The tobacco-specific nitrosamine N'-nitrosonornicotine (NNN) induces esophageal but not liver tumors in the rat. This may in part be due to tissue-specific differences in the activation of this nitrosamine. Therefore, the metabolism of NNN by microsomes from the mucosa of the rat esophagus was characterized and compared to its metabolism by liver microsomes. Esophageal microsomes metabolized NNN to both 4-hydroxy-l-(3-pyridyl)-l-butanone and 2-hydroxy-5-(3-pyridyI)tetrahydrofuran, the products of 2'- and 5'-hydroxylation of the pyrrolidine ring, respectively. This activity required an NADPH-generating system and was inhibited by carbon monoxide, suggesting that it s mediated by a cytochrome P450 enzyme. The apparent tfM for total a-hydroxylation of NNN by esophageal microsomes was 49 ± 6.5 μM and Vmax, was 113 ± 3.7 pmol/mg/min. The ratio of 2'-hydroxylation to 5'-hydroxylation was 3.2 ± 0.5 when the NNN concentration was varied from 1 μM to 2 mM. 2'-Hydroxylation is believed to be the activation pathway responsible for the tumorigenicity of NNN. In contrast, the ratio of 2'- to 5'-hydroxylation of NNN by liver microsomes was between 0.71 and 0.23 depending on the concentration of NNN used. Hepatic microsomal metabolism of NNN was not saturated at 2 mM NNN, the highest concentration of NNN used. These results confirm the existence of an esophageal enzyme with high affinity for a-hydroxylation of NNN; it is probably a cytochrome P450. If this enzyme exists in the liver its activity is masked by high mM, high Vmax, enzymes which also a-hydroxylate NNN. These enzymes are not present in the esophagus. The presence of a low KM esophageal enzyme that 2-hydroxylates NNN is consistent with the hypothesis that NNN esophageal tumorigenicity is at least in part due to the efficient activation of NNN in this tissue.
Bibliographical noteFunding Information:
We thank the Research Animal Facility and the Organic Synthesis Facility of the American Health Foundation for contributing to the work presented in this manuscript. These facilities are supported by NCI Cancer Center Support Grant CA-17613. This research was supported by grant CA-29580 from the US National Cancer Institute.