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Veterans Administration Medical Center, White River Junction, Vermont (S.G.W., J.L.B., P.R.S., J.F.S.); Departments of Pharmacology/Toxicology (K.K.W., P.R.S., J.F.S.) and Biochemistry (P.R.S., J.F.S.), Dartmouth Medical School, Hanover, New Hampshire; Department of Drug Disposition, Lilly Research Laboratories, Indianapolis, Indiana (S.A.W.); Department of Food Science and Human Nutrition, University of Illinois, Urbana, Illinois (E.J.); Laboratory of Metabolism, National Cancer Institute, Bethesda, Maryland (F.J.G.)
(Received December 22, 2003; accepted March 25, 2004)
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Abstract |
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; Morimoto et al., 1995; Kessova et al., 1998; Longo et al., 2000; Allis et al., 2001; Sapone et al., 2003). However, this activity is also catalyzed by expressed forms of human and rat CYP3A (Zerilli et al., 1998; Kobayashi et al., 2002) and rat CYP1A2 (Kobayashi et al., 2002). A role for CYP3A in this reaction was confirmed in intact rats and rat hepatocytes by studies showing that dexamethasone treatment, which induces CYP3A but not CYP2E1 (Sinclair et al., 1991; Zerilli et al., 1998), results in a marked increase in PNPH (Zerilli et al., 1998; DiPetrillo et al., 2002). Dexamethasone-induced PNPH activity is inhibited by troleandomycin, a specific inhibitor of CYP3A (Zerilli et al., 1998; DiPetrillo et al., 2002). Thus, the findings that rat CYP3A can catalyze PNPH indicate that this reaction cannot be used to specifically measure CYP2E1 enzymatically in rats when CYP3A is also present. However, the possible role of murine CYP3A in catalyzing PNPH activity has not been determined.
A recent study has shown considerably lower PNPH activity in hepatic microsomes from untreated Cyp2e1(-/-) mice compared with wild-type mice, suggesting that CYP2E1 has a major role in catalyzing this activity in mice (Carlson, 2003). However, that study analyzed microsomes from adult male mice only. We have previously found that hepatic amounts of CYP3A in adult male Cyp2e1(-/-) mice in the 129 strain are about 20% of those observed in female mice (Sinclair et al., 2000), similar to the findings of Sakuma et al. (2000) in a different mouse strain. This gender difference in expression of CYP3A in the liver could be used to assess the contribution of constitutive forms of CYP3A to PNPH activity. At this time, six forms of CYP3A have been identified in mice (Yanagimoto et al., 1997; Sakuma et al., 2000, 2002). CYP3A11 is a form of CYP3A expressed at similar amounts in the livers of untreated adult male and female mice (Sakuma et al., 2000). Two murine forms of CYP3A, CYP3A41 and CYP3A44, are expressed at high levels in adult female mice and are absent in adult male mice (Sakuma et al., 2002).
To assess the relative contributions of murine CYP2E1 and CYP3A to PNPH activity, we compared PNPH activity in hepatic microsomes from both genders in untreated Cyp2e1(-/-) and wild-type mice. We also investigated the effect of alcohol treatment, which induces both CYP3A and CYP2E1 in wild-type mice, on PNPH activity.
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Materials and Methods |
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,b). The polyclonal rabbit antibody prepared against human CYP2E1 that detects mouse CYP2E1 was purchased from Oxford Biomedical Research (Oxford, MI).
Alcohol Treatment. Cyp2e1(-/-) mice and wild-type mice in a 129SV background (Lee et al., 1996) were housed in a controlled environment with a light/dark cycle of 12 h. Mice were genotyped by the genetic testing services at Charles River Laboratories, Inc. (Wilmington, MA), using DNA extracted from tails. Three hundred ninety-four of 398 markers tested were identical in wild-type and Cyp2e1(-/-) mice. All animal protocols were approved by the internal animal review boards at the Department of Veterans Affairs Medical and Regional Office Center and at Dartmouth College. At the commencement of the experiment, mice weighed 18 to 26 g and were 3 months old. All mice were fed control liquid diet (Lieber-DeCarli) containing maltose-dextrin for an initial 2-day period. Animals to be treated with the alcohols were then administered 2.8% (w/v) ethanol and 0.4% (w/v) isopentanol for 7 days in a liquid diet. This diet was made isocaloric with the control diet by adjusting the maltose-dextrin content in the alcohol-containing diet, as directed by the manufacturer. Carbon dioxide was used to anesthetize the mice before they were sacrificed by cervical dislocation. Microsomes were prepared from liver homogenates as described (Sinclair et al., 2000a).
Immunochemical Analyses of Cytochromes P450. CYP2E1 was measured immunochemically in hepatic microsomes, as described (Sinclair et al., 2000a), using a commercially available polyclonal antibody against human CYP2E1 (Oxford Biomedical Research). Hepatic microsomes from each Cyp2e1(-/-) mouse used in this study were analyzed for CYP2E1 to confirm the genotype. CYP3A was measured immunochemically, as described (Sinclair et al., 2000a). The immunoblots were scanned into Adobe Photoshop with an HP Precision Scanner (Hewlett Packard, Palo Alto, CA) and quantified using OneDScan (Scanalytics Inc., Fairfax, VA). When samples were analyzed on separate gels, an identical sample was included in each gel, and the values were normalized to this sample. Each band was scanned in triplicate. The error bars in the densitometric graphs represent the range of mean values from two animals.
