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A Mechanistic Study on the Inhibition of Nicotine Metabolism by Trans ‐Cinnamaldehyde
Author(s) -
Espiritu Michael,
Black Stephen,
Johnston Casey,
Chan Jeannine,
Harrelson John
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.833.11
Subject(s) - chemistry , cyp2a6 , cinnamaldehyde , heme , catalase , microsome , biochemistry , reactive oxygen species , coumarin , nicotine , enzyme , cyp1a2 , organic chemistry , biology , neuroscience , catalysis
Trans ‐cinnamaldehyde, a natural product found in cinnamon oil, has previously shown to selectively inhibit the major nicotine metabolizing enzyme cytochrome P450 2A6 (CYP2A6) via metabolism based inactivation using assays involving coumarin as a reporter substrate. In this study we aimed to investigate the mechanism by which CYP2A6 is inactivated by trans‐ cinnamaldehyde, with the ultimate goal of developing trans ‐cinnamaldehyde as a smoking cessation agent. We have also determined the level of inhibition of nicotine metabolism when nicotine is incubated with trans ‐cinnamaldehyde, human liver microsomes and cytosol. CYP2A6 activity and heme loss were monitored in the presence of trans ‐cinnamaldehyde and NADPH in vitro using human liver microsomes (HLM) and reconstituted CYP2A6 respectively. The effect of reactive oxygen species (ROS) generation on CYP2A6 activity and heme loss was studied using the reactive oxygen scavengers: N‐acetylcysteine, glutathione, catalase and superoxide dismutase. In an attempt to determine if heme loss was observed due to heme destruction or adduct formation, predictive multiple reaction monitoring (MRM) was performed using an optimized liquid chromatography tandem mass spectrometry (LC‐MS/MS) protocol. In activity studies using HLM and coumarin all reactive oxygen scavengers protected against activity loss, with N‐acetylcysteine and catalase offering the most protection (20% respectively) which, using a two tailed t‐test was found to be statistically significant with 99% confidence. Heme monitoring using LC‐MS/MS resulted in a significant amount of heme loss (97%) with a standard deviation (SD) of 3% after 18 minute incubations with trans ‐cinnamaldehyde and NADPH in comparison to enzyme only controls. Inclusion of glutathione resulted in approximately 46% (SD of 7%) protection of the heme. Incubations with an NADPH regenerating system yielded a 41% (SD of 9%) loss of heme in comparison to controls without trans ‐cinnamaldehyde. Predictive MRM yielded no detectable alkylated or acylated heme adducts or their respective oxidized forms. Results indicated an average inhibition of nicotine metabolism of 74.68% in the presence of trans‐ cinnamaldehyde. This study demonstrated for the first time that CYP2A6‐mediated nicotine metabolism is inhibited by trans‐ cinnamaldehyde and that ROS generation and heme destruction are major contributors to this process. Support or Funding Information Research reported here was supported by the National Institute on Drug Abuse of the National Institutes of Health under award number R15DA042341. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .