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The Centre for Addiction and Mental Health, and the Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada
(Received March 31, 2006; Accepted June 8, 2006)
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Abstract |
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). CYP2E1 also metabolizes ethanol (Lieber, 1999) and can be induced by ethanol in rats (Howard et al., 2001), rabbits (Lieber, 1999), and humans (Oneta et al., 2002). CYP2E1 contributes to ethanol metabolism at high blood alcohol concentrations in humans (Salaspuro and Lieber, 1978) and rats (Matsumoto et al., 1996). Many low molecular weight compounds such as benzene and carbon tetrachloride are also CYP2E1 substrates (Caro and Cederbaum, 2004). CYP2E1 produces a high level of reactive oxygen species, without need of a ligand, that can result in cell damage from lipid peroxidation and DNA strand breaks (Caro and Cederbaum, 2004).
Chlorzoxazone (CZN), a clinically used muscle relaxant, is metabolized by CYP2E1 to 6-hydroxychlorzoxazone (6OHCZN), and this reaction has been established as a phenotypic measure of CYP2E1 activity in vivo in humans (Lucas et al., 1999) and in vitro in rats (Kobayashi et al., 2002) and cynomolgus monkeys (Amato et al., 1998). No studies linking in vivo CZN disposition, in vitro hepatic CZN metabolism, and hepatic CYP2E1 protein levels have been published in any species. The in vivo CZN disposition, in vitro CZN metabolism, and hepatic CYP2E1 protein levels have not yet been investigated in African green monkeys (vervets, Cercopithecus aethiops). This monkey is a useful model of human metabolism; it has been established as a model of CYP2A6 (Schoedel et al., 2003) and CYP2B6 (Lee et al., 2006) isozyme metabolism. Our model provides an opportunity to investigate the relationship between these three variables in a nonhuman primate. African green monkey CYP2E1 has not yet been sequenced, but it is expected to have similar amino acid homology to that of human CYP2E1. Other monkeys such as the cynomolgus monkey (Macaca fascicularis) and the rhesus monkey (Macaca mulatta) have 92 and 95% amino acid homology to human CYP2E1, respectively [cynomolgus accession number: P33266, GI: 461827 (Komori et al., 1992), rhesus accession number: AAT49269.1, GI:49066335 (unpublished data)].
Cigarette smoking can alter the activity of CYP2E1. In humans, periods of cigarette smoking increase the in vivo CZN clearance compared with nonsmoking periods (Benowitz et al., 2003). In mice, exposure to cigarette smoke increases CYP2E1 protein, mRNA, and activity in lung, kidney, and liver (Villard et al., 1994; Seree et al., 1996; Villard et al., 1998). CZN metabolism is not affected by carbon monoxide (Benowitz et al., 2003), which is produced by burning cigarettes, nor by cotinine, the primary metabolite of nicotine (Micu et al., 2003). The increase in CYP2E1 is important because it may contribute to the harmful effects of smoking. CYP2E1 activates many procarcinogens found in cigarette smoke, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N-nitrosodimethylamine, and N-nitrosodiethylamine (Kushida et al., 2000), and is associated with the development of some types of cancer (Caro and Cederbaum, 2004). Nicotine could be the inducing agent responsible for the increase in CZN clearance in smokers. Nicotine has been shown to increase CYP2E1 protein and in vitro activity in rats (Anandatheerthavarada et al., 1993; Bhagwat et al., 1998; Howard et al., 2001; Micu et al., 2003). The effect of nicotine on CYP2E1 levels is of interest because current and former smokers may be exposed to nicotine via smoking or as a smoking cessation drug.
We hypothesize that there will be a significant relationship between in vivo CZN disposition, in vitro CZN metabolism, and hepatic CYP2E1 levels in monkeys. In addition, we hypothesize that chronic nicotine treatment will increase the rate of in vivo CZN disposition and the rate of in vitro CZN metabolism in hepatic microsomes, via an increase in hepatic CYP2E1 protein levels in monkeys.
