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Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan
(Received March 11, 2004; accepted June 6, 2004)
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Abstract |
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; Mechan et al., 2002). Although they did not study diazepam metabolism, the authors of these studies suggested that inter- or intrastrain variation in diazepam metabolism may be the cause of these differences. Generally, variations in drug metabolism among individuals or among strains of animals or different human races are caused by polymorphic expression of one of the enzymes involved in the metabolism. Such polymorphisms in drug metabolism have been reported in the metabolism of debrisoquine, a number of ß-blockers, and other drugs metabolized by cytochrome P450 (P450) CYP2D6 in humans and CYP2D2 in rats (Marandi et al., 1996; Yamamoto et al., 1998; Schulz-Utermoehl et al., 1999; Vorhees et al., 1999) and in the metabolism of S-mephenytoin and other drugs metabolized by CYP2C19 in humans (Roh et al., 1996; Xie et al., 1996, 1999; Marandi et al., 1997; Adedoyin et al., 1998; Ferguson et al., 1998; Zhou, 2001; He et al., 2002). Inaba et al. (1988) reported interindividual variability of diazepam metabolism. Accumulating reports suggest the possible correlation of diazepam metabolism with the S-mephenytoin hydroxylation phenotype (Bertilsson et al., 1989). These polymorphisms in drug metabolism of a drug, i.e., the existence of extensive and poor metabolizers (EM and PM, respectively), are of clinical importance. Adverse effects typical of overdosage of the drug are the consequences of administration of a "normal" dosage to individuals expressing defective or low levels of metabolizing enzymes (PM).
Neville et al. (1993) reported that p-hydroxylation, 3-hydroxylation, and N-desmethylation are the pathways of diazepam metabolism in adult male W rat liver microsomes and that they were catalyzed by the respective P450 isoforms of CYP2D1, CYP3A2, and CYP2C11. In our previous report, we found that about 17% of W rats had markedly (more than 200-fold) higher activities of diazepam p-hydroxylation (EM of diazepam metabolism) than the rest of the W rat population in liver microsomes at low substrate concentrations. The expression levels of CYP2D1 in W rats did not show such dimorphism, but a kinetic study suggested the presence of a high-affinity and high-capacity diazepam p-hydroxylation enzyme other than CYP2D1 in the liver microsomes of EM rats from W strain (EM W) (Saito et al., 2004).
In this study, we investigated strain differences in diazepam metabolism with particular attention to the metabolic activities of primary pathways of diazepam metabolism at low substrate concentrations using SD, Brown Norway (BN), DA, and W adult male rats. The results showed good agreement with the strain differences in the aforementioned pharmacodynamic studies (Bert et al., 2001; Mechan et al., 2002).
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Materials and Methods |
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; Masubuchi et al., 1993; Nakamura et al., 1995; Yamamoto et al., 1996). Horseradish peroxidase-labeled anti-goat IgG and anti-rabbit IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The other chemicals and reagents used were of analytical and biochemical grade.
Rat Liver Microsomes. Ten-week-old SD, BN, DA (n = 4 each), and W (n = 6) male rats were supplied by Nihon SLC Co. (Shizuoka, Japan). All experiments using animals were performed with the supervision and approval of the Institutional Animal Care and Use Committee of Hokkaido University. Liver microsomes were prepared from the rats according to the method of Omura and Sato (1964). The liver samples were homogenized with three volumes of ice-cold 1.15% KCl. Each homogenate was centrifuged at 9000g at 4°C for 20 min to remove nuclear materials and then ultracentrifuged two times at 105,000g at 4°C for 70 min to attain a mitochondrial-free microsomal pellet. The pellet was resuspended in 0.1 M potassium phosphate buffer (pH 7.4) and separated into small vials, frozen in liquid nitrogen, and stored in the freezer at -80°C until use. Total protein contents were determined according to the method of Lowry et al. (1951). Total microsomal cytochrome P450 was quantified from the CO difference spectrum of the dithionite-reduced proteins between 450 and 490 nm, using an extinction coefficient of 91 mM-1 cm-1 (Omura and Sato, 1964).
