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

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 1 mu M 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 (0.3 ng/mL), cotinine and NNK (0.11 ng/mL). The LC-MS/MS results showed that nicotine is steadily metabolized to cotinine and NNK from 0.5 to 4 h. 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. (C)2012 Elsevier Ireland Ltd. All rights reserved.