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Materials and Methods |
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Animals. The study consisted of 12 male adult African green monkeys (vervets, C. aethiops) housed at Caribbean Primates Ltd. (St. Kitts) and three untreated adult African green monkey livers. The 12 African green monkeys were drawn from a large, isolated, and nonendangered Caribbean population and housed outdoors in social groups (Ervin et al., 1990). Monkeys were given standard rations of Purina monkey chow supplemented with fresh fruit and vegetables. Drinking water was available ad libitum. The nicotine-treated monkeys (n = 6) were given nicotine bitartrate (milligram base in saline, pH 7.0) at 0.05 mg/kg (all the injections s.c., twice daily) for 2 days, then 0.15 mg/kg for 2 days, followed by 0.3 mg/kg for 18 days. The control-treated monkeys (n = 6) received sham nicotine injections (saline s.c., twice daily). On day 14, all the monkeys received 7 mg/kg CZN intragastrically under ketamine anesthesia instead of nicotine or saline treatment, and a 6-ml blood sample was drawn at baseline, 10, 20, 30, 60, 120, 240, and 360 min after CZN administration. Blood samples were centrifuged, and the plasma removed and frozen for drug analyses. All the animals were sacrificed on day 22, 6 h after AM drug treatments under ketamine anesthesia. At the time of sacrifice, nicotine levels are estimated to be less than 5 ng/ml (Lee et al., 2006), and we have shown that nicotine does not interact with CYP2E1 (Howard et al., 2001), suggesting no interference in subsequent assays. Body weights of the monkeys did not decrease as a result of treatment. Organs were immediately dissected and flash-frozen in liquid nitrogen and stored at –80°C until further use. The experimental protocol was reviewed and approved by the Institutional Review Board of Behavioral Sciences Foundation and the University of Toronto Animal Care Committee. All the procedures were conducted in accordance with the guidelines of the Canadian Council on Animal Care.
In Vivo CZN and 6OHCZN Plasma Assessments. Plasma CZN and 6OHCZN levels were assayed based on the methods in Howard et al. (2001) with minor modifications. Briefly, plasma was centrifuged at 3000g for 10 min, and 0.5 ml of the supernatant was removed for analysis. Internal standard of 2.0 mM 2-benzoxazolinone (25 µl) and ß-glucuronidase (10 mg/ml, 250 µl) were added to 0.2 M acetate buffer (pH 5.0, 1.0 ml) and incubated overnight at 37°C. Following incubation, 10% perchloric acid (600 µl) and ethyl acetate/hexane (5 ml) were added, the sample was shaken for 30 min, centrifuged at 3500g for 15 min, and the organic phase evaporated to dryness at 37°C. The sample was reconstituted into 200 µl of mobile phase consisting of 19% acetonitrile in 10 mM sodium acetate buffer (pH 4.5). CZN and 6OHCZN were measured by high-performance liquid chromatography with UV detection at 295 nm. A Waters Spherisorb S5 ODS2 column (4.6 x 150 mm) (Waters, Bedford, MA) was used to separate CZN, 6OHCZN, and 2-benzoxazolinone using a mobile phase performed with isocratic elution at a flow rate of 1 ml/min. The retention times for CZN, 6OHCZN, and 2-benzoxazolinone were 19.6, 4.7, and 6.5 min, respectively.
Microsomal Membrane Preparation and Protein Assay. Monkey or human liver tissue was homogenized in 100 mM Tris, 0.1 mM EDTA, 0.1 mM DDT, and 0.32 M sucrose (pH 7.4) for immunoblotting or in 1.15% w/v KCl for in vitro metabolism assessments and then centrifuged at 12,500g for 30 min at 4°C. The supernatant was then centrifuged at 110,000g for 90 min at 4°C, and the pellet was resuspended in 100 mM Tris, 0.1 mM EDTA, 0.1 mM DDT, 1.15% w/v KCl, and 20% v/v glycerol for immunoblotting or 1.15% w/v KCl for in vitro metabolism assays. Microsomes were aliquoted and stored at –80°C. The protein content of liver microsomes was assayed with the Bradford technique using a Bio-Rad Protein Assay kit.