Assay for Diazepam-Metabolizing Activity of Liver Microsomes. Diazepam metabolism was assayed according to the method described previously, with slight modifications (Fujita et al., 1990a,b). The reaction mixture (in a final volume of 0.5 ml) contained 3 µM diazepam, 3 mM MgCl2, 5 mM G-6-P, and 0.25 mg of liver microsomes in 0.1 M potassium phosphate buffer (pH 7.4). The reaction was started by adding 1 mM ß-NADPH and 1 enzyme unit of G-6-PDH after 3 min of preincubation at 37°C. The reaction was carried out for 10 min. The rate of disappearance of diazepam was determined by the same HPLC protocol as the following experiments. For the determination of the rates of formation of primary metabolites, the incubation time was shortened to 3 min. During this period, the formation of the two secondary metabolites 3-hydroxy-N-desmethyl-diazepam and p-hydroxy-N-desmethyl-diazepam was negligible, and the formation of the three primary metabolites was linear with time. The reaction was terminated by the addition of 1.5 ml of ethyl acetate. Nitrazepam was added to the mixture as an internal standard, and it was extracted in the organic phase (ethyl acetate) with diazepam and its metabolites by mixing sufficiently. The mixture was centrifuged at 1200g for 20 min to separate the organic phase from the aqueous phase. The organic layer was transferred to another tube, evaporated under vacuum, and the residue was dissolved in the HPLC mobile phase to yield the final sample for the HPLC analysis. HPLC was performed using a Shimadzu LC-6AV spectrophotometric detector and data processor (Chromatopac C-R6A) and an Inertosil ODS-2 column (5 µm, 25 cm x 4.6 mm i.d.) (GL Science Inc., Tokyo, Japan). The mobile phase consisted of 10 mM potassium phosphate buffer (pH 3.0)/acetonitrile/methanol (30:15:12 by volume) at a flow rate of 0.9 ml/min; the wavelength of detection absorbance was set at 230 nm. Retention times for p-hydroxy-N-desmethyl-diazepam, p-hydroxy-diazepam, nitrazepam (the internal standard), 3-hydroxy-N-desmethyl-diazepam, 3-hydroxy-diazepam, N-desmethyl-diazepam, and diazepam were 9.9, 11.5, 14.9, 16.6, 23.6, 26.0, and 36.7 min, respectively. For the kinetic studies, substrate concentrations of 0.4 to 40 µM were used. Liver microsomes from SD, BN, DA, and W adult male rats were used for the kinetic studies. Enzyme kinetics parameters (Km and Vmax) were analyzed according to the Michaelis-Menten equation, using a computer-assisted nonlinear regression program (Simplex) (Fujita et al., 1985). The values obtained in this experiment were considered to be statistically significant when p was less than 0.05 by Scheffe's test.
Immunoinhibition Study of Diazepam Metabolism Using CYP2D2 Antibody. The immunoinhibition study with antibody against CYP2D2 was carried out by preincubating anti-CYP2D2 antiserum with liver microsomes of adult male SD and BN rats (0.1 mg of protein) at room temperature for 30 min prior to the measurement of diazepam metabolite formation. Control incubations contained an equivalent amount of preimmune rabbit serum proteins. The concentration of substrate (diazepam) was 25 µM. Other experimental details are described above.
Assay for Debrisoquine 4-Hydroxylation Activity of Liver Microsomes. Debrisoquine 4-hydroxylase activity was assayed by HPLC methods as described previously (Suzuki et al., 1992; Hiroi et al., 2002), with some modifications. The assay mixtures contained 50 µM debrisoquine, 10 mM MgCl2, 0.5 mg of microsomes, 10 mM G-6-P, and 0.1 M potassium phosphate buffer (pH 7.4) for a total volume of 0.5 ml. The reaction was started after 3 min of preincubation at 37°C by adding 1 mM ß-NADPH and 1 enzyme unit of G-6-PDH; mixtures were incubated in a shaker bath at 37°C for 5 min. The reaction was stopped with 0.5 ml of 12 N NaOH. After the extraction of debrisoquine and its metabolites with ethyl acetate, glycinexylidide (as the internal standard) and 0.01 N H2SO4 were added. After vortex mixing and centrifugation, the upper layer was removed. It was neutralized by 0.01 N NaOH, evaporated to dryness, and dissolved in HPLC mobile phase (10 mM potassium phosphate buffer, pH 3.0; acetonitrile = 85:15 by volume). The sample was applied to an Inertosil ODS column (5 µm, 25 cm x 4.6 mm i.d.) (GL Sciences Inc., Tokyo, Japan). The UV absorption intensity was monitored at 210 nm.
