Inactivation of the Hepatic Cytochrome P450 System by Conditional Deletion of Hepatic Cytochrome P450 Reductase*

Colin J. HendersonDagger, Diana M. E. OttoDagger, Dianne Carrie, Mark A. Magnuson§, Aileen W. McLaren, Ian Rosewell, and C. Roland Wolf||

From the Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Centre, Level 5, Ninewells Hospital & Medical School, Dundee DD1 9SY, United Kingdom

Received for publication, November 27, 2002, and in revised form, January 23, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of a large number of endogenous compounds and the majority of ingested environmental chemicals, leading to their elimination and often to their metabolic activation to toxic products. This enzyme system therefore provides our primary defense against xenobiotics and is a major determinant in the therapeutic efficacy of pharmacological agents. To evaluate the importance of hepatic P450s in normal homeostasis, drug pharmacology, and chemical toxicity, we have conditionally deleted the essential electron transfer protein, NADH:ferrihemoprotein reductase (EC 1.6.2.4, cytochrome P450 reductase, CPR) in the liver, resulting in essentially complete ablation of hepatic microsomal P450 activity. Hepatic CPR-null mice could no longer break down cholesterol because of their inability to produce bile acids, and whereas hepatic lipid levels were significantly increased, circulating levels of cholesterol and triglycerides were severely reduced. Loss of hepatic P450 activity resulted in a 5-fold increase in P450 protein, indicating the existence of a negative feedback pathway regulating P450 expression. Profound changes in the in vivo metabolism of pentobarbital and acetaminophen indicated that extrahepatic metabolism does not play a major role in the disposition of these compounds. Hepatic CPR-null mice developed normally and were able to breed, indicating that hepatic microsomal P450-mediated steroid hormone metabolism is not essential for fertility, demonstrating that a major evolutionary role for hepatic P450s is to protect mammals from their environment.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The hepatic cytochrome P450 (CYP)1-dependent monoxygenase system plays a central role in mammalian defense against harmful environmental chemicals (1); it is also a major determinant of the half-life and pharmacological properties of therapeutic drugs and in certain cases, mediates the activation of drugs, toxins, and carcinogens to their ultimate toxic species (2, 3). Several other functions have been ascribed to hepatic P450s, including control of cholesterol and steroid hormone metabolism and bile acid biosynthesis (4). However, for certain of these pathways, the exact role of P450s in normal homeostasis is unknown.

Over the last four decades, there have been significant advances in understanding the functions, genetics, and regulation of these enzymes and more recently their structure (5). However, a great deal remains to be learned about the expression and regulation of P450s, and their endogenous function(s), particularly in individual tissues. The size and diversity of the P450 multigene family results in great difficulties in dissecting out the function(s) of individual enzymes, particularly as many of those involved in foreign compound metabolism exhibit overlapping substrate specificities and may be expressed to a greater or lesser extent in almost every cell and tissue. The contribution that P450s in any particular tissue make to the overall pharmacokinetics of a drug is still, in the majority of cases, unknown. For example, the precise role of P450s in the metabolism of chemotherapaeutic agents, and the generation of side effects, remains unclear, as does the site of metabolism (either systemic (hepatic) or local (tumor site)), or both.

To study further the involvement of P450s in the tissue specificity of drug metabolism would require the simultaneous deletion of multiple P450 genes, a process rendered impractical both by the large number of such genes and their location throughout the genome and our lack of knowledge about the regulation of tissue-specific P450 expression. However, all cytochrome P450s receive electrons from a single donor, cytochrome P450 reductase (CPR, NADPH:ferrihemoprotein reductase, EC 1.6.2.4) (6, 7). Deletion of this protein would therefore inactivate all the P450s located in the endoplasmic reticulum. In view of the known requirement for P450 expression during development, a complete deletion of CPR was anticipated and has recently been shown to be embryonic lethal (8). We therefore generated mice where CPR could be deleted in the postnatal period in any tissue, using the Cre/loxP system (9). Below, we describe the generation and characterization of mice carrying a deletion of hepatic CPR, and thus lacking P450 activity in the liver. Such mice exhibited many intriguing phenotypes, and remarkably they were viable and healthy. In addition, hepatic CPR-null mice had a severely compromised ability to metabolize the narcotic drug pentobarbital or the analgesic acetaminophen, demonstrating the predominant role of the hepatic P450 enzymes in the pharmacology and toxicology of these compounds and also demonstrating the power of this model for understanding P450 functions.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of CPR Floxed and Knockout Mice-- A replacement-type targeting vector was constructed from a 12-kb SalI fragment, isolated from a mouse 129/Ola genomic library and containing exons 3-16 of the mouse CPR gene. A cassette, flanked by same orientation loxP sites and containing a selectable marker (neomycin (neo)), driven by the herpes simplex thymidine kinase (hsv-tk) promoter, was inserted into an XhoI site in intron 4. A third loxP site was cloned into a BamHI site in intron 15. The correct arrangement of the construct and orientation of all three loxP sites was confirmed by detailed restriction mapping and sequence analysis. The construct was transfected into GK129/1 embryonic (ES) cells by electroporation, and the ES cells were subsequently cultured in 96-well plates and G418 selection applied. Eight G418-resistant clones were found to have undergone specific homologous recombination as demonstrated by Southern analysis, digesting genomic DNA with KpnI and using a 600-bp PCR fragment generated with primers 1105 (5'-GACCCTGAAGAGTATGACTTG-3') and 1184 (5'-GCTTCCTCTTGCAAAACCACACTGC-3') (Fig. 1a), and two of these correctly targeted ES cell clones (CPRlox/+) were expanded, injected into C57BL/6 blastocysts, and transferred into 2.5-day post-coitum (dpc) recipient pseudopregnant mice. Male chimera were bred to C57BL/6 mice, and heterozygous offspring were screened by Southern analysis to confirm germline transmission of the CPRlox/+ genotype. Five of these ES clones containing the CPRlox locus were transiently transfected with a vector containing the Cre recombinase gene (pMC1Cre). Colonies were obtained without selection and were isolated into a 96-well plate; after 3-4 days the plates were split into two duplicate plates, and after an additional 3-4 days one plate was frozen and the other further split. G418 selection was applied for up to 5 days to one of these plates in order to identify sensitive colonies. DNA was prepared from the duplicate plate after 3-4 days. ES clones (800) were tested by Southern blot analysis as described above. Nine of those clones screened showed excision of the floxed sequence including exons 5 to 15. Two of these ES cell clones (CPR+/-) were expanded and chimeric mice generated. Male chimera were bred to C57BL/6 mice and heterozygous offspring were screened by Southern blot analysis to confirm germline transmission.