In Vitro CZN and 6OHCZN Assessments. CZN 6-hydroxylation was assayed according to the method of Leclercq et al. (1998) for rat liver microsomes with minor modifications for use with monkey tissues (Leclercq et al., 1998). African green monkey hepatic microsomes were mixed with 0.1 M Tris buffer at pH 7.6 containing 10 mM magnesium chloride and 5 mM NADPH to a final volume of 500 µl. Incubations were carried out with CZN in a shaking water bath at 37°C. Different protein concentrations and incubation times were tested to establish the linear incubation conditions for CZN metabolism. Final reactions included 0.4 mg of protein incubated for 20 min. Zinc sulfate (15% w/v, 0.2 ml) was added to stop the reaction, and 6.4 µg of the internal standard, 2-benzoxazolinone in Tris buffer, was added per reaction. Following centrifugation for 10 min at 12,700g, the supernatant was injected onto an Agilent Zorbax SB-C18 column (5 µm, 4.6 x 250 mm) (Agilent Technologies, Palo Alto, CA) with UV detection at 287 nm. Separation of peaks was performed using a mobile phase of 50 mM ammonium acetate (adjusted to pH to 4.0 with 1 M glacial acetic acid)/acetonitrile (65:35) at a flow rate of 0.7 ml/min. The retention times for CZN, 6OHCZN, and 2-benzoxazolinone were 18.5, 7.5, and 10.1 min, respectively. Kinetic parameters (Km and Vmax) in untreated monkey liver microsomes were obtained by incubating with CZN at various concentrations.
Immunohistochemistry. Frozen monkey liver, fixed with 4% paraformaldehyde, was cut into 16-µm sections. Sections were washed twice for 5 min with phosphate-buffered saline (PBS) (10 mM sodium phosphate buffer, 0.9% sodium chloride, pH 7.4) and incubated for 1 h in a blocking solution [PBS with 1% w/v skim milk powder, 0.1% bovine serum albumin (BSA), 0.01% Triton X-100 and 2% normal horse serum]. Tissue sections were then incubated for 48 h at 4°C with polyclonal goat anti-rat CYP2E1 antibody diluted 1:1000 with 0.1% BSA, 0.01% Triton X-100, and 2% normal horse serum in PBS. Tissue sections were washed three times for 5 min each with 0.01% Triton X-100 in PBS and then reblocked for 30 min with 0.1% BSA, 2% normal horse serum, and 0.01% Triton X-100 in PBS. Sections were then incubated for 1 h with biotinylated anti-goat secondary antibody diluted 1:1500 with 0.1% BSA, 2% normal horse serum, and 0.01% Triton X-100 in PBS, followed by three washes of 5 min. Tissue sections were then quenched for 10 min with 0.3% hydrogen peroxide in PBS, followed by two washes of 5 min each with 0.01% Triton X-100 in PBS. The antigen-antibody complex was visualized using the avidin-biotin complex, followed by a reaction with 3,3'-diaminobenzidine and hydrogen peroxide. Tissue sections were then dehydrated and mounted onto silane-coated slides with Permount. Immunohistochemical control sections were incubated without primary antibody.
Immunoblotting. Monkey liver microsomes and cDNA expressed CYP2E1 were serially diluted to generate standard curves and establish the linear detection range for the assays. Liver microsomal proteins (3 µg) were separated with SDS-polyacrylamide gel electrophoresis using a 10% separating gel and a 4% stacking gel. Proteins were transferred overnight onto nitrocellulose membranes. To detect CYP2E1, membranes were incubated for 1 h in a blocking solution of 1% w/v skim milk powder, 0.1% BSA, 0.1% Triton X-100, and Tris-buffered saline (TBS) (50 mM Tris, 150 mM NaCl, pH 7.4). Membranes were then incubated for 1 h with polyclonal goat anti-human CYP2E1 antibody diluted 1:3000 in 0.1% BSA, 0.1% Triton X-100, and TBS followed by three washes of 5 min each with TBS and 0.1% Triton X-100. Membranes were reblocked with the same initial blocking solution for 45 min. To detect CYP2E1, membranes were then incubated for 1 h with horseradish peroxidase-conjugated anti-goat secondary antibody diluted 1:2500 in 0.1% BSA, 0.1% Triton X-100, and TBS followed by three washes for 5 min each with 0.1% Triton X-100 and TBS. CYP2E1 proteins were visualized on immunoblots using chemiluminescence, followed by exposure to autoradiographic film for 1 to 5 min. MCID Elite imaging software (Imaging Research, Inc., St. Catherines, ON, Canada) was used to analyze films. Baseline corrections for band intensities were made by subtracting the film background from the band intensity. Using a standard curve of cDNA-expressed CYP2E1, band intensities were calculated and expressed as amount of CYP2E1.
