From the Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York, Albany, New York 12201
Received for publication, March 26, 2003
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ABSTRACT |
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INTRODUCTION |
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In contrast to the multiplicity of microsomal CYP genes, there is only one CPR gene in mammals (6). CPR also functions as a redox partner for several other enzymes, including HO (7), cytochrome b5 (8, 9), squalene monooxygenase (10), and fatty acid elongase (11). A critical role of microsomal CYP enzymes in embryonic development was recently demonstrated in a knockout mouse model with germ line deletion of the Cpr gene (12). However, the impact of CPR loss on biological function and xenobiotic metabolism has not been examined in adult animals, which can be studied using a tissue-specific knockout approach (13). We have reported recently2 the preparation and initial characterization of a mouse strain with a floxed Cpr allele (Cprlox); the mutant allele has the exons 315 of the Cpr gene flanked by two loxP sites, which permits Cre-mediated excision of the Cpr gene. Homozygous Cprlox mice are fertile and without any histological abnormality or any change in CPR expression. In the present study, we have prepared a mouse model with liver-specific deletion of the Cpr gene (designated Alb-Cre/Cprlox mice) by cross-breeding the Cprlox mice with transgenic mice having liver-specific Cre expression (Albumin-Cre or Alb-Cre; see Ref. 14). The Alb-Cre/Cprlox mice were characterized for general health status, potential embryonic lethality, and fertility. The impact of liver-specific Cpr gene deletion on liver microsomal CYP and HO activities, in vivo drug clearance, plasma cholesterol level, and liver morphology was examined. The impact of CPR loss on microsomal CYP expression in liver and extrahepatic tissues was also characterized. Our data indicate the absence of significant alternative redox partners for liver microsomal CYP and HO, provide in vivo evidence for the significance of hepatic CPR-dependent enzymes in regulating plasma cholesterol level and systemic drug clearance, and reveal novel regulatory pathways of CYP expression associated with altered cellular homeostasis. We believe that the Alb-Cre/Cprlox mouse represents a unique model for studying the in vivo function of hepatic HO and of microsomal CYP-dependent pathways in the biotransformation of endogenous and xenobiotic compounds.
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EXPERIMENTAL PROCEDURES |
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Immunoblot AnalysisImmunoblots were analyzed with an ECL kit from Amersham Biosciences, with use of the following polyclonal antibodies: anti-mouse CYP2A5 (15); anti-rat CYP1A1/2, CYP2B1, -2E1, -3A2, and CPR (BD Biosciences); anti-rat HO-1 and anti-rat HO-2 (Stressgen); and anti-rat mEH (a gift from Dr. Charles Kasper of University of Wisconsin). Immunoblot quantification was carried out using a densitometer.
Enzyme Activity AssaysAll assays were carried out in duplicate. CPR activity was determined using cytochrome c as an electron acceptor (16). Metabolic activation of NNK was assayed by determination of the rates of formation of keto alcohol and keto aldehyde, as described previously (17). Metabolic activation of AP was assayed by a determination of the rates of formation of acetaminophen-glutathione conjugate (15). For NNK, the combined rates of formation of keto alcohol and keto aldehyde were determined. The reaction mixtures contained 100 mM sodium phosphate, pH 7.4, 1.0 mM EDTA, an NADPH-generating system (5 mM glucose 6-phosphate, 3 mM MgCl2, 1 mM NADP+, and 1.5 units of glucose-6-phosphate dehydrogenase), 10 µM [3H]NNK (250 Ci/mol), 5 mM sodium bisulfite, and 0.5 mg/ml microsomal protein. For AP, the reaction mixtures contained 50 mM potassium phosphate, pH 7.6, 0.5 mM AP, 10 mM reduced glutathione, 1 mM NADPH, and 1.0 mg/ml liver microsomes. For both substrates, reactions were carried out for 30 min at 37 °C.