Mouse Breeding and Maintenance-- CPR+/- mice were maintained by random breeding with CPR+/+ mice on a 129P2xC57BL/6 genetic background. CPRlox/+ mice were crossed to produce homozygous CPRlox/lox mice and maintained by random breeding on a 129P2 × C57BL/6 genetic background. The CPRlox/lox line was bred with CPR+/- to generate a CPRlox/- line.

A transgenic mouse line expressing Cre recombinase under control of the rat albumin promoter (10) was obtained from Dr. Mark Magnuson, Vanderbilt University School of Medicine, Nashville, TN and crossed onto both the CPRlox/lox (CPRlox/lox + CreALB) and CPRlox/- (CPRlox/- + CreALB) lines to generate liver-specific CPR conditional knockout mice. The presence of the CreALB transgene was determined by PCR (data not shown).

All mice were maintained under standard animal house conditions, with free access to food and water, and 12-h light/12-h dark cycle. All mouse work was carried out in accordance with the Animal Scientific Procedures Act (1986), and after local ethical review.

Drug Treatment of Mice-- Pentobarbital sleep time-adult male CPRlox/lox + CreALB, CPRlox/- + CreALB, CPRlox/- and CPR+/+ + CreALB mice (n = 3) were given a single intraperitoneal dose of pentobarbital (Sagatal) at 20 mg/kg of body weight. The time taken for the mice to lose, and subsequently to regain, their righting reflex was measured (11). Mice still asleep 2 h after treatment were sacrificed as required by Home Office License.

Acetaminophen treatment-adult male CPRlox/- + CreALB and CPR+/+ + CreALB mice were administered acetaminophen intraperitoneally at 300 mg/kg of body weight in phosphate-buffered saline. At 1, 5, 7, 17, and 24 h after treatment, mice were sacrificed (n = 4) by a rising concentration of CO2, and blood and tissues were taken for analysis.

Immunoblotting and Biochemical Assays-- Microsomal fractions were prepared from frozen tissues by differential centrifugation (12) and protein concentration determined as previously described (13). Western blots were carried out as described previously (14) using 9% SDS/PAGE gels and electroblotted onto nitrocellulose membranes. Polyclonal antisera raised against human CPR (7) and rats P450s (15) were used as primary antibodies, and a donkey anti-rabbit horseradish peroxidase IgG as secondary antibody (Scottish Antibody Production, Carluke, UK). Immunoreactivity was determined by chemiluminiscence (ECL Plus, Amersham Biosciences) and XAR5 autoradiographic film (Eastman Kodak).

Glutathione levels were measured by the method of Sen et al., (16). Cytochrome P450 reductase activity was determined as previously described (7).

Blood Chemistry-- Blood was collected by cardiac puncture into heparinized tubes, and serum prepared by centrifugation. Serum was either analyzed immediately or stored at -70 °C for a period not exceeding 4 weeks. Analysis for serum alanine aminotransferase and cholesterol was carried out using commercially available kits (Infinity Reagents, Sigma, Poole, UK) on a Cobas Fara II centrifugal analyser (Roche Molecular Biochemicals).

Histopathology-- Tissue samples were either fixed in formalin/phosphate-buffered saline for 24 h and transferred to 80% ethanol for storage, or snap-frozen embedded in Cryo-M-Bed (Bright Instrument Co., Huntingdon) on cork discs and stored at -70 °C. Formalin-fixed samples were sectioned and stained with hematoxylin and eosin, or processed for immunostaining with a polyclonal antibody against rat CPR or various P450s (15, 17). Snap-frozen tissue samples were cryosectioned and processed for staining with Oil Red O to determine lipid content.

Statistical Analysis-- Statistical analysis was carried out using the Statview program (v4.5) for Macintosh, Abacus Concepts, Berkeley, CA.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of Conditional CPR Knockout Mice-- Fig. 1a illustrates the targeting strategy adopted in this study. Correct integration of the floxed CPR targeting construct was confirmed by Southern analysis (Fig. 1b) of genomic DNA isolated from CPRlox/+ mice (lanes 1 and 5 (9.2/8-kb bands), while the pattern obtained from a wild-type mouse (CPR+/+) is shown in lane 2 (8-kb band only). Genomic DNA of mice generated from ES cells in which Cre recombinase had been transiently expressed, resulted in deletion of the CPR gene between the first and third loxP sites (Fig. 1b, lane 6 (CPR+/-, 8/3.2-kb bands)). Mice homozygous for the floxed CPR locus (CPRlox/lox) or with one floxed and one deleted CPR allele (CPRlox/-) were generated by mating of appropriate mouse lines. Southern analysis of genomic DNA from these mice is shown in lanes 7 and 8 (CPRlox/lox, 9.2-kb band only) and in lanes 3 and 4 CPRlox/-, 9.2/3.2 kb). These latter mice were apparently completely normal, displaying no phenotypic differences from wild-type littermates: their growth and development, blood chemistry, organ size and structure, and fertility were identical (data not shown).


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 1.   Targeting of the mouse CPR gene. a, maps of the wild-type, floxed, and disrupted CPR alleles. A 12-kb SalI fragment containing exons 3-16 of the mouse CPR gene was cloned and used for gene targeting. Exon 1 (untranslated) and exon 2 reside ~30-kb upstream, and are shown only for information. LoxP sites are indicated by triangles, and the selectable marker (hsv-tk-neo) is indicated by the hatched box in intron 4. b, Southern analysis of tail DNA from CPR+/+, CPR+/-, CPRlox/+, CPRlox/-, and CPRlox/lox mice. Genomic DNA was digested with KpnI and hybridized with a 600-bp PCR fragment generated using primers 1105 and 1184, as shown in a and detailed under "Materials and Methods." The wild-type allele (CPR+) is represented by a fragment of 8 kb, the targeted allele (CPRlox, which also contains the selectable marker cassette), by a fragment of ~9.2 kb, while the deleted allele (CPR-) is represented by a fragment size of 3.2 kb. Lanes 1 and 5, CPRlox/+; lanes 3 and 4, CPRlox/-; lane 2, CPR+/+; lane 6, CPR+/-; lanes 7 and 8, CPRlox/lox.

Generation of Hepatic CPR-null Mice-- Specific hepatic deletion of CPR was achieved by crossing CPRlox/lox mice into a line where Cre expression was regulated by the rat albumin promoter (10). Mice identified as CPRlox/+ + CreALB were either backcrossed with CPRlox/lox mice to generate a CPRlox/lox + CreALB line, or crossed with CPR heterozygous nulls (CPR+/-) to generate CPRlox/- + CreALB mice. The presence of the CreALB transgene was determined by PCR (data not shown). Offspring born from either of these crosses were found in Mendelian proportions as predicted from parental genotype, indicating there was no embryonic lethality from opportunistic expression of the Cre transgene during development.

As the albumin promoter becomes active neonatally, (10) we investigated hepatic CPR levels in adult mice, i.e. from 6 to 8 weeks of age. CPRlox/- + CreALB and CPRlox/lox + CreALB mice displayed no overt phenotypic differences from their wild-type littermates in the postnatal period: mice of these genotypes grew and developed normally. In mice where one CPR allele had been deleted, only half the expression of CPR was found in both males and females (Fig. 2a, liver, tracks 2 and 3 versus 4 and 5 and tracks 7 and 8 versus 9 and 10). In mice of genotype CPRlox/- + CreALB, an immunoreactive CPR protein band was essentially absent in both males and females (Fig. 2a, lanes 1 and 6, respectively), indicating an almost complete lack of CPR protein in the microsomal fractions of the livers of these animals. Upon prolonged exposure (>10-fold normal) of the immunoblot shown in Fig. 2a, a very faint band corresponding to the correct molecular weight for CPR could be seen (not shown). Hepatic CPR activity in CPRlox/- + CreALB mice was reduced by more than 90% in both males (92.5%) and females (94.5%), and similar reductions were observed in hepatic microsomes from CPRlox/lox + CreALB mice of both sexes (Fig. 2b). The activity of cytochrome b5 reductase, an enzyme that could conceivably transfer electrons from NADH to the P450 system (18), was unchanged (not shown). In liver sections from CPR hepatic-null mice stained with a polyclonal antiserum to CPR, only a very few cells contained immunoreactive protein (Fig. 2c), in contrast to the wild-type mice where CPR immunostaining was extensive across the entire section.


View larger version (61K):
[in this window]
[in a new window]
 
Fig. 2.   Liver-specific deletion of CPR in hepatic CPR-null mice. a, representative immunoblot showing CPR protein levels in liver and kidney of the following mice: lanes 1 and 6, CPRlox/- + CreALB male, female; lanes 2 and 7, CPRlox/- male, female; lanes 3 and 8, CPRlox/+ + CreALB male, female; lanes 4 and 9, CPRlox/+ male, female; lanes 5 and 10, CPR+/+ + CreALB male, female; S, CPR standard. 5 µg of protein were run in each lane, and the anti-CPR antiserum was diluted 1:1000. b, hepatic and renal CPR activity in male (white bar) and female (gray bar) wild-type (CPR+/+ + CreALB) and hepatic CPR-null mice (CPRlox/lox + CreALB and CPRlox/- + CreALB) (n = 3-5). Values are expressed as nmol of cytochrome c reduced/min/mg of microsomal protein ± S.E. *, p < 0.05; ***, p < 0.001 using an unpaired Student's t test, comparing hepatic CPR-null mice (CPRlox/lox + CreALB or CPRlox/- + CreALB) with wild-type. c, immunostaining of liver, kidney, and lung sections from wild-type (CPR+/+ + CreALB) and hepatic CPR-null mice (CPRlox/- + CreALB) with anti-CPR antiserum (diluted 1:100). Data shown are representative of that found in each group (n = 3).

The specificity of the CPR deletion was demonstrated by showing that no change was observed in CPR expression in the kidneys of CPRlox/+ + CreALB, CPRlox/+ and CPR+/+ + CreALB mice in both sexes (Fig. 2a, kidney; lanes 3-5 male  and 8-10 female ). A reduction in CPR protein level of ~50% was seen in CPRlox/- + CreALB and in CPRlox/- mice was observed (Fig. 2a, lanes 1 and 2 male  and 6 and 7 female ) in offspring missing only one CPR allele. Generally, protein expression reflected the CPR activity (Fig. 2b) in kidney microsomal fractions, with no significant difference in CPR activity between CPR+/+ + CreALB and CPRlox/lox + CreALB mice of either sex, while CPR activity was lower in CPRlox/- + CreALB mice, significantly so in males. Further confirmation of the tissue-specific nature of the CPR deletion was demonstrated by immunostaining kidney and lung sections from CPRlox/- + CreALB and CPR+/+ + CreALB mice with CPR antiserum (Fig. 2c), where the staining was the same in both lines.

Characterization of Hepatic CPR-null Mice-- Hepatic CPR-null mice exhibited no overt phenotypic differences to controls. The mice grew at the same rate as their wild-type counterparts, and there was no change in survival rates or behavior (data not shown). Interestingly, although CPR hepatic-null mice exhibited normal fertility when mated to wild-type (CPR+/+ or CPRlox/lox) mice, when crossed with each other (CPRlox/- + CreALB × CPRlox/- + CreALB or CPRlox/lox + CreALB × CPRlox/lox + CreALB), both fertility and litter size were reduced (data not shown): the reason(s) for this remain unclear.

