Identification of the enzyme isoforms responsible for the N‐oxide detoxification pathway in tobacco specific nitrosamines (TSNAs)

Abstract

Tobacco‐specific nitrosamines (TSNAs), are among the most potent carcinogens found in tobacco products. Among them are N′‐nitrosonornicotine (NNN), N′‐nitrosoanabasine (NAB), N′‐nitrosoanabatine (NAT), 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) and 4‐ (methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL), a major metabolite of NNK. TSNAs are detoxified by a number of metabolic pathways, including glucuronidation by the UDP‐glucuronosyltransferase (UGT) family of enzymes, as well as N‐oxidation by an as yet unidentified enzyme or family of enzymes. TSNAs share a pyridine ring in their chemical structure that can be further metabolized to a detoxification N‐oxide product. TSNA‐N‐oxides have been identified in the urine of rodents exposed to TSNAs and NNAL‐N‐oxide was found in the urine of smokers, however, the enzymes responsible for this conversion have not yet been determined. To identify enzymes that catalyze TSNA‐N‐oxide formation, we used specific inhibitors for the major hepatic cytochrome P450 (CYP) enzymes (CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) in oxidation reactions with pooled human liver microsomes, including phenacetin, coumarin, bupropion, amodiaquine, diclofenac, omeprazole, dextrometrphan, chlorzoxazone and midazolam. Inhibition of NNK‐N‐oxide and NNAL‐N‐oxide formation was observed when using inhibitors for CYPs 1A2, 2A6, 2E1, 2C19 and 3A4. To confirm results from these inhibition studies, NNK‐N‐oxide and NNAL‐N‐oxide formation was examined using microsomal fractions from CYP over‐expressing HEK293 cells. Microsomes from cells over‐expressing CYPs 1A2, 2C19, 2E1 and 3A4 isoforms exhibited TSNA‐N‐oxide formation. The isoforms 2C19 and 2E1 presented an approximate two‐fold higher activity than the other active isoforms. These data suggest that several cytochrome P450 enzymes may play an important role in the N‐oxidation of TSNAs, and that inter‐individual variability in the activity of these enzymes could potentially be important in TSNA‐induced cancer risk.Support or Funding Information National Institutes of Health, National Institutes of Environmental Health Sciences [Grant R01‐ES025460] Health Sciences and Services Authority of Spokane, WA [Grant WSU002292] This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.