Microsomal HO activities were assayed by determination of the formation of bilirubin according to a protocol described by Maines (18). Bilirubin concentration was measured by a determination of spectral differences between reaction mixtures containing NADPH and reference samples incubated without NADPH, with the use of a Cary 3E dual-beam spectrophotometer (Varian) and an extinction coefficient of 40 mM1 cm1 for absorbance differences between 464 and 530 nm. The reaction mixtures contained 0.1 M potassium phosphate buffer, pH 7.4, 10 µM hemin (Sigma), 0.5 mg/ml liver microsomes, 1 mM NADPH (excluding reference samples), and 0.5 mg/ml mouse liver cytosol in a final volume of 1.0 ml. The cytosol fraction, which was used as the source of biliverdin reductase, was prepared from saline-perfused C57BL/6 mouse liver according to a protocol originally described for rats (18). Reactions were initiated by the addition of NADPH, carried out at 37 °C for 30 min, and terminated by placing samples on ice.
Pathology ProcedureTissues were collected promptly after euthanasia and placed in at least 10 volumes of 10% neutral buffered formalin for 24 h. The tissues were then sectioned at 23 mm and processed for histologic examination in a VIP Tissue Tek processor (Sakura Finetech). After dehydration through a series of alcohols, tissues were cleared in xylene and impregnated with paraffin wax prior to embedding in blocks of paraffin wax. Sections were then cut at 34 µm and floated onto glass slides, baked at 60 °C overnight, and stained with hematoxylin and eosin.
RNA Preparation and Quantitative RNA-PCRTotal RNA was isolated from mouse liver with Tri-reagent (Molecular Research Center) according to the manufacturer's instructions. RNA concentrations were determined spectrally. The integrity of the RNA samples was assessed by the A260/A280 ratio and by ethidium bromide staining after electrophoretic analysis on denaturing gels. RT reactions were performed in a PE9600 PCR machine (Applied Biosystems) with an RNA-PCR kit from PerkinElmer Life Sciences. First strand cDNAs were synthesized at 42 °C with use of 1 µg of total RNA, 2.5 µM (dT)16 primer, 1 mM each of dNTP, and 5 mM MgCl2 in a total volume of 20 µl. The relative levels of various CYP transcripts in the liver of ``wild-type'' and ``homozygous'' mice were determined using real time RT-PCR, with a LightCyclerTM (Roche Applied Science).
Real time PCRs were performed according to the instructions in the
LightCyclerTM FastStart DNA Master SYBR Green I Kit (Roche Applied
Science), using gene-specific PCR primers for CYP1A1, CYP1A2, CYP2A4/5,
CYP2B10, CYP2B20, CYP3A11, and
CYP3A13.3 PCR mixtures
contained 2 µl of FastStart DNA Master SYBR Green I, 4 mM
MgCl2, 0.5 µM each primer, and 4 µl of undiluted
or diluted (101000-fold) RT product in a total volume of 20 µl. PCR
was monitored for 45 cycles with annealing temperature at 60 °C. At the
end of the PCR cycles, melting curve analysis was performed according to
LightCyclerTM kit instructions, to assess the purity of the PCR products.
PCR products were also analyzed by electrophoresis on agarose gels to confirm
PCR specificity. Negative control reactions (no template) were routinely
included to monitor potential contamination of reagents. The relative levels
of -actin mRNA in various RNA samples were determined as described
elsewhere,3 and the
results were used as an internal reference for the actual amounts of RNA added
to each RT reaction. For each primer pair, a plot of the threshold cycle value
versus the log of the amount of input RNA equivalent (0.1100
ng, calculated from the dilution factor of the RT product) was generated,
which was used to calculate the fold of difference in the levels of a CYP mRNA
between the wild-type and the homozygous mice.