Post-mortem examination revealed that both male and female hepatic CPR-nulls displayed hepatomegaly, the liver being almost doubled in size, in relation to body weight (7.5%), compared with wild-type mice (4%). Furthermore, the liver was pale in color, and the tissue was mottled and friable (Fig. 3, a and b). Microscopic examination revealed the presence of microvisicular and macrovisicular fatty changes, the former predominating (Fig. 3c, plates i and v versus iii and vii). Otherwise, hepatocytes in both the centrizonal and periportal regions appeared normal, with no apparent increase in hepatocyte proliferation or apoptosis.


View larger version (57K):
[in this window]
[in a new window]
 
Fig. 3.   Characterization of hepatic CPR-null phenotype. a, photographs show typically enlarged size and pale appearance of liver from hepatic CPR-null mouse (right) and wild-type mouse (left). b, liver/body weight ratio in adult CPR+/+, CPRlox/-, and CPRlox/lox mice with and without the CreALB transgene (n = 4-12). Values are expressed as mean ± S.E. ***, p < 0.001 using an unpaired Student's t test, comparing genotypes ± CreALB transgene. c, representative sections from livers of male (left) and female (right) CPR+/+ + CreALB (top) (control) and CPRlox/- + CreALB CPR-null, (bottom) mice, stained with hematoxylin and eosin (i, ii, v, vi) or Oil Red O (iii, iv, vii, viii). d, bile volume in adult hepatic CPR-null and wild-type mice (n = 5-8). Values are expressed as mean ± S.E. *, p < 0.05 using an unpaired Student's t test. e, serum cholesterol in adult hepatic CPR-null and wild-type mice (n = 4-12). Values are expressed as mean ± S.E. ***, p < 0.001 using an unpaired Student's t test, comparing genotypes ± CreALB transgene. f, serum triglycerides in adult hepatic CPR-null and wild-type mice (n = 4-12). Values are expressed as mean ± S.E. ***, p < 0.001 using an unpaired Student's t test, comparing genotypes ± CreALB transgene.

The livers of mice lacking hepatic CPR were hyperlipidaemic, as evidenced by staining with Oil Red O (Fig. 3c, plates ii and vi versus iv and viii). The increase in hepatic lipid was accompanied by a 90% reduction in the volume of bile acids in the gall bladder in hepatic CPR-null mice, compared with wild-type animals (Fig. 3d), and a 65% reduction in the level of serum cholesterol (Fig. 3e) and a 50% reduction in serum triglycerides (Fig. 3f). It is interesting to note that these mice also had a slight but significantly elevated serum ALT, indicating a low level of liver damage or an altered rate of hepatocyte turnover (data not shown).

P450 Activity in Hepatic CPR-null Mice-- In order to determine the effect of CPR deletion on hepatic P450 monoxygenase activities, we measured the microsomal hydroxylation of testosterone (Table I). When expressed as pmol of metabolite per nmol of P450, a 90% reduction in the formation of 6beta -hydroxytestosterone, an activity associated with CYP3A proteins, was measured in both males and females, indicating that a lack of CPR in this tissue resulted in severely compromised P450 function. The 7alpha -hydroxylation of testosterone, catalyzed by P450s of the CYP2A subfamily, was decreased by >99% in both males and females, while 16beta -hydroxylation was undetectable in hepatic CPR-nulls of both sexes. In addition to the marked reduction in testosterone metabolism, the O-dealkylation of 7-methoxyresorufin was also almost completely ablated in CPRlox/- + CreALB mice, being reduced by 99.5% in males and 98.5% in females, when expressed as nmol of metabolite per nmol of P450 (not shown). It is thus clear that hepatic deletion of CPR results in the inactivation of multiple P450s in the liver.


                              
View this table:
[in this window]
[in a new window]
 
Table I
Testosterone hydroxylation by hepatic microsomes from hepatic CPR-null and wild-type mice
Testosterone 6beta , 7alpha , and 16beta hydroxylation in liver microsomes from CPR+/+ + CreALB and CPRlox/lox + CreALB mice. Activities were determined as described under "Materials and Methods" and are expressed in pmol/min/nmol P450, mean ± S.E. Numbers in parentheses represent % activity in samples relative to wild-type, which is 100%. Assay carried out in duplicate (n = 4).

Intriguingly, mice lacking hepatic CPR exhibited a profound increase in cytochrome P450 content; in males the increase was 450%, while in females it was greater than 500% (Fig. 4a). This large increase in P450 expression was further demonstrated by immunoblotting of hepatic microsomes with antisera to P450s from different gene families (Fig. 4b). For certain of the enzymes, e.g. members of CYP2B and CYP3A gene families, the induction was at least as much as observed when potent exogenous P450 inducers are administered to mice. Hepatic immunostaining clearly showed that while expression of CYP3A P450s was confined to the perivenous or centrilobular area in wild-type mice, as has been previously reported (19, 20), in null mice CYP3A was localized throughout the section at high levels (Fig. 4c).


View larger version (49K):
[in this window]
[in a new window]
 
Fig. 4.   CPR and P450 expression in hepatic CPR-null mice. a, hepatic cytochrome P450 content in adult male and female CPR+/+ + CreALB (white bars) and CPRlox/-+ CreALB (gray bars) mice (n = 3). Values are expressed in nmol/mg of protein, mean ± S.E. *, p < 0.05; **, p < 0.005 using an unpaired Student's t test, hepatic CPR-null versus wild-type mice. b, immunoblotting of hepatic microsomes from adult male and female CPR+/+ + CreALB and CPRlox/-+ CreALB mice with polyclonal antisera to CPR and various P450s. std, protein standard. 5 µg of protein was run in each lane, and the anti-CYP antisera were diluted 1:1000. c, immunostaining of liver sections from female CPRlox/-+ CreALB (i) and CPR+/+ + CreALB (ii) mice with polyclonal antisera to CYP3A1, phase contrast, magnification ×4. Antiserum was diluted 1:100.

Hepatic Drug Metabolism in CPR Hepatic-null Mice-- Acetaminophen was administered as a single intraperitoneal dose (300 mg/kg of body weight) to male wild-type and hepatic CPR-null mice (Fig. 5, a and b). In wild-type mice, a 90% decrease in hepatic glutathione occurred within one hour of administration (Fig. 5a), whereas the level remained unchanged in hepatic CPR-nulls. Furthermore, 24 h after treatment, wild-type mice showed a marked rise in serum ALT, indicative of extensive, potentially fatal, liver damage, whereas in mice lacking hepatic CPR ALT remained unchanged.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of acetaminophen or pentobarbital treatment of hepatic CPR-null mice. a, hepatic glutathione and b, serum alanine aminotransferase levels in adult male CPR+/+ + CreALB (closed squares) and CPRlox/-+ CreALB (open circles) mice at 1, 5, 7, 17, and 24 h following treatment with acetaminophen at 300 mg/kg intraperitoneal (n = 3). Values are expressed as mean ± S.E. c, time taken for regain of righting reflex in adult male mice of various genotypes following treatment with pentobarbital at 20 mg/kg intraperitoneal (n = 3). Values are expressed as mean ± S.E.

Intraperitoneal administration of pentobarbital at a dose of 20 mg/kg of body weight failed to induce sleep in wild-type mice; however, mice lacking hepatic CPR, of genotype CPRlox/- + CreALB or CPRlox/lox + CreALB, slept for a period in excess of 2 h (Fig. 5c). At this point it was necessary to humanely cull the mice under the terms of our Home Office License granted under the Animal (Scientific Procedures) Act (1986). Pentobarbital treatment of CPR heterozygous null mice (CPR+/-), i.e. lacking one CPR allele and therefore having ~50% less CPR activity, resulted in an average sleeping period of ~50 min.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CPR is a multidomain protein containing NADPH/flavinadeninedinucleotide (FAD) and flavinmononucleotide (FMN) binding domains (7, 21). When expressed in Escherichia coli, these domains fold to form a functional polypeptide, which can, in the case of the FAD domain, independently catalyze the one electron reduction of a number of foreign compounds (7). For this reason, the targeting construct was designed to ensure deletion of both of these domains (Fig. 1a), and also utilized Cre/loxP to allow the conditional deletion of CPR and circumvent the anticipated embryonic lethality of a complete CPR knockout (8). The data presented in the current study is based on the generation of mice from targeted ES cells such that these mice retain all three loxP sites and the selectable marker, as shown in Fig. 1a, either at both CPR alleles (CPRlox/lox) or at one allele with the other CPR allele deleted (CPRlox/-), tissue-specific deletion being achieved by crossing in the CreALB transgene.

The CreALB transgene has previously been shown to work with high efficiency in other systems, and in this study resulted in a reduction in hepatic CPR activity of more than 90% in both males and females (Fig. 2b) by 6-8 weeks postpartum. The residual CPR activity observed in hepatic microsomal preparations from CPR hepatic-null mice could be explained by the restriction of albumin promoter activity (and thus Cre recombinase expression) to hepatocytes (10) leaving CPR activity undiminished in other liver cell types. This would also account for the CPR immunostaining observed in individual cells in liver sections (Fig. 2c). A further explanation could lie with an alternative electron donor to P450s, i.e. the cytochrome b5/NADH b5 reductase system. CPR has recently been reported to be deleted in Saccharomyces cerevisiae and such altered yeast are apparently able to survive by using the cytochrome b5/NADH reductase system to supply both electrons to the P450 during the catalytic cycle (18). However, although cytochrome b5 has been implicated as participating in the electron transfer cycle for P450s, often resulting in higher P450 activity (22), cytochrome b5 has classically been deemed capable of undertaking transfer of the second, but not the first, electron in the P450 catalytic cycle, and this would seem to be confirmed by the embryonic lethality observed in homozygous CPR-null mice (8).

Intriguingly, hepatic CPR-null mice were found to have an appreciably lower level of serum cholesterol, despite the profoundly raised lipid content of the liver (Fig. 3e). These phenomena may be rationalized by the inability of these mice both to synthesize cholesterol de novo, (resulting in reduced circulating levels) and to degrade cholesterol through the bile acid biosynthetic pathway (to generate increased hepatic levels). This is consistent with the observation that hepatic CPR-null mice have significantly reduced (~90%) bile acid production (Fig. 3d). Both of these processes involve P450 enzymes at key stages, i.e. CYP51 (sterol 14-demethylase) (23, 24) in cholesterol biosynthesis, and CYP7A1 (cholesterol 7alpha -hydroxylase) (25-27) at the rate-limiting step of cholesterol metabolism in the classical or neutral bile acid biosynthetic pathway. Under normal circumstances, cholesterol accumulation in the liver would trigger the feedback down-regulation of cholesterol uptake and biosynthesis by inhibiting the action of the SREBP transcription factors on a number of key lipogenic genes such as HMG CoA reductase, HMGCoA synthase, and squalene synthase, and also the LDL receptor (28). It is interesting to note that transgenic mice expressing a truncated dominant positive form of SREBP1 also displays hepatomegaly with elevated lipid content of the liver (29). However, these mice had essentially normal serum lipid chemistry, which was speculated to be due to an alteration in lipid metabolism such that lipids were stored in, rather than secreted from, the liver. Hayhurst et al. (30) recently reported the conditional deletion of HNF4alpha in mouse liver. This transcription factor, a member of the nuclear receptor superfamily, appears to play a key role in the maintenance of lipid homeostasis; mice lacking liver expression of HNF4alpha displayed a similar hepatic phenotype (increased liver/body weight, elevated hepatic lipids, reduced serum cholesterol) to that described for hepatic CPR-null mice in this study. Interestingly, this group and others (31, 32) have shown that HNF4 plays an important role in the regulation of cytochrome P450s in the CYP2 gene family.

Although the liver is considered to be the major organ for cholesterol biosynthesis, almost all tissues possess the capacity to produce cholesterol (33), reviewed in Ref. 34. In the rat, for example, the liver is responsible for approximately half of all de novo cholesterol synthesis, with the major extrahepatic tissues involved being small intestine and skin; however, the contribution of different organs to cholesterol homeostasis can vary significantly between species (35). The other significant source of cholesterol is dietary, although cholesterol itself is not an essential nutrient, and intestinal absorption is relatively inefficient, varying considerably even within species, including mice and humans (36, 37). A recent review of cholesterol and hepatic lipoprotein assembly proposed that the liver may be segregated into metabolic zones, with the periportal region undertaking the assembly of VLDL and being the major site of lipogenesis (anabolic phenotype), and the pericentral area exhibiting a catabolic phenotype, being the main location of LDL receptor and CYP7A1 expression and therefore undertaking the biosynthesis of bile acids from cholesterol (38). These authors also suggested a link between these two processes, with the induction of bile acid synthesis (increased CYP7A1 expression) leading to increased transcription of genes regulated by SREBP. CYP7A1-null mice (26) exhibit a complex phenotype including increased mortality in the postnatal period, although those which survive to 3 weeks of age thereafter develop normally by utilizing an alternative, acidic, bile acid biosynthetic pathway located in the mitochondrion, involving the sterol 27-hydroxylase (CYP27) (27). The fact that the hepatic CPR-null mice described in this study do not exhibit the same phenotype as the CYP7A1-null mice may be explained by the inactivation of hepatic P450s involved in the acidic pathway downstream from CYP27, i.e. CYP7B1 (39, 40). The change in circulating triglycerides in the hepatic CPR-null mice identifies a potentially novel function of hepatic P450 enzymes. Although P450s from the CYP4A gene family have been shown to metabolize lipids (41) this activity will be lost in hepatic CPR-null mice and cannot explain the changes observed. Hepatic CPR-null mice therefore constitute an ideal model with which to study this phenomenon.

The profound consequences for drug metabolism and toxic response in mice lacking hepatic CPR are illustrated both in vitro, with the almost complete ablation of testosterone hydroxylation (Fig. 4a), and in vivo with acetaminophen and pentobarbital (Fig. 5). The analgesic drug acetaminophen is activated by the P450 system to a highly reactive hepatotoxic intermediate, N-acetylbenzoquinonimine (NAPQI), a reaction undertaken mainly by CYP1A2 and CYP2E1 (42-44). Normally, NAPQI is subsequently detoxified by conjugation with glutathione, leading to a significant decrease in hepatic levels of this thiol (45, 46), and this was indeed observed in wild-type mice (Fig. 5a). In contrast, no such change was seen in hepatic CPR-null mice, indicating that little or no NAPQI had been formed due to the lack of a functional hepatic microsomal P450 system.

The activity of the cytochrome P450 system can also be assessed by measuring the sleeping time of animals exposed to barbiturate drugs, the length of sleep reflecting the rate of P450-mediated metabolism (11). In order to establish the role of hepatic P450s in the disposition of pentobarbital, wild-type and hepatic CPR-null mice were administered an intraperitoneal dose of 20 mg/kg of body weight. Such a dose was non-narcotic to wild-type mice with normal levels of CPR (Fig. 5c); however, hepatic-null mice, lacking a functional hepatic microsomal P450 system, slept for a period in excess of 2 h. Mice which were heterozygous null for CPR slept for less than 1 h, not only demonstrating a clear gene-dosage effect, but also that CPR activity is rate-limiting in vivo for the metabolism of this substrate.

Cytochrome P450s involved in drug metabolism are distributed throughout the tissues of the body; it has been estimated that a significant proportion of drug metabolism (up to 30%) may be extrahepatic. The profound changes in drug response described above show that extrahepatic metabolism plays only a minor role in the disposition of these compounds, at least in the mouse. The data also further demonstrate that the potential alternate electron transport pathway for P450s, from NADH via cytochrome b5/b5 reductase, plays only a minimal role, if any, in drug disposition.

One unexpected consequence of deleting hepatic CPR was the finding that the P450 content of the liver was increased by a factor of approximately five (Fig. 4a). This increase, which was found in both male and female mice, was evident in several different P450 subfamilies (Fig. 4b), with expression going from essentially undetectable in wild-type mice to significant levels for CYP2B and CYP3A proteins in hepatic CPR-nulls, and resulted in pan-lobular P450 expression rather than the zonal P450 expression usually found (Fig. 4c) (19, 20). Cytochrome P450 isozymes provide an adaptive response to environmental challenge, and certain isozymes can be induced in the liver by a wide range of endogenous agents (47, 48). In most cases, enzymes are induced in a specific manner according to the inducing agent, which results in an increased rate of disposition of the compound. Apart from the role of hormones in the regulation of certain hepatic P450s (49, 50) essentially nothing is known about the factors that regulate the endogenous levels of these enzymes. The profound induction of P450s in the hepatic CPR-nulls, across a range of P450 subfamilies, is therefore intriguing and suggests that there may be a single key endogenous regulatory pathway that becomes activated (or inactivated) in the absence of P450 activity. Although it is feasible that in the absence of metabolism specific endogenous agents, such as glucocorticoids, may accumulate in the liver, such a phenomenon would not explain the generalized P450 induction seen in the hepatic CPR-null mice. To our knowledge, the only similar pan-family induction of the P450 system observed in mice is that seen following administration of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) (51, 52). Since this compound does not inhibit CPR activity, it will be intriguing to establish whether a common mechanism is involved.

