A LC-MS/MS Method for Concurrent Determination of Nicotine Metabolites and Role of CYP2A6 in Nicotine Metabolism in U937 Macrophages: Implications in Oxidative Stress in HIV + Smokers

Abstract

Nicotine, the major constituent of tobacco, is predominantly metabolized by liver CYP2A6 into cotinine and many other compounds, including nicotine-derived nitrosamine ketone (NNK), which is known to cause oxidative stress. We have recently shown that CYP2A6 is highly expressed in U937 monocyte-derived macrophages. In this study we investigated the role of CYP2A6 in nicotine metabolism and oxidative stress in U937 macrophages. To study nicotine metabolism, we developed a highly sensitive LC-MS/MS method for simultaneous quantitative determination of nicotine, cotinine, and NNK. The LC-MS/MS analysis was carried out by multiple reaction monitoring mass transitions with m/z of 163.2/130.1, 177.4/98.3, and 208.4/122.1 for nicotine, cotinine, and NNK, respectively. The calibration curves were linear within 3.3–1028.1 ng/ml for nicotine and 0.3–652.6 ng/ml for cotinine and NNK. This novel method was then applied to quantify nicotine metabolites, cotinine and NNK, in nicotine-treated U937 macrophages. Cotinine and NNK initially formed at 30 min, followed by a peak at 2–3 h. The role of CYP2A6 in nicotine metabolism in U937 macrophages was further confirmed by using CYP2A6-selective inhibitor, tryptamine, which significantly decreased cotinine (70%) and completely inhibited NNK formations. Finally, we showed that nicotine-treated macrophages increase the formation of oxidant at 30–60 min, which is consistent with the initial formation of cotinine and NNK. In conclusion, we have developed a new LCMS/MS method for concurrent determination of nicotine metabolites and analyzed the role of CYP2A6 in nicotine metabolism and oxidative stress in U937 macrophages, which may have implications in viral replication among HIV+smokers.

Enhanced Nicotine Metabolism in HIV-1–Positive Smokers Compared with HIV-Negative Smokers: Simultaneous Determination of Nicotine and its Four Metabolites in Their Plasma Using a Simple and Sensitive Electrospray Ionization Liquid Chromatography–Tandem Mass Spectrometry Technique

“…The recovery efficiency of this method is higher than the previous methods (Miller et al, 2010;Jacob et al, 2011;Ande et al, 2012;Jin et al, 2012a), and the peak response is also reproducible. We previously developed a liquid-liquid extraction method for nicotine and its metabolites in astrocytic and monocytic cell lines (Ande et al, 2012;Jin et al, 2012a).…”

“…Furthermore, we have shown that the ROS generated through ethanol metabolism by CYP2E1 induces the expression of CYP2A6 in monocytes (Jin et al, 2012b) Earlier findings have shown that oxidative stress increases HIV-1 replication in alveolar macrophages from smokers (Boelaert et al, 1996;Israel and Gougerot-Pocidalo, 1997;Aquaro et al, 2007). Since our recent findings show the role of nicotine metabolism by CYP2A6 in ROS production (Ande et al, 2012;Jin et al, 2012a), we sought to determine the plasma levels of nicotine and four of its metabolites that are formed by CYP2A6 in HIV-1-positive smokers. Nicotine and its metabolites have primarily been studied in urine and to some extent in other biologic matrices such as serum, plasma, hair, and nails using immunoassay, high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry with protein precipitation, liquid extraction methods, and solid phase extraction (SPE) methods (Jarvis et al, 2003;Byrd et al, 2005;Shu and Wang, 2013).…”

“…We previously developed a liquid-liquid extraction method for nicotine and its metabolites in astrocytic and monocytic cell lines (Ande et al, 2012;Jin et al, 2012a). However, in the present method, we used strong cation exchange SPE cartridges for plasma sample analysis.…”

“…In addition, nicotine is known to be toxic to other cells, including brain (Bhagwat et al, 1998). We recently showed the expression of CYP2A6 in astrocytes and monocytes, as well as the role of CYP2A6 in nicotine metabolism to cotinine that induces the formation of reactive oxygen species (ROS) (Jin et al, 2011(Jin et al, , 2012aAnde et al, 2012). Furthermore, we have shown that the ROS generated through ethanol metabolism by CYP2E1 induces the expression of CYP2A6 in monocytes (Jin et al, 2012b) Earlier findings have shown that oxidative stress increases HIV-1 replication in alveolar macrophages from smokers (Boelaert et al, 1996;Israel and Gougerot-Pocidalo, 1997;Aquaro et al, 2007).…”

Reduced biological effect of e-cigarette aerosol compared to cigarette smoke evaluated in vitro using normalized nicotine dose and RNA-seq-based toxicogenomics

“…A Waters Acquity UPLC (Waters, Milford, MA, USA) connected to an Applied Biosciences 4000QTrap mass spectrometer (Applied Biosystems, Foster City, CA, USA) was used for the quantification of nicotine. The UPLC method was adapted from Onoue et al 44 and the quantitative MS/MS method used the settings described by Jin et al 45 .…”