Additional Assays. PNPH activity was measured in liver microsomes, as described (Louis et al., 1994). Proteins were determined by the procedure of Lowry et al. (1951), using bovine serum albumin as a standard.
Statistical Analyses. Values represent the means and S.E.M. or range, as indicated in the legends. PNPH data from four or more mice per treatment were first analyzed by analysis of variance, followed by the Student Newman-Keuls multiple comparisons test. p Values <0.05 indicated significance.
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Results and Discussion |
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), hepatic levels of CYP3A protein were very low in adult male Cyp2e1(-/-) mice (Fig. 1). Similar low hepatic levels were also observed in adult male wild-type mice (Fig. 1). Hepatic levels of CYP3A were over 4-fold greater in females than in males, in both Cyp2e1(-/-) and wild-type mice (Fig. 1). Yet, in untreated mice, PNPH activity was similar in hepatic microsomes from male and female mice (Fig. 2). These findings suggest that constitutive, female-specific forms of CYP3A, CYP3A41 and CYP3A44 (Sakuma et al., 2000, 2002), do not contribute to PNPH activity. The PNPH activities observed for untreated male mice in this study (Fig. 2) are similar to those reported by Carlson (2003), in studies comparing hepatic PNPH activities in untreated wild-type and Cyp2e1(-/-) male mice.
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Treatment with a combination of ethanol and isopentanol for 7 days increased PNPH activity in hepatic microsomes from wild-type mice of both genders (Fig. 2, p < 0.001). In contrast, treatment of Cyp2e1(-/-) mice with the alcohols resulted in no increase in PNPH activity. As found previously in Cyp2e1(-/-) mice (Sinclair et al., 2000a), the alcohol treatment increased a higher-molecular-weight CYP3A protein (Fig. 3A), which is most likely CYP3A11 (Sakuma et al., 2000). Our findings in adult male Cyp2e1(-/-) mice, that the alcohol treatment did not increase PNPH activity (Fig. 2) despite a 5-fold increase in CYP3A protein (Fig. 3, A and B) suggest that CYP3A induced by the alcohols has no appreciable PNPH activity. In male wild-type mice, as in the knockout mice, the alcohol treatment increased only the higher molecular weight CYP3A protein, whereas in female wild-type mice, the alcohol treatment increased both the higher- and lower-molecular-weight forms of CYP3A (Fig. 3A). In mice of both genders, the alcohol treatment appeared to cause a greater increase in CYP3A in wild-type mice compared with Cyp2e1(-/-) mice (Fig. 3, A and B).
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We confirmed by immunoblots that there was no CYP2E1 protein in any of the microsomal samples from Cyp2e1(-/-) mice used in these studies (Fig. 3, C and D for two representative mice of eight total). In both male and female wild-type mice, pretreatment with ethanol plus isopentanol for 7 days resulted in 3- to 4-fold increases in hepatic microsomal CYP2E1 protein (Fig. 3, C and D). Our findings that the fold increases in CYP2E1 protein (Fig. 3D) are similar to the fold increases in PNPH activity (Fig. 2) and that the alcohol treatment did not increase PNPH activity in Cyp2e1(-/-) mice suggest that CYP2E1 is the major catalyst of PNPH activity in hepatic microsomes from alcohol-treated mice.
In contrast to the findings in rats (Zerilli et al., 1998; Kobayashi et al., 2002; DiPetrillo et al., 2002) and humans (Zerilli et al., 1998), our findings that female-specific and alcohol-induced forms of mouse CYP3A do not contribute appreciably to PNPH activity suggest that minor differences between species in the amino acid sequences of CYP3A are responsible. Differences in substrate specificities of CYP3A have been detected for different forms of CYP3A within the same species. Although 6ß-hydroxylation of testosterone is widely used as a measure of CYP3A in several species, a newly identified human form of CYP3A, CYP3A43, when expressed in baculovirus, has very low activity (Domanski et al., 2001). Even single amino acid changes within cytochromes P450 have been shown to dramatically alter their activities. For example, in CYP2E1, a Leu209Ile mutation caused a major decrease in its intrinsic activity for PNPH (Spatzenegger et al., 2003).
In summary, we have found that hepatic PNPH activities were similar in untreated male and female mice, despite dramatically greater levels of CYP3A in females. Alcohol treatment caused an increase in PNPH activity in wild-type mice, but not in Cyp2e1(-/-) mice, despite induction of CYP3A in both lines. The results suggest that constitutively expressed and alcohol-induced forms of mouse CYP3A have little to no role in hepatic PNPH activity. Therefore, in microsomes isolated from the livers of untreated and alcohol-treated mice, PNPH activity can be used as a specific probe for CYP2E1.
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Footnotes |
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ABBREVIATIONS: PNPH, p-nitrophenol O-hydroxylation.
Address correspondence to: Jacqueline Sinclair, Research 151, Veterans Administration Medical Center, White River Junction, VT 05009. E-mail: JSINC{at}dartmouth.edu
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