Other Methods and MaterialsMicrosomes or postmitochondrial S9 fractions from liver and other tissues were prepared as described previously (15, 19). Protein concentration was determined by the bicinchoninic acid method (Pierce) with bovine serum albumin as the standard. Microsomal CYP concentration was determined by CO difference spectroscopy (20). Pentobarbital clearance test was performed essentially as described by Tsuji et al. (21). Mice were given pentobarbital (Sigma), intraperitoneally, as a 10 mg/ml solution in phosphate-buffered saline (2.7 mM KCl, 1.5 mM KH2PO4, 134 mM NaCl, and 8.2 mM Na2HPO4·7H2O), at a dose of 60 mg/kg. Plasma total cholesterol level was determined using the Infinity Cholesterol Reagent kit from Sigma (Procedure 401), with use of 10 µl of plasma per assay.
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RESULTS |
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Time Course and Tissue Specificity of Cpr Gene Deletion Liver-specific expression of the Cre transgene is expected to result in targeted deletion of the floxed Cpr allele, leading to deletion of the Cpr gene in Cre-expressing cells (Fig. 1A). Deficiency of hepatic CPR expression was confirmed by immunoblot analysis (Fig. 1B). Decreases in microsomal CPR protein level were observed in both heterozygous and homozygous animals, at either 3 weeks or 2 months of age. Densitometric analysis of immunoblot data indicated that, at 3 weeks, hepatic microsomal CPR level was about 59 ± 9% in heterozygotes, compared with that of the wild-type littermates, and 18 ± 10% in homozygotes (means ± S.D., n = 6, males and females combined). The decrease in CPR expression was markedly more pronounced in the 2-month-old than in the 3-week-old mice; at 2 months, the level of CPR protein in liver microsomes from heterozygous mice was about 50% that in wild-type littermates and, as shown in Fig. 1, was not detected in homozygous mice (although, on longer exposure, a very faint band was detected in a sample from a male mouse; data not shown). Thus, the loss of CPR expression was essentially complete in the homozygous mice at 2 months after birth. On the other hand, in experiments not presented, no change in CPR protein level was observed in fetal liver at gestational day 14. The age-dependent increase in recombination efficiency, which reflects the age-dependent increase in Cre expression and accumulation of Cre protein in the nucleus, is consistent with previous observations on the Alb-Cre mice (22). Additional immunoblot studies (examples are shown in Fig. 1C) with microsomes from lung, kidney, brain, and olfactory mucosa of 2-month-old mice indicated that there was no significant difference in CPR protein level among the three haplotypes, confirming tissue specificity of Cre expression (14).
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The Impact of a Decreased CPR Expression on Microsomal CYP and HO ActivitiesDifferences among the three haplotypes, and between the two age groups, in liver microsomal CPR activity toward cytochrome c paralleled the differences in the levels of CPR protein (Fig. 2). Similarly, microsomal CYP activities toward NNK and AP, as well as HO activities, were also decreased in agreement with the decreases in CPR activity, except that the 3-week-old heterozygous mice did not show a decrease in HO activity and that the 2-month-old heterozygous mice did not show a significant decrease in the rates of NNK metabolism. At the age of 2 months, the homozygotes had 58% of wild-type activities toward NNK and AP and barely detectable HO activity (Fig. 2). At 3 weeks, the residual CYP activities were more substantial in the homozygotes, about 25%, whereas the HO activity was below 5%. These data confirm that CPR is the predominant, if not the only, electron donor for microsomal CYP as well as HO.
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The Impact of a Decreased CPR Expression in the Liver on Systemic Clearance of PentobarbitalPentobarbital is a sedative known to be metabolized by CYPs. The rates of in vivo pentobarbital clearance were monitored using the pentobarbital sleeping test (Table I). After treatment with a single dose of pentobarbital (60 mg/kg, intraperitoneal), all mice in the three haplotype groups lost righting reflex within 5 min. Whereas mice in the wild-type and heterozygous groups regained righting reflex after about 30 min, the homozygotes were still unconscious after 10 h, when the experiment was terminated. Thus, the loss of CPR activity in the homozygotes had a profound effect on in vivo drug metabolism and clearance, although smaller decreases in CPR activity in the heterozygotes did not have a significant impact on sleeping time.
