Chronic exposure to e-cigarettes elevates CYP2A5 activity, protein expression, and cotinine-induced production of reactive oxygen species in mice.

Coumarin 7'-hydroxylase activity, a specific marker of CYP2A5 activity, and the protein level were measured in liver microsomes of male mice after chronic exposure to e-cigarettes (2.4% nicotine). After exposure for 240 min/day for 5 days, the activity and the protein level in preproenkephalin (ppENK) heterozygous (ppENK (+/-)) mice were significantly elevated ( p< 0.05) compared to the untreated control. This elevation was not due to deletion of the ppENK gene, because the activity did not differ among untreated ppENK (+/-), ppENK (-/-) and wildtype ppENK (+/+) controls. Hence the elevation can reasonably be attributed to nicotine exposure. The production of reactive oxygen species (ROS) upon incubation of the hepatic microsomes of these mice with cotinine, was higher in microsomes from the e-cig-treated mice, compared to the untreated controls ( p <0.01). Liquid-chromatography/mass-spectrometry assay showed three oxidation products of cotinine, viz trans 3'-hydroxycotinine (3'-HC), 5'-hydroxycotinine (5'-HC) and cotinine N-oxide (CNO) in the plasma of these mice. The result identifies these three oxidation reactions as the source of the observed ROS and also shows that, in nicotine-treated mice, the appropriate "nicotine metabolite ratio" is (3'-HC + 5'-HC + CNO)/cotinine. The results suggest intriguing possibilities that (i) this metabolite ratio may correlate with plasma nicotine clearance and hence impact nicotine's psychoactive effects and (ii) chronic e-cig treatment causes ROS-induced oxidative stress which may play a major role in the regulation of CYP2A5 expression. Our present results clearly show that both the activity and the protein level of CYP2A5 are elevated by repeated exposure to nicotine. Significance Statement Nicotine, the psychoactive ingredient of tobacco, is eliminated as the oxidation products of cotinine in reactions catalyzed by the enzymes CYP2A5 in mice and CYP2A6 in humans. This study shows that repeated exposure to e-cigarettes elevates the level of CYP2A5 and the formation of reactive oxygen species. The results suggest an intriguing possibility that CYP2A5 may be up-regulated by chronic nicotine exposure due to oxidative stress caused by the oxidation of cotinine, in this preclinical model of human smokers.