Statistical Analysis. In vivo CZN and 6OHCZN pharmacokinetic parameters were calculated for each treatment group. Pharmacokinetic results are expressed as mean ± S.D. (range). Parameters measured were area under the curve to the last quantifiable concentration (AUCT,), terminal disposition rate constant calculated from last 3 to 4 points on the log-linear end of the concentration versus time curve (Kel), measurement of area under the curve up to the last quantifiable concentration plus additional area extrapolated to infinity calculated using Kel (AUCinf), Cmax, Tmax, and t1/2. All the pharmacokinetic statistical analyses were carried out using SAS software version 8.2 (SAS Institute, Cary, NC). Differences between groups were evaluated using a one-tailed Student's t test and were deemed significant if p 
0.05. Before statistical analysis, plasma levels were corrected for any baseline values by subtraction of time 0 values from all the subsequent time points.
For in vitro CZN metabolism, the calibration curves were constructed by using peak area ratios (6OHCZN/internal standard), and least square linear regression analysis was used to determine slope, intercept, and correlation coefficients. The absolute recovery was calculated by comparing the peak area obtained with the standards in the Tris-HCl buffer, and relative recovery was obtained by calculating the ratio of the additional amount to the value calibrated by the curve. The apparent Vmax and Km were calculated using nonlinear regression. The 6OHCZN formation activities between the treatment groups were evaluated using unpaired, one-tailed Student's t tests. In vitro pharmacokinetics and the Hill coefficient were calculated using GraphPad Prism version 4.0 (GraphPad Software, Inc., San Diego, CA). CYP2E1 femtomole protein levels per microgram microsomal protein were extrapolated from expressed cDNA CYP2E1 and control-treated monkey microsomal protein dilution curves. CYP2E1 content of microsomes prepared from livers of control- and nicotine-treated monkeys were compared using unpaired one-tailed Student's t tests. For in vitro kinetics and CYP2E1 protein measurements, results are expressed as mean ± S.D., which represents the average of the six animals per group from at least four different experiments. Groups were deemed significantly different if p 0.05. Correlations between groups were calculated with Pearson correlation coefficients, and correlations were deemed significant if p 0.05.
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The in vitro pharmacokinetic parameters of CZN metabolism were determined in hepatic microsomes prepared from three untreated African green monkey livers. The absolute and relative recoveries of both CZN and 6OHCZN were superior when centrifugation was used compared with liquid-liquid extraction with ethyl acetate and filtration. The average absolute and relative recoveries of 6OHCZN were 90.4 and 99.4%, respectively. The linear detection range for 6OHCZN formation was 0.5 to 16 µg/ml, and intraday and interday S.D. were less than 1%. During assay optimization, we established that the formation of 6OHCZN was linear up to microsomal protein concentrations of 800 µg/ml (Fig. 2A) and up to an incubation time of 40 min (Fig. 2B). Incubations lacking NADPH, or using heat denatured monkey microsomes, produced no detectable 6OHCZN (data not shown).
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Hepatic CYP2E1 Expression and Protein Levels in Control-Treated Monkeys. CYP2E1 immunoreactivity was primarily found around the central hepatic vein with lower CYP2E1 immunoreactivity in midzonal areas and surrounding the hepatic portal vein (Fig. 4A). No immunoreactivity was found in sections incubated without primary antibody (Fig. 4C).
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Monkey CZN AUCinf values correlated significantly with the log plasma metabolic ratio (6OHCZN/CZN) calculated at 0.5, 1, 2, and 4 h (Table 2; Fig. 1C), indicating a strong relationship between CZN AUCinf and the metabolic ratio. The in vivo CZN disposition and hepatic CYP2E1 protein levels in control- and nicotine-treated monkeys were significantly correlated (r = –0.62, p = 0.03) (Fig. 6A), with higher CYP2E1 protein levels associating with lower CZN AUCinf. Nicotine treatment resulted in a 1.35-fold increase in CYP2E1 levels per microgram microsomal protein compared with control treatment (62 ± 21 versus 46 ± 11 fmol/µg microsomal protein, p = 0.07), although this was not significant (Fig. 6B). There was large interanimal variation in CYP2E1 levels within the treatment groups shown in a representative immunoblot (Fig. 5D).