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The Impact of a Decreased CPR Expression in the Liver on Cholesterol Homeostasis and Liver MorphologySeveral CPR-supported enzymes are involved in cholesterol synthesis, and microsomal CYPs are also involved in cholesterol metabolism. As shown in Table II, the decreases in hepatic CPR activity in the homozygous mice led to an 80% decrease in plasma total cholesterol levels at 2 months of age and about 30% decrease at 3 weeks of age, when they were compared with their wild-type littermates. On the other hand, cholesterol levels were not changed in heterozygotes, which had lesser decreases in microsomal CPR activity.
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At 2 months, the decreased cholesterol level in the homozygous mice was accompanied by a significant increase in liver weight/body weight ratio in both males and females (Table III); there was no significant change in body weight (Table III) or in the weights of other organs, including kidney, lung, heart, and brain (not shown). The liver enlargement was not observed at 3 weeks or in 2-month-old heterozygotes. The enlarged liver had a lighter red color than did the normal liver (Fig. 3A). Microscopic changes in homozygote livers consist of centrilobular and midzonal fine and coarse vacuolar changes (hepatic lipidosis; Fig. 3C). In addition, hepatocellular swelling without lipidosis, characterized by abundant lightly granular, pale, eosinophilic cytoplasm, as well as individual hepatocyte necrosis or apoptosis, and few scattered foci of coagulative necrosis (Fig. 3D), sometimes with secondary neutrophilic cell infiltrates, or hepatitis was observed in some homozygotes. In other experiments not presented, microscopic examination of livers from 3-week-old male and female mice showed mild microvesicular vacuolar changes in centrilobular hepatocytes of homozygotes, with rare occurrence of individual cell to focal necrosis of hepatocytes, which indicated early, subtle changes preceding the pathological changes seen in the older mice.
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Induction of Hepatic CYP and HO-1, but Not HO-2 or Epoxide Hydrolase, by the Decreased CPR Expression in the LiverAs shown in Table IV, the drastic reduction in CPR expression in the homozygous mice led to significant increases in microsomal total CYP content at both 3 weeks (about 2-fold) and 2 months of age (3-fold). However, there was no significant change in microsomal CYP content in heterozygous mice. CYP content was also not increased in kidney microsomes of 2-month-old homozygous mice (data not shown). The induction of CYP was confirmed in 2-month-old mice by immunoblot analysis with antibodies specific for various CYP subfamilies (Fig. 4A). Quantitative immunoblot analysis revealed that there was a 1-fold increase in the levels of CYP1A, CYP2E1, and CYP3A; a 3-fold increase in the level of CYP2B; and a 5-fold increase in the level of CYP2A proteins (Fig. 4B). The level of HO-1 was also increased, by about 9-fold, whereas the levels of HO-2 and microsomal epoxide hydrolyase were not affected. The induction of the CYP2B proteins was particularly intriguing. Two bands were detected in liver microsomes with an antibody to rat CYP2B1 (Fig. 4A), which is expected to cross-react with multiple mouse CYP2B proteins. Although the intensities of both bands were increased in the homozygotes, the upper band, which appeared to be a minor component in the wild-type group, was induced to a much greater extent than was the lower band. A significant, but small, induction was observed in heterozygotes for CYP3A, but not for the other proteins examined (Fig. 4B).
