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J. Biol. Chem., Vol. 278, Issue 15, 13480-13486, April 11, 2003
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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
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.
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.
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+/ Mouse Breeding and Maintenance--
CPR+/
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/
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/
Acetaminophen treatment-adult male CPRlox/ 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 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 Statistical Analysis--
Statistical analysis was carried out
using the Statview program (v4.5) for Macintosh, Abacus Concepts,
Berkeley, CA.
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+/ 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+/
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/
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 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/
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.
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 6
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).
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.
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/ 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/ 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 7 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.
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.
*
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.
§
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.
Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M212087200
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.
Inactivation of the Hepatic Cytochrome P450 System
by Conditional Deletion of Hepatic Cytochrome P450 Reductase*
,,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) 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.
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.
(CPRlox/ + CreALB) lines to generate liver-specific CPR conditional
knockout mice. The presence of the CreALB transgene was
determined by PCR (data not shown).
+ 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.
+ 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.
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).
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, 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 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.
) 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.
+ 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 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).
and 8-10
). 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 and 6 and 7 ) 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.
+ 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.
View larger version (57K):
[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.
-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 7
-hydroxylation of testosterone, catalyzed by P450s of
the CYP2A subfamily, was decreased by >99% in both males and females,
while 16-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.
Testosterone hydroxylation by hepatic microsomes from hepatic CPR-null
and wild-type mice
, 7, and 16 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).
View larger version (49K):
[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.
View larger version (15K):
[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.
+ 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
), tissue-specific deletion being
achieved by crossing in the CreALB transgene.
-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
HNF4 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
HNF4 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.
ACKNOWLEDGEMENTS
FOOTNOTES
These authors contributed equally to the work.
To whom correspondence should be addressed. Tel.:
01382-632621; Fax: 01382-669993; E-mail:
roland.wolf@cancer.org.uk.
ABBREVIATIONS
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 Y. Weng, C. Fang, R. J. Turesky, M. Behr, L. S. Kaminsky, and X. Ding Determination of the Role of Target Tissue Metabolism in Lung Carcinogenesis Using Conditional Cytochrome P450 Reductase-Null Mice Cancer Res., August 15, 2007; 67(16): 7825 - 7832. [Abstract] [Full Text] [PDF]
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 R. D. Finn, A. W. McLaren, D. Carrie, C. J. Henderson, and C. R. Wolf Conditional Deletion of Cytochrome P450 Oxidoreductase in the Liver and Gastrointestinal Tract: A New Model for Studying the Functions of the P450 System J. Pharmacol. Exp. Ther., July 1, 2007; 322(1): 40 - 47. [Abstract] [Full Text] [PDF]