Immunological Analysis of CYP2D1, CYP3A2, CYP2C11, and CYP2D2. The expression levels of CYP2D1, CYP3A2, and CYP2C11, which are reported to catalyze diazepam p-hydroxylation, 3-hydroxylation, and N-desmethylation in the liver microsomes of adult male W rats, respectively (Neville et al., 1993), and CYP2D2 were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (1970), with some modifications, and immunoblot technique according to Towbin et al. (1979). Liver microsomal proteins (10 µg of protein/well) from rats were applied to 12% SDS-polyacrylamide gels and separated by electrophoresis using a Mini-PROTEAN 2 1-D cell (Bio-Rad, Noordwijkerhout, The Netherlands). The proteins were electrophoretically transferred to nitrocellulose membranes and probed with antibodies against rat CYP2C11 (goat antiserum), CYP2D1, CYP2D2, and CYP3A2 (rabbit antiserum). Horseradish peroxidase-labeled anti-goat IgG was used as the secondary antibody for CYP2C11 detection, and horseradish peroxidase-labeled anti-rabbit IgG was used for CYP2D1, CYP2D2, and CYP3A2 detection. Immunoreactive protein bands were revealed colorimetrically by oxidation of 3,3'-diaminobenzidine tetrahydrochloride dehydrate with hydrogen peroxide and catalyzed by peroxidase in Tris-HCl buffer (pH 7.4). The color intensities of the bands that emerged were measured using NIH Image v. 1.61 (National Institutes of Health, Bethesda, MD) (Lennard, 1990).
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Results |
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), a dimorphic distribution of diazepam-metabolizing activities was observed in the W strain of rats, with about 17% being EM and 83% being PM of diazepam. In this study, only the data from most of the population of W rats (PM) are indicated. The disappearance rate of diazepam in W rats was one-half to one-third of those of other rat strains (Fig. 1). DA rats had the highest diazepam-metabolizing activity at this concentration of diazepam.
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Rates of Formation of Primary Metabolites. To elucidate the diazepam metabolic pathways responsible for these strain differences, rates of formations of the primary metabolites of diazepam, p-hydroxy-diazepam, N-desmethyl-diazepam, and 3-hydroxyl-diazepam were determined by the use of HPLC. With a short (3 min) incubation period to avoid the formation of the secondary metabolites, it was found that the amount of diazepam that disappeared during the incubation period equaled the sum of the amounts of these three primary metabolites. Figure 2 shows the elution profiles of diazepam and its primary metabolites from the HPLC column after the incubation of diazepam with liver microsomes from SD, BN, DA, and W adult male rats. Although metabolic profiles of diazepam in liver microsomes from SD and BN rats showed peaks representing all three metabolites (3-hydroxy-diazepam, N-desmethyl-diazepam, and p-hydroxy-diazepam), those from DA and W rats showed only two peaks representing 3-hydroxy-diazepam and N-desmethyl-diazepam. The peaks of p-hydroxy-diazepam in DA and W rats are not detectable at the sensitivity shown in Fig. 2. As indicated in Table 1, diazepam p-hydroxylation activity was very low in DA and W rats compared with that in SD and BN rats (300-fold difference). DA rats showed 3-fold higher diazepam 3-hydroxylation and 2-fold higher diazepam N-desmethylation activities than those in the other strains.
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Kinetic Studies of Diazepam Metabolite Formation. The kinetics of diazepam metabolism in the incubation mixture containing the NADPH generating system, 0.4 to 40 µM diazepam, and liver microsomes from SD, BN, DA, and W strains of adult male rats are plotted in Fig. 3. The kinetic parameters obtained are listed in Table 2. Kinetic parameters of EM W rats obtained in our previous study (Saito et al., 2004) are also included for comparison. These studies revealed that p-hydroxylation in SD and BN rats proceeds with lower Km and higher Vmax values than those in DA and W rats, just like the activity observed in EM W. The Vmax of the diazepam 3-hydroxylation in DA rats is 2 times that of other strains. The sum of the CLint (Vmax/Km), which reflects the metabolic activity at low substrate concentrations in each strain, was in the order of DA > SD = BN >> W. The CLint also indicates that p-hydroxylation is the preferred pathway of diazepam metabolism in SD and BN rats, whereas 3-hydroxylation is the preferred pathway in DA and W strains of rats at the low substrate concentrations. The sum of Vmax was in the order of DA > SD > BN = W.