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The log plasma metabolic ratio at 1 h approached a significant correlation with hepatic CYP2E1 levels (p = 0.07) and 6OHCZN AUCinf (r = 0.53, p = 0.17). The 6OHCZN clearance increased in nicotine-treated monkeys, indicated by a 29% decrease in 6OHCZN AUCinf (p < 0.01) (Fig. 1B; Table 1). One nicotine-treated and one control-treated monkey were outliers (greater than 2 S.D. away from the mean of their group). When their values were omitted (n = 10), significant correlations existed between both the log plasma metabolic ratio at 0.5 and 1 h with 6OHCZN AUCinf (r =–0.61, p = 0.03; r = –0.83, p < 0.001, respectively) and hepatic CYP2E1 protein content (r = 0.88, p < 0.0001; r = 0.58, p = 0.05, respectively). These data indicated a significant relationship existed between the log plasma metabolic ratio, hepatic CYP2E1 protein levels, and in vivo CZN and 6OHCZN disposition in most monkeys.
The in vitro CZN velocity at 950 µM CZN (approximate Vmax) and hepatic CYP2E1 protein levels in control- and nicotine-treated monkeys were significantly correlated (r = 0.74, p = 0.004) (Fig. 7A), with higher CYP2E1 protein levels associated with higher rates of CZN oxidation in vitro. Nicotine treatment resulted in a 1.27-fold increase in velocity at 950 µM CZN (approximate Vmax) compared with control treatment (2.13 ± 0.83 versus 1.67 ± 0.04 pmol/min/µg microsomal protein, p = 0.14), but this did not reach significance (Fig. 7B), likely because of the large variation within the treatment groups. There was also no significant difference in velocity at 95 µM CZN (approximate Km) between nicotine- and control-treated groups (0.95 ± 0.37 versus 1.20 ± 0.47 pmol/min/µg, p = 0.34), suggesting that nicotine increased the amount of CYP2E1 rather than altered the affinity of the interaction of CZN with the enzyme. The increases in CYP2E1 protein levels and velocity at 950 µM CZN (approximate Vmax) in the nicotine-treated monkeys were similar (1.35-fold increase in CYP2E1 levels and 1.27-fold increase in CZN metabolism) (Figs. 6B and 7B).
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For hepatic CYP2E1 protein, chronic nicotine treatment did not alter the location of CYP2E1 immunoreactivity in monkey liver slices (Fig. 4B). CYP2E1 immunoreactivity appeared to be more intense in nicotine-treated monkey liver compared with controls (Fig. 4A).
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; Dreisbach et al., 1995; Frye et al., 1998). The African green monkeys were treated with the higher dose of 7 mg/kg because of the faster in vitro CZN metabolism previously observed for the cynomolgus monkey (Court et al., 1997).
Control-treated African green monkeys had similar CZN disposition compared with humans. Studies that administered 500-mg CZN doses (approximately 7 mg/kg for an average 70-kg person) to healthy human volunteers reported AUCinf from 25 to 34 µg x h/ml and a Cmax of 11 µg/ml (Girre et al., 1994; Dreisbach et al., 1995). Control-treated monkeys given a 7-mg/kg CZN dose had an average AUCinf of 19.7 ± 4.5 µg x h/ml and an average Cmax of 9.2 ± 2.1 µg/ml. Monkey CZN t1/2 (0.57 ± 0.07 h) was faster than humans (1.45 ± 0.44 h) (Dreisbach et al., 1995), and monkey CZN and 6OHCZN Tmax values were 2-fold smaller than values reported for humans (Frye et al., 1998). The African green monkeys studied here have a 1.5-fold higher turnover rate for CYP2E1-mediated CZN metabolism compared with humans (Court et al., 1997), which can contribute to the faster half-life and Tmax. In humans, the metabolic ratio (6OHCZN/CZN) is optimal for detection of CZN metabolism at 2 through 4 h post-CZN (Frye et al., 1998). In African green monkeys, we observed significant correlations between the log metabolic ratio calculated at 0.5, 1, 2, and 4 h with CZN AUCinf (p = 0.01–0.0001) (Table 2), further indicating that these monkeys are a good model of human CZN metabolism.