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J. Gu, C.-S. Chen, Y. Wei, C. Fang, F. Xie, K. Kannan, W. Yang, D. J. Waxman, and X. Ding A Mouse Model with Liver-Specific Deletion and Global Suppression of the NADPH-Cytochrome P450 Reductase Gene: Characterization and Utility for in Vivo Studies of Cyclophosphamide Disposition J. Pharmacol. Exp. Ther., April 1, 2007; 321(1): 9 - 17. [Abstract] [Full Text] [PDF]
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 D. M. Mutch, V. Crespy, J. Clough, C. J. Henderson, S. Lariani, R. Mansourian, J. Moulin, C. R. Wolf, and G. Williamson Hepatic cytochrome P-450 reductase-null mice show reduced transcriptional response to quercetin and reveal physiological homeostasis between jejunum and liver Am J Physiol Gastrointest Liver Physiol, July 1, 2006; 291(1): G63 - G72. [Abstract] [Full Text] [PDF]
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Y. Weng, C. C. DiRusso, A. A. Reilly, P. N. Black, and X. Ding Hepatic Gene Expression Changes in Mouse Models with Liver-specific Deletion or Global Suppression of the NADPH-Cytochrome P450 Reductase Gene: MECHANISTIC IMPLICATIONS FOR THE REGULATION OF MICROSOMAL CYTOCHROME P450 AND THE FATTY LIVER PHENOTYPE J. Biol. Chem., September 9, 2005; 280(36): 31686 - 31698. [Abstract] [Full Text] [PDF]
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W. L. Miller Minireview: Regulation of Steroidogenesis by Electron Transfer Endocrinology, June 1, 2005; 146(6): 2544 - 2550. [Abstract] [Full Text] [PDF]
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G. J. Pass, D. Carrie, M. Boylan, S. Lorimore, E. Wright, B. Houston, C. J. Henderson, and C. R. Wolf Role of Hepatic Cytochrome P450s in the Pharmacokinetics and Toxicity of Cyclophosphamide: Studies with the Hepatic Cytochrome P450 Reductase Null Mouse Cancer Res., May 15, 2005; 65(10): 4211 - 4217. [Abstract] [Full Text] [PDF]
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 M. Liao, V. G. Zgoda, B. P. Murray, and M. A. Correia Vacuolar Degradation of Rat Liver CYP2B1 in Saccharomyces cerevisiae: Further Validation of the Yeast Model and Structural Implications for the Degradation of Mammalian Endoplasmic Reticulum P450 Proteins Mol. Pharmacol., May 1, 2005; 67(5): 1460 - 1469. [Abstract] [Full Text] [PDF]
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V. M. Arlt, M. Stiborova, C. J. Henderson, M. R. Osborne, C. A. Bieler, E. Frei, V. Martinek, B. Sopko, C. R. Wolf, H. H. Schmeiser, et al. Environmental Pollutant and Potent Mutagen 3-Nitrobenzanthrone Forms DNA Adducts after Reduction by NAD(P)H:Quinone Oxidoreductase and Conjugation by Acetyltransferases and Sulfotransferases in Human Hepatic Cytosols Cancer Res., April 1, 2005; 65(7): 2644 - 2652. [Abstract] [Full Text] [PDF]
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 M. P. Duarte, B. B. Palma, A. A. Gilep, A. Laires, J. S. Oliveira, S. A. Usanov, J. Rueff, and M. Kranendonk The stimulatory role of human cytochrome b5 in the bioactivation activities of human CYP1A2, 2A6 and 2E1: a new cell expression system to study cytochrome P450 mediated biotransformation Mutagenesis, March 1, 2005; 20(2): 93 - 100. [Abstract] [Full Text] [PDF]
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J. Gu, H. Cui, M. Behr, L. Zhang, Q.-Y. Zhang, W. Yang, J. A. Hinson, and X. Ding In Vivo Mechanisms of Tissue-Selective Drug Toxicity: Effects of Liver-Specific Knockout of the NADPH-Cytochrome P450 Reductase Gene on Acetaminophen Toxicity in Kidney, Lung, and Nasal Mucosa Mol. Pharmacol., March 1, 2005; 67(3): 623 - 630. [Abstract] [Full Text] [PDF]
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L. Wu, J. Gu, H. Cui, Q.-Y. Zhang, M. Behr, C. Fang, Y. Weng, K. Kluetzman, P. J. Swiatek, W. Yang, et al. Transgenic Mice with a Hypomorphic NADPH-Cytochrome P450 Reductase Gene: Effects on Development, Reproduction, and Microsomal Cytochrome P450 J. Pharmacol. Exp. Ther., January 1, 2005; 312(1): 35 - 43. [Abstract] [Full Text] [PDF]
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 C. J. Henderson and C. R. Wolf Transgenic Analysis of Human Drug-Metabolizing Enzymes: Preclinical Drug Development and Toxicology Mol. Interv., September 1, 2003; 3(6): 331 - 343. [Abstract] [Full Text] [PDF]
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J. Gu, Y. Weng, Q.-Y. Zhang, H. Cui, M. Behr, L. Wu, W. Yang, L. Zhang, and X. Ding Liver-specific Deletion of the NADPH-Cytochrome P450 Reductase Gene: IMPACT ON PLASMA CHOLESTEROL HOMEOSTASIS AND THE FUNCTION AND REGULATION OF MICROSOMAL CYTOCHROME P450 AND HEME OXYGENASE J. Biol. Chem., July 3, 2003; 278(28): 25895 - 25901. [Abstract] [Full Text] [PDF]
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