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Western Blot Analyses Using CYP3A2, 2C11, and 2D1 Antibodies. Western blot analysis revealed that DA rat liver microsomes have a higher expression level of CYP3A2 protein, which is reported to be responsible for diazepam 3-hydroxylation and partly responsible for diazepam N-desmethylation (Fig. 4). This is in agreement with the high diazepam 3-hydroxylation and N-desmethylation activities in this strain of rats (Tables 1 and 2). The expression levels of CYP2C11, which is responsible for diazepam N-desmethylation activity, are similar in all rat strains tested. In the CYP2D1 expression level, the blot from DA rats was more intense than those from SD, W, and BN rats (DA > SD > W = BN). This result was in agreement with the previous study (Schulz-Utermoehl et al., 1999) but not with the observations of the high diazepam p-hydroxylation activities in SD and BN rats and low diazepam p-hydroxylation activities in DA and W rats (Tables 1 and 2).
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Inhibition Study of Diazepam Metabolism Using CYP2D2 Antibody. CYP2D1 has been reported to be responsible for debrisoquine 4-hydroxylation, but later it was corrected to be CYP2D2 (Suzuki et al., 1992; Yamamoto et al., 1996, 1998). Thus, we carried out an inhibition study on diazepam metabolism in the liver microsomes of SD and BN rat strains using an antibody against CYP2D2. An anti-rat CYP2D2 caused about 80% inhibition of the diazepam p-hydroxylation, indicating that the enzyme responsible for diazepam p-hydroxylation cross-reacts with CYP2D2 antibody. It did not inhibit the diazepam 3-hydroxylation and N-desmethylation (Fig. 5).
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Debrisoquine 4-Hydroxylation Activity and Expression Level of CYP2D2. If CYP2D2 is the enzyme responsible for diazepam p-hydroxylation, species differences in a known CYP2D2-dependent activity such as debrisoquine 4-hydroxylation and the expression levels of CYP2D2 should cosegregate with that of diazepam p-hydroxylation. We examined debrisoquine 4-hydroxylation activity (Fig. 6) and Western blot analysis for CYP2D2 in the liver microsomes of those rat strains (Fig. 7). Only DA rats showed low debrisoquine 4-hydroxylation activity as expected. We could not find any differences in the activity of debrisoquine 4-hydroxylation among the liver microsomes from SD, BN, and W rats. These results were in agreement with the results of Western blotting analysis for CYP2D2 expression level. The species differences in debrisoquine 4-hydroxylation and CYP2D2 expression level did not cosegregate with that of diazepam p-hydroxylation, indicating that CYP2D2 is not responsible for the high-affinity enzyme activity of diazepam p-hydroxylation.
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Discussion |
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As summarized in Table 2, p-hydroxylation is the preferred pathway of diazepam metabolism in SD and BN rats, whereas 3-hydroxylation is the preferred pathway in DA and W rats at low substrate concentrations. DA rats have a higher rate of diazepam metabolism than SD or BN rats (Fig. 1). In spite of the very low activity of diazepam p-hydroxylation in DA rats, the high activities of 3-hydroxylation and N-desmethylation might be sufficient to compensate for the difference in p-hydroxylation activity in DA rats (Table 1). Significant differences were observed in the metabolism of diazepam among these strains of rats at the low substrate concentrations, which is represented by the total CLint (DA > SD = BN >> W). The results of these studies revealed that the strain difference in diazepam metabolism between DA and other strains of rats (DA > other strains) was mainly due to the differences in diazepam 3-hydroxylation and N-desmethylation activities, whereas the strain differences between SD and W (SD > W) or BN and W rats (BN > W) are due to differences in the activity of diazepam p-hydroxylation. SD and BN rats showed a similar metabolic profile to EM W rats (Saito et al., 2004) in the present study.
Mechan et al. (2002) reported strain differences in the pharmacodynamics of diazepam between SD and DA rat strains (the SD rat is more responsive to diazepam than the DA rat) and indicated that it may be due to the differences in diazepam metabolism between these strains of rats. Here we show that DA rats eliminate diazepam more quickly from the body with a higher CLint than SD rats. The quicker metabolic elimination of diazepam in DA rats may result in lower sensitivity to diazepam in these strains of rats than SD. In addition to the above, the fact that the major metabolite in SD rats is p-hydroxy-diazepam, whereas in DA rats it is 3-hydroxy-diazepam, may contribute to the pharmacodynamic difference. Unfortunately, no pharmacodynamic data are available for p-hydroxy-diazepam. If p-hydroxy-diazepam is pharmacodynamically more potent than 3-hydroxy-diazepam, this contributes to the difference. Similar explanations are applicable for the intrastrain pharmacodynamic difference among different outbred W rats (Bert et al., 2001). As has been stated earlier, we found that there is a dimorphic variation in diazepam p-hydroxylation in Wistar rats (Saito et al., 2004).
