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E-Cigarette Flavoring Compounds Inhibit the Primary Nicotine-Metabolizing Enzyme in a Novel Cell Culture System
Citation:
Winters, B., A. Wallace, P. Clapp, J. Pleil, Steve Simmons, M. Madden, AND I. jaspers. E-Cigarette Flavoring Compounds Inhibit the Primary Nicotine-Metabolizing Enzyme in a Novel Cell Culture System. Society of Toxicology Annual Meeting, Baltimore, MD, March 10 - 14, 2019.
Impact/Purpose:
This study demonstrates the inhibition of CYP2A6 activity by some e-cigarette flavorings and some of the components of the flavorings. Such alteration of CYP2A6 activity may alter nicotine pharmacokinetics in e-cigarette users. A purpose of the study was to demonstrate CYP2A6 inhibition by some agents.
Description:
E-Cigarette Flavoring Compounds Inhibit the Primary Nicotine-Metabolizing Enzyme in a Novel Cell Culture System Brett R. Winters1, Ariel M. Wallace2, Phillip W. Clapp1, Joachim D. Pleil2, Steven Simmons2, Michael C. Madden2, and Ilona Jaspers1 1Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC, 2US EPA, Research Triangle Park, NC Flavorings in e-cigarette liquids are a primary driver of e-cigarette use among teens. While recent studies have investigated pulmonary toxicities associated with exposure to flavorings in e-cigarettes, little research has been conducted on the impact of flavorings on the pharmacokinetics of nicotine, the primary addictive compound of e-cigarettes. Utilizing recombinant CYP2A6, the major enzyme responsible for the metabolism of nicotine, we screened 3 e-liquids, Strawberry Poptart (SP), Apple Watermelon (AW), and Flamethrower (FT) at concentrations ranging from 0.00025% to 0.25% (v/v), for CYP2A6 activity inhibition. CYP2A6 activity was determined by measuring the formation of the fluorescent metabolite of 3-cyano-7-hydroxycoumarin, a substrate of CYP2A6. Incubation with FT and SP strongly inhibited CYP2A6 as low as at 0.0025%, while AW exhibited no CYP2A6 inhibition. GC-MS analysis was conducted on FT and SP to identify compounds responsible for CYP2A6 inhibition. Trans-cinnamaldehyde, a potential inhibitor of CYP2A6, was identified in FT, while benzaldehyde was identified in SP. We subsequently screened both aldehydes using recombinant CYP2A6 at concentrations ranging from 0.25¿M to 250¿M. Trans-cinnamaldehyde and benzaldehyde exhibited near maximal inhibition of CYP2A6 at 2.5¿M and 25¿M, respectively. To confirm our findings in a cell-based system, we developed a novel in vitro cell culture apparatus capable of screening volatile compounds when heated in an incubator-something not feasible in traditional cell culture systems. In the novel culture system, BEAS-2B cells transduced to overexpress CYP2A6 were cultured and subsequently exposed to e-liquids and flavoring compounds in the presence of nicotine. We quantified nicotine levels using LC-MS. Exposure to SP, FT, and both flavoring aldehydes significantly reduced the metabolism of nicotine by CYP2A6 at concentrations consistent with what was observed using recombinant CYP2A6. In summary, these data indicate that some flavoring compounds in e-cigarettes have the capacity to inhibit CYP2A6 activity, the enzyme responsible for catabolizing nicotine. This may increase serum concentrations of nicotine and have implications for the risks associated with e-cigarette use. [This abstract may not reflect official US EPA policy.]