title: 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.
abstract: 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 μM 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 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.
authors.affiliation: Pharmacology and Toxicology, School of Pharmacy, 3253 Health Sciences Building, University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, MO 64108, USA.
authors.forename: Anusha
authors.initials: A
authors.lastname: Ande
authors.forename: Ravinder
authors.initials: R
authors.lastname: Earla
authors.forename: Mengyao
authors.initials: M
authors.lastname: Jin
authors.forename: Peter S
authors.initials: PS
authors.lastname: Silverstein
authors.forename: Ashim K
authors.initials: AK
authors.lastname: Mitra
authors.forename: Anil
authors.initials: A
authors.lastname: Kumar
authors.forename: Santosh
authors.initials: S
authors.lastname: Kumar
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis / Q000378:metabolism*; D001253:Astrocytes / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003367:Cotinine / Q000032:analysis; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis / Q000378:metabolism; D065637:Cytochrome P-450 CYP2A6; D004790:Enzyme Induction / Q000187:drug effects; D006801:Humans; D007659:Ketones / Q000032:analysis; D057230:Limit of Detection; D009538:Nicotine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000032:analysis* / Q000494:pharmacology; D009602:Nitrosamines / Q000032:analysis; D018384:Oxidative Stress / Q000502:physiology*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D017382:Reactive Oxygen Species / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
chemical_list: D007659:Ketones; D018722:Nicotinic Agonists; D009602:Nitrosamines; D017382:Reactive Oxygen Species; D012313:RNA; D009538:Nicotine; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D019363:Cytochrome P-450 CYP1A1; D065637:Cytochrome P-450 CYP2A6; D003367:Cotinine