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. 2012 Sep 1;125(1-2):49-59.
doi: 10.1016/j.drugalcdep.2012.03.015. Epub 2012 Apr 11.

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

Anusha Ande  1 , Ravinder Earla, Mengyao Jin, Peter S Silverstein, Ashim K Mitra, Anil Kumar, Santosh Kumar
Affiliations

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

Anusha Ande et al. Drug Alcohol Depend. .

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.

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Conflict of interest statement

Conflict of interest

The authors report no conflicts of interest regarding this manuscript. The authors alone are responsible for the content and writing of the paper.

Figures

Figure 1
Figure 1
LC-MS/MS MRM chromatograms of nicotine (A), cotinine (B), and NNK (C) along with the internal standard ritonavir in SVGA astrocytes. The level of the blank, the lower limit of quantification, and upper limit of quantification for nicotine (top three panels), cotinine (middle three panels), and NNK (bottom three panels) are shown in the left panels. Ritonavir is shown in the right column except for the blank for each compound. The intensity (cps) is presented in the Y-axis and time (min) is presented in the X-axis. The standard linear curve of nicotine, cotinine, and NNK are shown from top to bottom in the right side of the chromatograms.
Figure 1
Figure 1
LC-MS/MS MRM chromatograms of nicotine (A), cotinine (B), and NNK (C) along with the internal standard ritonavir in SVGA astrocytes. The level of the blank, the lower limit of quantification, and upper limit of quantification for nicotine (top three panels), cotinine (middle three panels), and NNK (bottom three panels) are shown in the left panels. Ritonavir is shown in the right column except for the blank for each compound. The intensity (cps) is presented in the Y-axis and time (min) is presented in the X-axis. The standard linear curve of nicotine, cotinine, and NNK are shown from top to bottom in the right side of the chromatograms.
Figure 1
Figure 1
LC-MS/MS MRM chromatograms of nicotine (A), cotinine (B), and NNK (C) along with the internal standard ritonavir in SVGA astrocytes. The level of the blank, the lower limit of quantification, and upper limit of quantification for nicotine (top three panels), cotinine (middle three panels), and NNK (bottom three panels) are shown in the left panels. Ritonavir is shown in the right column except for the blank for each compound. The intensity (cps) is presented in the Y-axis and time (min) is presented in the X-axis. The standard linear curve of nicotine, cotinine, and NNK are shown from top to bottom in the right side of the chromatograms.
Figure 2
Figure 2
Effect of nicotine on mRNA expression of CYP1A1 and CYP2A6 in SVGA astrocytes. The SVGA astrocytes were treated with 1 μM nicotine for 0.5, 1, 3, 6, and 12 h. The percentage mRNA levels were calculated using qRT-PCR, with 100% expression normalized for the control at every time point. Expression of both the CYP genes was normalized to glyceraldehyde 3-phosphate dehydrogenase. In the above figures, the X-axis represents treatment time (hours) and Y-axis represents % mRNA expression. * Represents p ≤ 0.05 compared to respective controls.
Figure 3
Figure 3
Effect of nicotine on levels of protein expression of CYP1A1 and CYP2A6. The SVGA astrocytes were treated with 1 μM nicotine for 0.5, 1, 3, 6, and 12h and the protein was isolated. The percentage protein expression was calculated by quantifying the immunoblots, with 100% expression normalized to the untreated cells (control) at every time points. Expression of CYP1A1 and CYP2A6 was normalized against β-actin. In the above figures, X-axis represents treatment time (hours) and Y-axis represents % protein expression. * Represents p ≤ 0.05 compared to respective controls.
Figure 4
Figure 4
Metabolism of nicotine in SVGA astrocytes. The cells were incubated with 1 μM nicotine for 0.5, 1, 2, and 4h and cotinine and NNK levels were measured using the LC-MS/MS method. A. Diagram depicting the metabolism of nicotine and the metabolic products cotinine and NNK. B. Representative MS2 profiles of nicotine, cotinine, and NNK with parent molecular ion transition and its fragmentation pattern in MS2 of nicotine, cotinine, and NNK. C. Kinetic profiles of cotinine and NNK formation. In the figures, X-axis represents the treatment time (hours) and Y-axis represents concentration of cotinine and NNK. The graphs were plotted as mean ± SD using three replicates for each time point.
Figure 4
Figure 4
Metabolism of nicotine in SVGA astrocytes. The cells were incubated with 1 μM nicotine for 0.5, 1, 2, and 4h and cotinine and NNK levels were measured using the LC-MS/MS method. A. Diagram depicting the metabolism of nicotine and the metabolic products cotinine and NNK. B. Representative MS2 profiles of nicotine, cotinine, and NNK with parent molecular ion transition and its fragmentation pattern in MS2 of nicotine, cotinine, and NNK. C. Kinetic profiles of cotinine and NNK formation. In the figures, X-axis represents the treatment time (hours) and Y-axis represents concentration of cotinine and NNK. The graphs were plotted as mean ± SD using three replicates for each time point.
Figure 4
Figure 4
Metabolism of nicotine in SVGA astrocytes. The cells were incubated with 1 μM nicotine for 0.5, 1, 2, and 4h and cotinine and NNK levels were measured using the LC-MS/MS method. A. Diagram depicting the metabolism of nicotine and the metabolic products cotinine and NNK. B. Representative MS2 profiles of nicotine, cotinine, and NNK with parent molecular ion transition and its fragmentation pattern in MS2 of nicotine, cotinine, and NNK. C. Kinetic profiles of cotinine and NNK formation. In the figures, X-axis represents the treatment time (hours) and Y-axis represents concentration of cotinine and NNK. The graphs were plotted as mean ± SD using three replicates for each time point.
Figure 5
Figure 5
Effect of tryptamine (20 μM), a CYP2A6 selective inhibitor, on the formation of cotinine and NNK in 1 μM nicotine treated SVGA astrocytes. In the figures, percent of cotinine and NNK formation are presented in Y-axis. The time points of nicotine treatment are presented on the X-axis. The graphs were plotted as mean ± SD using three replicates for each time point. * Represents p ≤ 0.05 compared to respective controls.
Figure 6
Figure 6
Effect of nicotine on CYP2A6 mediated oxidative stress in SVGA astrocytes. The ROS production was measured using a flow cytometer from untreated and 1 μM nicotine treated cells from 15, 30, 60, 90, and 120 min. A. Bar graphs representing mean fluorescence intensity (MFI) from control and nicotine-treated cells. MFI is presented in Y-axis and the time points of nicotine treatment are presented in X-axis. The graphs were plotted as mean ± SD from three replicates. B. Effect of 20 μM tryptamine, a CYP2A6 selective inhibitor on the formation of oxidant contents in untreated and 1 μM nicotine treated cells for 30 min. * Represents p value ≤ 0.05 and # represents p value ≤ 0.1.
Figure 7
Figure 7
Effect of 1 μM nicotine on the mRNA expression of SOD and catalase in SVGA astrocytes. The SVGA astrocytes were treated with 1 μM nicotine for 1, 3, 6, and 12h. The percentage mRNA levels of both the enzymes were calculated using qRT-PCR, with 100% expression normalized to the control. . Expression of both the genes was normalized against glyceraldehyde 3-phosphate dehydrogenase. X-axis represents the treatment time and Y-axis represents % mRNA expression. * Represents p value ≤ 0.05 and # represents p value ≤ 0.1.
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