The Hill coefficient of 0.99 for in vitro CZN metabolism suggested that only one catalytic site was involved in CZN metabolism, providing further support for the specificity of CYP2E1-mediated CZN metabolism in these monkeys. In vitro CZN metabolism is mediated by CYP2E1 in humans (Lucas et al., 1999), rats (Kobayashi et al., 2002), and cynomolgus monkeys (Amato et al., 1998), consistent with the single-site kinetics observed in these African green monkeys and further supporting the role of CYP2E1 as the sole enzyme importantly involved in CZN metabolism.
The characteristics of in vitro CZN metabolism in African green monkeys are similar to humans. In humans, the Km ranges from 77 to 149 µM (Court et al., 1997; Lejus et al., 2002), and in African green monkeys, the Km was 95.4 µM. In humans, the Vmax ranges from 1.43 to 3.19 pmol/min/µg (Court et al., 1997; Lejus et al., 2002; Muzeeb et al., 2005); the Vmax in African green monkeys was 3.5 pmol/min/µg, and in cynomolgus monkeys it ranges from 0.52 to 3.38 pmol/min/µg (Court et al., 1997; Tanaka et al., 2000). The amount of CYP2E1 protein detected in microsomes from African green monkeys is similar to the levels of CYP2E1 in human microsomes, assuming equal detection of the two proteins by the antibody, consistent with the similar Vmax values for CZN metabolism between African green monkeys and humans (Court et al., 1997; Lejus et al., 2002; Muzeeb et al., 2005). Monkey CYP2E1 is mainly expressed around the central veins in liver tissue (Fig. 4), similar to the expression in humans (Cohen et al., 1997).
There were significant correlations between in vitro CYP2E1 protein content with in vivo CZN AUCinf (p = 0.03) (Fig. 6) and between in vitro CYP2E1 protein content with velocity at 950 µM CZN (approximate Vmax) (p = 0.004) (Fig. 7), indicating a consistent relationship between in vivo CZN AUCinf, in vitro CZN velocity, and CYP2E1 protein levels among the monkeys and strongly suggesting that the increase in in vivo CZN disposition is mediated by an increase in hepatic CYP2E1 protein, which is also reflected by the increased in vitro CZN metabolism.
Nicotine-treated monkeys had significantly increased in vivo CZN disposition. The amount of nicotine a 70-kg smoker receives per day is estimated to range from 0.2 to 1.1 mg/kg (Benowitz and Jacob, 1984). The total daily dose of nicotine administered to the monkeys (0.6 mg/kg/day) is similar to the average amount received by a smoker. Although the frequency of administration differs from smoking, this dosing regimen has previously been shown to alter other P450 to a similar extent to that seen in smokers (Schoedel et al., 2003).
In humans, periods of smoking increase in vivo CZN clearance compared with nonsmoking periods (Benowitz et al., 2003). Previous studies have shown that carbon monoxide does not increase CZN metabolism (Benowitz et al., 2003). During cigarette smoking, the CZN clearance increases by 24% (p < 0.05), whereas the half-life does not change, suggesting that cigarette smoking increases CZN first-pass metabolism (Benowitz et al., 2003). Chronic nicotine treatment in monkeys resulted in similar kinetic changes to those seen in humans with cigarette smoking, with a 52% decrease in CZN AUCinf, a decreased time to Tmax, and no change in half-life (Table 1). These data suggested that nicotine increased the first-pass metabolism and decreased the oral bioavailability of CZN, as seen in human smoking, where CZN clearance increased without a change in half-life. The decrease in CZN Tmax and nonsignificant decrease in Cmax are consistent with a larger first-pass metabolism.
Differences in hepatic enzyme levels have been previously shown to alter these pharmacokinetic aspects of p.o. administered drugs. For example, subjects with higher hepatic CYP2D6 metabolism showed a decrease in debrisoquine AUCinf but no change in half-life compared with subjects with lower CYP2D6 metabolism (Sloan et al., 1983; Dalen et al., 1999). This indicates that having more functional CYP2D6 increased debrisoquine first-pass metabolism after p.o. administration without altering systemic metabolism. The increases in velocity at 950 µM CZN (approximate Vmax) and CYP2E1 protein levels in the nicotine-treated monkeys were similar to each other (1.27-fold compared with 1.35-fold, respectively) and to the increase in CZN clearance seen in smokers (1.24-fold) (Benowitz et al., 2003). There was no difference in velocity at 95 µM CZN (approximate Km) between control- and nicotine-treated monkeys, suggesting that nicotine treatment does not alter the affinity of the enzyme for CZN, but rather acts via an increase in hepatic CYP2E1 levels. The removal of nicotine during smoking cessation may reverse the induction of CYP2E1 protein and activity such that CYP2E1-metabolized drugs, such as acetaminophen, may require dose adjustments because of the decreased level of hepatic CYP2E1 (Frishman et al., 2006).