DA rats had a higher Vmax in diazepam N-desmethylation activity than other strains of rats, whereas the expression levels of CYP2C11 were similar in all four strains. This may be due to the fact that CYP3A2 is partially involved in this reaction and because DA rats express a higher level of CYP3A2, which contributes to diazepam N-desmethylation, and show a higher Vmax in N-desmethylation activity. Interestingly, the expression levels of CYP2D1, supposedly responsible for diazepam p-hydroxylation activity (Neville et al., 1993), did not cosegregate with the p-hydroxylation activities in these strains of rats. The blots from DA rats were more intense than those from SD, BN, and W rats. Since the original study showing that CYP2D1 is the enzyme responsible for diazepam p-hydroxylation was carried out using only W rats, the presence of the lower Km and higher Vmax p-hydroxylation enzyme in SD and BN rats shown in this study was not observed. Therefore, this is the first study to report the presence of this enzyme that should be identified by further study. Previously, we have reported that CYP2D2, but not CYP2D1, contributed to the metabolism of debrisoquine, bunitrolol, and bufuralol, which are known as typical CYP2D substrates, in rat liver microsomes (Suzuki et al., 1992; Yamamoto et al., 1996, 1998). Thus, we examined the possibility that CYP2D2 is the catalyst for diazepam p-hydroxylation. We carried out an inhibition study on diazepam metabolism in the liver microsomes of SD and BN rat strains using an antibody against CYP2D2. An anti-rat CYP2D2 caused about 80% inhibition of the diazepam p-hydroxylation, whereas it did not inhibit the diazepam 3-hydroxylation and N-desmethylation (Fig. 5). Therefore, we presumed that the rat liver microsomal diazepam p-hydroxylation is mediated largely by CYP2D2 or an enzyme that cross-reacts with anti-CYP2D2 antibody. We also examined CYP2D2-dependent activity, debrisoquine 4-hydroxylation, and Western blotting analysis for CYP2D2 expression levels in the liver microsomes of these rat strains. Debrisoquine 4-hydroxylation activity in DA rats was the lowest among liver microsomes from these strains of rats. We could not find any differences in the activity of debrisoquine 4-hydroxylation among the liver microsomes from SD, BN, and W rats. These results were in agreement with the results of Western blotting analysis for CYP2D2 expression level. Thus, the patterns of debrisoquine 4-hydroxylation activity and CYP2D2 expression coincided with each other as expected, but they did not coincide with diazepam p-hydroxylation activity in the liver of the four rat strains. We concluded that CYP2D2 is not the main contributor to diazepam p-hydroxylation in liver microsomes of adult male rats. But the result of the inhibition study using CYP2D2 antibody suggested that the enzyme that contributed to this pathway in SD and BN rats is closely related to CYP2D2 and may possibly be an isoform of the CYP2D subfamily.
The current study demonstrated that the strain difference in diazepam metabolism is unique in that it is not due to a difference in the expression level of a single enzyme but to two enzymes. The enzymes responsible for the activities of diazepam p-hydroxylation and diazepam 3-hydroxylation are involved in these species differences. Western blot analysis revealed that CYP3A2 is expressed more highly in DA rats than in the other rat strains. Kinetic analysis revealed the presence of a high-affinity, high-capacity diazepam p-hydroxylation enzyme in SD and BN rats. Due to these differences, the major metabolic pathways of diazepam differ among rat strains at low diazepam concentrations. These differences in drug metabolism, in which major metabolites differ depending on the strain of the animal, may cause significant differences not only in the results of pharmacokinetics of the drug, but also in pharmacological or toxicological studies, especially when the metabolites of the test compound have a pharmacological or toxicological effect.
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ABBREVIATIONS: SD, Sprague-Dawley; DA, Dark Agouti; W, Wistar; EM, extensive metabolizer(s); PM, poor metabolizer(s); EM W, EM rats from W strain; P450, cytochrome P450; BN, Brown Norway; G-6-P, glucose-6-phosphate; G-6-PDH, glucose-6-phosphate dehydrogenase; HPLC, high-performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis.
Address correspondence to: Dr. Shoichi Fujita, Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, N18W9 North Ward, Sapporo 060-0818, Japan. E-mail: fujita{at}vetmed.hokudai.ac.jp
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, diazepam p-hydroxylation; 
, diazepam 3-hydroxylation; 
, diazepam N-desmethylation.