The data presented in this study demonstrate unequivocally that we have specifically deleted hepatic CPR in the mouse. One of the most intriguing aspects of this model is that through the deletion of a single gene the functions of an entire multigene family (microsomal cytochrome P450s) have been ablated in a tissue-specific manner, resulting not only in a profound reduction in bile acid production but also in the metabolism of a hormone, testosterone, and drugs such as pentobarbital and acetaminophen. Despite the hepatic phenotype, hepatic CPR-null mice live and reproduce normally; indeed, we now have mice over 18 months of age. This unexpected finding means, at least in mice, that the hepatic P450 system in adults is not essential for life and furthermore accentuates its fundamental role in providing protection from toxic environmental agents. Currently, the hepatic metabolism of endogenous molecules such as steroid hormones does not appear to be of major significance in endogenous hormone homeostasis. However, the P450 system does appear to play a major role in regulating lipid homeostasis and hepatic lipid levels.

    ACKNOWLEDGEMENTS

We thank Steve Wilson and Mary Ann Haskins (Cancer Research UK Transgenic Services, Clare Hall, Hertfordshire) for transfection of ES cells and generation of chimeric mice.

    FOOTNOTES

* This work was supported by Cancer Research UK.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger These authors contributed equally to the work.

§ Present address: Dept. of Molecular Physiology and Biophysics, Vanderbilt School of Medicine, 702 Light Hall, Nashville, TN 37232. E-mail: mark.magnuson@vanderbilt.edu.

Present address: Cancer Research UK Transgenic Services, Clare Hall Laboratories, Blanche Lane, South Mimms, Potters Bar, Herts, EN6 3LD, UK. E-mail: ian.rosewell@cancer.org.uk.

|| To whom correspondence should be addressed. Tel.: 01382-632621; Fax: 01382-669993; E-mail: roland.wolf@cancer.org.uk.

Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M212087200

    ABBREVIATIONS

The abbreviations used are: CYP, cytochrome P450; ALT, alanine aminotransferase; CMV, cytomegalovirus; CPR, NADPH; ferrihemoprotein reductase, ES, embryonic stem; hsv-tk, herpes simplex virus-thymidine kinase; i.p., intraperitoneal; neo, neomycin; pfu, plaque-forming units; SREBP, sterol regulatory element-binding protein; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Wolf, C. R. (1986) Trends Genet. 2, 209-214[CrossRef]
2. Raucy, J. L., and Allen, S. W. (2001) Pharmacogen. J. 1, 178-186
3. Nelson, D. R., Koymans, L., Kamataki, T., Stegeman, J. J., Feyereisen, R., Waxman, D. J., Waterman, M. R., Gotoh, O., Coon, M. J., Estabrook, R. W., Gunsalus, I. C., and Nebert, D. W. (1996) Pharmacogenetics 6, 1-42[Medline] [Order article via Infotrieve]
4. Pikuleva, I., and Waterman, M. (1999) Mol. Aspects Med. 20, 33-42[Medline] [Order article via Infotrieve],43-37
5. Omura, T. (1999) Biochem. Biophys. Res. Commun. 266, 690-698[CrossRef][Medline] [Order article via Infotrieve]
6. Goeptar, A. R., Scheerens, H., and Vermeulen, N. P. (1995) Crit. Rev. Toxicol. 25, 25-65[Medline] [Order article via Infotrieve]
7. Smith, G. C., Tew, D. G., and Wolf, C. R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8710-8714[Abstract]
8. Shen, A. L., O'Leary, K. A., and Kasper, C. B. (2002) J. Biol. Chem. 277, 6536-6541[Abstract/Free Full Text]
9. Le, Y., and Sauer, B. (2001) Mol. Biotechnol. 17, 269-275[Medline] [Order article via Infotrieve]
10. Postic, C., Shiota, M., Niswender, K. D., Jetton, T. L., Chen, Y., Moates, J. M., Shelton, K. D., Lindner, J., Cherrington, A. D., and Magnuson, M. A. (1999) J. Biol. Chem. 274, 305-315[Abstract/Free Full Text]
11. Brochet, D., Chermat, R., DeFeudis, F. V., and Drieu, K. (1999) Gen. Pharmacol. 33, 249-256[CrossRef][Medline] [Order article via Infotrieve]
12. Meehan, R. R., Forrester, L. M., Stevenson, K., Hastie, N. D., Buchmann, A., Kunz, H. W., and Wolf, C. R. (1988) Biochem. J. 254, 789-797[Medline] [Order article via Infotrieve]
13. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
14. Henderson, C. J., and Wolf, C. R. (1992) in Immunochemical Protocols (Manson, M. M., ed), Vol. 10 , pp. 221-234, Humana Press, Totowa, NJ
15. Forrester, L. M., Henderson, C. J., Glancey, M. J., Back, D. J., Park, B. K., Ball, S. E., Kitteringham, N. R., McLaren, A. W., Miles, J. S., Skett, P., and Wolf, C. R. (1992) Biochem. J. 281, 359-368[Medline] [Order article via Infotrieve]
16. Sen, C. K., Marin, E., Kretzschmar, M., and Hanninen, O. (1992) J. Appl. Physiol. 73, 1265-1272[Abstract/Free Full Text]
17. Henderson, C. J., Scott, A. R., Yang, C. S., and Wolf, C. R. (1990) Biochem. J. 266, 675-681[Medline] [Order article via Infotrieve]
18. Lamb, D. C., Kelly, D. E., Manning, N. J., Kaderbhai, M. A., and Kelly, S. L. (1999) FEBS Lett. 462, 283-288[CrossRef][Medline] [Order article via Infotrieve]
19. Oinonen, T., and Lindros, K. O. (1998) Biochem. J. 329, 17-35[Medline] [Order article via Infotrieve]
20. Wolf, C. R., Moll, E., Friedberg, T., Oesch, F., Buchmann, A., Kuhlmann, W. D., and Kunz, H. W. (1984) Carcinogenesis 5, 993-1001[Abstract]
21. Modi, S., Lian, L. Y., Roberts, G. C., Smith, G. C., Paine, M., and Wolf, C. R. (1995) Biochem. Soc. Trans. 23, 476S[Medline] [Order article via Infotrieve]
22. Voice, M. W., Zhang, Y., Wolf, C. R., Burchell, B., and Friedberg, T. (1999) Arch. Biochem. Biophys. 366, 116-124[CrossRef][Medline] [Order article via Infotrieve]
23. Stromstedt, M., Rozman, D., and Waterman, M. R. (1996) Arch. Biochem. Biophys. 329, 73-81[CrossRef][Medline] [Order article via Infotrieve]
24. Kelly, S. L., Lamb, D. C., Cannieux, M., Greetham, D., Jackson, C. J., Marczylo, T., Ugochukwu, C., and Kelly, D. E. (2001) Biochem. Soc. Trans. 29, 122-128[CrossRef][Medline] [Order article via Infotrieve]
25. Schwarz, M., Russell, D. W., Dietschy, J. M., and Turley, S. D. (1998) J. Lipid Res. 39, 1833-1843[Abstract/Free Full Text]
26. Ishibashi, S., Schwarz, M., Frykman, P. K., Herz, J., and Russell, D. W. (1996) J. Biol. Chem. 271, 18017-18023[Abstract/Free Full Text]
27. Schwarz, M., Lund, E. G., Setchell, K. D., Kayden, H. J., Zerwekh, J. E., Bjorkhem, I., Herz, J., and Russell, D. W. (1996) J. Biol. Chem. 271, 18024-18031[Abstract/Free Full Text]
28. Brown, M. S., and Goldstein, J. L. (1997) Cell 89, 331-340[Medline] [Order article via Infotrieve]
29. Shimano, H., Horton, J. D., Hammer, R. E., Shimomura, I., Brown, M. S., and Goldstein, J. L. (1996) J. Clin. Invest. 98, 1575-1584[Abstract/Free Full Text]
30. Hayhurst, G. P., Lee, Y. H., Lambert, G., Ward, J. M., and Gonzalez, F. J. (2001) Mol. Cell. Biol. 21, 1393-1403[Abstract/Free Full Text]
31. Jover, R., Bort, R., Gomez-Lechon, M. J., and Castell, J. V. (2001) Hepatology 33, 668-675[CrossRef][Medline] [Order article via Infotrieve]
32. Cairns, W., Smith, C. A., McLaren, A. W., and Wolf, C. R. (1996) J. Biol. Chem. 271, 25269-25276[Abstract/Free Full Text]
33. Spady, D. K., and Dietschy, J. M. (1983) J. Lipid Res. 24, 303-315[Abstract]
34. Repa, J. J., and Mangelsdorf, D. J. (2000) Annu. Rev. Cell Dev. Biol. 16, 459-481[CrossRef][Medline] [Order article via Infotrieve]
35. Dietschy, J. M., Spady, D. K., and Stange, E. F. (1983) Biochem. Soc. Trans. 11, 639-641[Medline] [Order article via Infotrieve]
36. Carter, C. P., Howles, P. N., and Hui, D. Y. (1997) J. Nutr. 127, 1344-1348[Abstract/Free Full Text]
37. Bosner, M. S., Lange, L. G., Stenson, W. F., and Ostlund, R. E., Jr. (1999) J. Lipid Res. 40, 302-308[Abstract/Free Full Text]
38. Kang, S., and Davis, R. A. (2000) Biochim. Biophys. Acta 1529, 223-230[Medline] [Order article via Infotrieve]
39. Repa, J. J., Lund, E. G., Horton, J. D., Leitersdorf, E., Russell, D. W., Dietschy, J. M., and Turley, S. D. (2000) J. Biol. Chem. 275, 39685-39692[Abstract/Free Full Text]
40. Li-Hawkins, J., Lund, E. G., Turley, S. D., and Russell, D. W. (2000) J. Biol. Chem. 275, 16536-16542[Abstract/Free Full Text]
41. Simpson, A. E. (1997) Gen. Pharmacol. 28, 351-359[CrossRef][Medline] [Order article via Infotrieve]
42. Tonge, R. P., Kelly, E. J., Bruschi, S. A., Kalhorn, T., Eaton, D. L., Nebert, D. W., and Nelson, S. D. (1998) Toxicol. Appl. Pharmacol. 153, 102-108[CrossRef][Medline] [Order article via Infotrieve]
43. Lee, S. S. T., Buters, J. T. M., Oineau, T., Fernandez-Salguero, P., and Gonzalez, F. J. (1996) J. Biol. Chem. 271, 12063-12067[Abstract/Free Full Text]
44. Zhang, J., Huang, W., Chua, S. S., Wei, P., and Moore, D. D. (2002) Science 298, 422-424[Abstract/Free Full Text]
45. Coles, B., Wilson, I., Wardman, P., Hinson, J. A., Nelson, S. D., and Ketterer, B. (1988) Arch. Biochem. Biophys. 264, 253-260[Medline] [Order article via Infotrieve]
46. Henderson, C. J., Wolf, C. R., Kitteringham, N., Powell, H., Otto, D., and Park, B. K. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 12741-12745[Abstract/Free Full Text]
47. Hines, R. N., Luo, Z., Cresteil, T., Ding, X., Prough, R. A., Fitzpatrick, J. L., Ripp, S. L., Falkner, K. C., Ge, N. L., Levine, A., and Elferink, C. J. (2001) Drug Metab. Dispos. 29, 623-633[Abstract/Free Full Text]
48. Conney, A. H. (1982) Cancer Res. 42, 4875-4917[Medline] [Order article via Infotrieve]
49. Sundseth, S. S., Alberta, J. A., and Waxman, D. J. (1992) J. Biol. Chem. 267, 3907-3914[Abstract/Free Full Text]
50. Shapiro, B. H., MacLeod, J. N., Pampori, N. A., Morrissey, J. J., Lapenson, D. P., and Waxman, D. J. (1989) Endocrinology 125, 2935-2944[Abstract]
51. Tzameli, I., Pissios, P., Schuetz, E. G., and Moore, D. D. (2000) Mol. Cell. Biol. 20, 2951-2958[Abstract/Free Full Text]
52. Smith, G., Henderson, C. J., Parker, M. G., White, R., Bars, R. G., and Wolf, C. R. (1993) Biochem. J. 289, 807-813[Medline] [Order article via Infotrieve]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.