It is possible that the decrease in CZN AUCinf may be caused by nicotine's effect on hepatic blood flow, but this is unlikely. In rats, the decreased hepatic blood flow after intraportal nicotine administration returns to baseline after 15 min of ceasing treatment (Hashimoto et al., 2004). The monkeys were administered CZN more than 12 h after the last nicotine treatment; therefore, any effect on hepatic blood flow should have ceased.
In this study, we did not investigate the mechanism of CYP2E1 induction by nicotine in monkeys. Our laboratory has shown that in rats, nicotine does not increase hepatic CYP2E1 by increasing transcription (Howard et al., 2001), by protein stabilization, or by regulation via cotinine, the primary metabolite of nicotine (Micu et al., 2003). Kocarek et al. (2000) have shown that translational efficiency of CYP2E1 mRNA is increased if the 5'-UTR region of the CYP2E1 mRNA is deleted. It is possible that chronic nicotine treatment results in a direct or indirect interaction with the 5'-UTR region of CYP2E1 mRNA to increase translation.
Nicotine-treated monkeys also had a decrease in 6OHCZN AUCinf (Table 1), suggesting an increase in 6OHCZN disposition. 6OHCZN is glucuronidated and excreted quickly compared with its rate of formation (Dreisbach et al., 1995). Cigarette smoking can increase glucuronidation in humans (Benowitz and Jacob, 2000), suggesting that nicotine may increase 6OHCZN glucuronidation, leading to the observed increase in 6OHCZN disposition.
Nicotine induction of CYP2E1 is of interest because it could result in metabolic cross-tolerance via increased metabolism of alcohol by CYP2E1. In humans, smokers have faster alcohol metabolism than nonsmokers (Kopun and Propping, 1977; Morabia et al., 1999), and smoking increases alcohol consumption (Barrett et al., 2006). In rats, nicotine increases ethanol consumption and can result in reinstatement of alcohol-seeking behavior (Le et al., 2003). This suggests that nicotine, for example via cigarette smoking, can increase CYP2E1 levels, which may result in increased alcohol metabolism, possibly leading to increased consumption of alcohol.
In conclusion, we assessed in vivo CZN disposition, in vitro CZN metabolism, and hepatic CYP2E1 protein levels within 12 monkeys. Monkeys are good models of human CYP2E1 activity and protein because the in vivo CZN disposition, in vitro CZN metabolism, and CYP2E1 expression are highly related to each other and similar to that seen in humans. Chronic nicotine treatment increased in vivo CZN clearance by increasing first-pass metabolism, likely via increased hepatic CYP2E1, which is reflected in the increased in vitro metabolism. These effects of nicotine may be importantly increasing CYP2E1 metabolism of drugs and toxins such as alcohol, acetaminophen, and nitrosamines.
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Acknowledgments |
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Footnotes |
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Conflict of Interest Statement: R.F.T. is a shareholder in Nicogen, a company focused on novel treatment approaches. No support from Nicogen was used, and no benefit to the company was obtained.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: CZN, chlorzoxazone; 6OHCZN, 6-hydroxychlorzoxazone; PBS, phosphate-buffered saline; BSA, bovine serum albumin; TBS, Tris-buffered saline; AUCT, area under the curve to the last quantifiable concentration; Kel, terminal disposition rate constant calculated from last three to four points on the log-linear end of the concentration versus time curve; AUCinf, area under the curve extrapolated to infinity; P450, cytochrome(s) P450.
Address correspondence to: Rachel F. Tyndale, Department of Pharmacology, University of Toronto, 1 King's College Circle, Toronto, ON, Canada, M5S 1A8. E-mail: r.tyndale{at}utoronto.ca
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