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The mechanisms of induction of hepatic microsomal CYP by the loss of CPR were explored by determining the steady-state level of seven different CYP mRNAs. As shown in Fig. 5, four of the seven mRNAs examined, CYP2A4/5, CYP2B10, CYP2B20, and CYP3A11, had average increases of 10-fold or higher in the homozygous versus the wild-type group. The mRNAs detected with CYP2A4/5 primers were mostly CYP2A5, as confirmed by sequence analysis of PCR products (not shown), a finding consistent with the previous observation that CYP2A4 is not expressed to a significant extent in the livers of male mice (23). Changes in the levels of the other three transcripts were either small (CYP1A2 and CYP3A13) or not detected (CYP1A1). The greatest inductions were found for the Cyp2a and Cyp2b genes, a result consistent with the immunoblot data indicating that the folds of induction were the highest for CYP2A and CYP2B proteins (Fig. 4), even though the fold of increase in protein levels was much lower than the increases in mRNA levels.
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DISCUSSION |
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The dramatic increase in pentobarbital sleeping time in the homozygotes provide in vivo evidence for the role of hepatic CPR-dependent enzymes in drug clearance and illustrates the utility of this model for similar studies on other drugs and dietary chemicals. Although several Cyp knockout mouse models have been generated and have seen increasing applications in drug metabolism and toxicity studies (3), the liver-specific Cpr-null mice will be valuable for delineating the following: (i) the relative contributions of total microsomal CYP versus other biotransformation enzymes, such as flavin monooxygenases (27); (ii) the role of liver versus extrahepatic tissues; and (iii) the possible role of mitochondrial CYPs in the metabolism of endogenous as well as exogenous compounds. Several microsomal CYPs, such as CYP1A1 and CYP2E1, have been found to be transported to the mitochondria, where they function through the mitochondrial electron transfer system (2830). These CYPs are believed to contribute to drug metabolism and chemical toxicity, particularly in extrahepatic organs (31, 32). However, the results from the pentobarbital sleeping test suggest that mitochondrial contributions (if any) are limited for the hepatic metabolism of this drug.
The Alb-Cre/Cprlox mouse will also be useful as a model of liver-specific HO-null mice that can be used in the study of the systemic impact of hepatic heme metabolism, for example. HO oxidatively cleaves heme to generate biliverdin and carbon monoxide; biliverdin is converted to bilirubin, a potent antioxidant; and carbon monoxide may function as a signaling molecule (33, 34). At present, a mouse model with deletions of all three known HO enzymes, HO-1, HO-2, and HO-3, or one with liver-specific deletion of an HO gene, is not available.
The significant and age-dependent decreases in plasma cholesterol level in the homozygotes shed interesting light on the regulation of cholesterol homeostasis. CPR-dependent enzymes are involved in both the biosynthesis, by such enzymes as CYP51 (35) and squalene monooxygenase (10), and the degradation, by enzymes such as CYP7A1 (36), of cholesterol. As such, it was not clear whether a decrease or increase in plasma cholesterol would occur in the Cpr-null mice. The potential of CPR as a target for therapeutic modulation of systemic cholesterol level requires further investigation. The morphological and histopathological changes in the livers of homozygous mice at 2 months of age indicate that a complete inhibition of CPR, which led to substantial decreases in plasma cholesterol level, would not be ideal as a therapeutic strategy. However, a modest decrease of plasma cholesterol may be achieved by a partial inhibition of CPR, as was found in the homozygotes at 3 weeks of age, when the liver appeared to be essentially normal.
Finally, the general induction of microsomal CYP proteins as well as mRNAs in the homozygous mice warrants further investigation, which may reveal novel regulatory pathways of CYP expression by endogenous compounds associated with altered cellular homeostasis in the homozygotes. The induction in CYP expression appears to precede pathological changes in the liver, because a 2-fold increase in total CYP was detected at 3 weeks, when the liver weight was still normal. It is likely that the induction was an adaptive or compensatory response of the cells to a decrease in total CYP activities, which normally consume oxygen and NADPH, and which turn over a myriad of endogenous substrates such as eicosanoids, retinoids, and other fatty acids and steroid hormones, as well as dietary components (1). Accumulation of these chemicals in the hepatocytes may stimulate expression of CYP genes at the transcriptional and/or post-transcriptional stages. Nonetheless, the general induction of all of the examined CYPs, which are known to be regulated by diverse pathways, is intriguing, and may involve multiple mechanisms.
