presented in this report, we examined the effect of dietary phenethyl isothiocyanate (PEITC), an inhibitor of lung tumor induction in F344 rats by NNK, on O⁶-methyldeoxyguanosine (O⁶-mG) and pyridyloxobutyl DNA adducts in whole lung and lung cells of F344 rats treated with NNK. F344 rats were fed control or PEITC-containing diets (3 µmol/g diet) before and throughout NNK treatment (1.76 mg/kg, three times weekly for 4, 8, 12, 16, or 20 weeks). PEITC inhibited formation of pyridyloxobutyl DNA adducts in whole lung and all lung cells except macrophages. There was also inhibition of O⁶-mG, but it varied with cell type and length of NNK treatment. Overall, PEITC treatment decreased pyridyloxobutyl DNA adducts by 57% in Clara cells, 51% in Type II cells, 40% in small cells, and 44% in whole lung. PEITC treatment decreased O⁶-mG levels by 52% in Clara cells, 19% in Type II cells and small cells, and 36% in whole lung. These results support the hypothesis that PEITC inhibition of NNK-induced lung tumors is a result of decreased metabolic activation and DNA binding of NNK. The 50% reduction of pyridyloxobutyl DNA adducts in Type II cells agreed well with the 50% reduction of NNK-induced lung tumors by PEITC. Because NNK-induced tumors arise from Type II cells, these results suggest an important role for pyridyloxobutyl DNA adducts in NNK-induced rat lung tumorigenesis.