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1. Drug Alcohol Depend. 2012 Apr 10. [Epub ahead of print]

An LC-MS/MS method for concurrent determination of nicotine metabolites and the
role of CYP2A6 in nicotine metabolite-mediated oxidative stress in SVGA

Ande A, Earla R, Jin M, Silverstein PS, Mitra AK, Kumar A, Kumar S.

Pharmacology and Toxicology, School of Pharmacy, 3253 Health Sciences Building,
University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, MO 64108,

BACKGROUND: Nicotine is known to generate oxidative stress through cytochrome
P450 2A6 (CYP2A6)-mediated metabolism in the liver and other organs, including
macrophages. This study has been designed to examine the role of CYP2A6 in
nicotine metabolism and oxidative stress in SVGA cells, an immortalized human
astrocyte cell line. METHODS: SVGA astrocytes were treated with 1μM nicotine,
followed by determination of mRNA and protein levels of several CYPs using
quantitative RT-PCR and western blot analyses, respectively. Quantitation of
nicotine and the nicotine metabolites, cotinine and nicotine-derived nitrosamine
ketones (NNK), was performed using an LC-MS/MS method. The generation of reactive
oxygen species (ROS) was measured using flow cytometry. RESULTS: Nicotine
significantly upregulated mRNA and protein expression of the most abundantly
expressed CYPs in SVGA astrocytes, CYP2A6 and CYP1A1. To characterize the
metabolism of nicotine in astrocytes, a highly sensitive LC-MS/MS method was
developed which is capable of quantifying very low concentrations of nicotine
(0.3ng/mL), cotinine and NNK (0.11ng/mL). The LC-MS/MS results showed that
nicotine is steadily metabolized to cotinine and NNK from 0.5 to 4h. Finally, we
showed that nicotine initially causes an increase in ROS formation which is then
gradually decreased, perhaps due to the increase in superoxide dismutase level.
Nicotine metabolism and ROS formation by CYP2A6 were further confirmed by using
tryptamine, a selective inhibitor of CYP2A6, which significantly lowered the
levels of cotinine and NNK and inhibited ROS formation. CONCLUSIONS: CYP2A6 plays
a key role in nicotine metabolism and oxidative stress in astrocytes, and this
has implications in nicotine-associated brain toxicity.

Copyright © 2012. Published by Elsevier Ireland Ltd.

PMID: 22498344 [PubMed - as supplied by publisher]