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.

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 LCMS/MS results showed that nicotine is steadily metabolized to cotinine and NNK from 0.5–4h. 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.

No abstract

Smoking is approximately three times more prevalent in HIV-1-positive than HIV-negative individuals in the United States. Nicotine, which is the major constituent of tobacco, is rapidly metabolized mainly by cytochrome P450 (CYP2A6) to many metabolites. In this study, we developed a simple, fast, and sensitive electrospray ionization liquid chromatography-tandem mass spectrometry method using a strong cation solid phase extraction, and determined the concentration of nicotine and its four major metabolites (cotinine, nornicotine, norcotinine, and trans-39-hydroxycotinine) in the plasma of HIV-1-positive and HIV-negative smokers. The multiple reaction monitoring transitions for nicotine, cotinine, trans-39-hydroxycotinine, nornicotine, norcotinine, nicotine-d4, and cotinine-d3 were selected at mass-to-charge ratios of 163.3/117.1, 177.5/80.3, 193.2/80.1, 149.5/132.3, 163.4/80.3, 167.3/121.4, and 180.3/101.2, respectively. The lower limit of quantitation for nicotine and its metabolites was 0.53 ng/ml, which is relatively more sensitive than those previously reported. The concentration of nicotine was detected 5-fold lower in HIV-1-positive smokers (7.17 6 3.8 ng/ml) than that observed in HIV-negative smokers (33.29 6 15.4 ng/ml), whereas the concentration of the metabolite nornicotine was 3-fold higher in HIV-1-positive smokers (6.8 6 2.9 ng/ml) than in HIVnegative smokers (2.3 6 1.2 ng/ml). Although it was statistically nonsignificant, the concentration of the metabolite cotinine was also higher in HIV-1-positive smokers (85.6 6 60.5 ng/ml) than in HIV-negative smokers (74.9 6 40.5 ng/ml). In conclusion, a decrease in the concentration of nicotine and an increase in the concentration of its metabolites in HIV-1-positive smokers compared with HIV-negative smokers support the hypothesis that nicotine metabolism is enhanced in HIV-1-positive smokers compared with HIVnegative smokers.

Abstract. The objective of this study was to develop a clear, aqueous rapamycin-loaded mixed nanomicellar formulations (MNFs) for the back-of-the-eye delivery. MNF of rapamycin (0.2%) was prepared with vitamin E tocopherol polyethylene glycol succinate (TPGS) (Vit E TPGS) and octoxynol-40 (Oc-40) as polymeric matrix. MNF was characterized by various parameters such as size, charge, shape, and viscosity. Proton nuclear magnetic resonance ( 1 H NMR) was used to identify unentrapped rapamycin in MNF. Cytotoxicity was evaluated in human retinal pigment epithelial (D407) and rabbit primary corneal epithelial cells (rPCECs). In vivo posterior ocular rapamycin distribution studies were conducted in male New Zealand white rabbits. The optimized MNF has excellent rapamycin entrapment and loading efficiency. The average size of MNF was 10.98±0.089 and 10.84±0.11 nm for blank and rapamycin-loaded MNF, respectively. TEM analysis revealed that nanomicelles are spherical in shape. Absence of free rapamycin in the MNF was confirmed by 1 H NMR studies. Neither placebo nor rapamycin-loaded MNF produced cytotoxicity on D407 and rPCECs indicating formulations are tolerable. In vivo studies demonstrated a very high rapamycin concentration in retina-choroid (362.35±56.17 ng/g tissue). No drug was identified in the vitreous humor indicating the sequestration of rapamycin in lipoidal retinal tissues. In summary, a clear, aqueous MNF comprising of Vit E TPGS and Oc-40 loaded with rapamycin was successfully developed. Back-of-the-eye tissue distribution studies demonstrated a very high rapamycin levels in retina-choroid (place of drug action) with a negligible drug partitioning into vitreous humor.