The large increase in the level of the CYP2A5 transcript may be stimulated by an increase in intracellular heme concentration, which is expected to result from the loss of HO function in the homozygotes. Heme and other oxidative stress-inducing compounds are known to induce the expression of HO-1, but not HO-2, in the liver (37). Exogenous heme has been shown to induce CYP2A5 mRNA in the livers of C57BL/6 mice (38); this is one of the parental strains of the Alb-Cre/Cprlox mice. Furthermore, a more recent study (39) using porphobilinogen deaminase-deficient mice, which have deficient heme synthesis, showed that a reduced heme level had an inhibitory effect on phenobarbital-induced CYP2A5 expression; this effect can be overcome by addition of exogenous heme. CYP2A5 can also be induced by a number of porphyrinogenic compounds, such as pyrazole, through a mechanism involving RNA stabilization (40, 41), and by liver cell injury associated with hepatitis (e.g. see Ref. 42). The latter, however, causes a significant decrease in liver microsomal total P450.
Large increases in CYP2B expression have been found previously to be induced by phenobarbital and other compounds, which increase transcriptional activation through the nuclear orphan receptor constitutive androstane receptor (for a recent review, see Ref. 43). Thus, it will be interesting to determine whether the CYP2B induction associated with Cpr gene deletion is a consequence of feedback regulation by endogenous constitutive androstane receptor ligands, such as estrogen and isoprenoid (44, 45). On the other hand, the lack of increase in CYP1A1 mRNA suggests lack of activation of the aryl hydrocarbon receptor-mediated pathway that is essential for CYP1A1 induction by polycyclic aromatic hydrocarbons (46, 47). Notably, although the lack of a transcriptional activation of CYP1A1 in the homozygotes does not appear to support the hypothesis that endogenous CYP substrates serve as ligands of aryl hydrocarbon receptor and regulate basal expression of CYP1A1 in vivo (48, 49), the Alb-Cre/Cprlox may not be an ideal model in which to test this hypothesis, because of the multitude of pathways affected by the loss of CPR.
The induction of microsomal CYPs in the homozygotes may also involve increased stability of the CYP proteins as a result of an accumulation of endogenous substrates, which leads to reduced rates of CYP inactivation and proteolytic degradation. Substrate-mediated stabilization of CYP2E1 is well documented (50). Microsomal CYPs are also known to generate reactive oxygen species in CPR-dependent reactions (for a recent review, see Ref. 51). However, the induction of hepatic CYP and HO-1 may not be because of the altered plasma cholesterol level or changes in other systemic parameters, because an induction was not seen in the kidneys of homozygous mice. To that end, although the effects of CPR loss on circulating hormones have not been determined, the normal fertility of the homozygous mice suggests that gross alterations in serum sex hormone levels did not occur.
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FOOTNOTES |
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* This work was supported in part by United States Public Health Service
Grant ES07462 from the NIEHS, National Institutes of Health. The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
To whom correspondence should be addressed: Wadsworth Center, New York State
Dept. of Health, Empire State Plaza, Box 509, Albany, NY 12201-0509. Tel.:
518-486-2585; Fax: 518-486-1505; E-mail:
xding{at}wadsworth.org.
1 The abbreviations used are: CYP, cytochrome P450; CPR, NADPH-cytochrome
P450 reductase; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; AP,
acetaminophen; Cre, Cre recombinase; HO, heme oxygenase; mEH, microsomal
epoxide hydrolase; RT, reverse transcription.
2 L. Wu, J. Gu, Y. Weng, K. Kluetzman, P. Swiatek, M. Behr, Q.-Y. Zhang, X.
Zhuo, Q. Xie, and X. Ding, submitted for publication.
3 Q.-Y. Zhang, D. Dunbar, and L. S. Kaminsky, submitted for publication.
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ACKNOWLEDGMENTS |
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REFERENCES |
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