(Received for publication, January 29, 1996)
From the
CYP2E1, a cytochrome P-450 that is well conserved across mammalian species, metabolizes ethanol and many low molecular weight toxins and cancer suspect agents. The cyp2e1 gene was isolated, and a mouse line that lacks expression of CYP2E1 was generated by homologous recombination in embryonic stem cells. Animals deficient in expression of the enzyme were fertile, developed normally, and exhibited no obvious phenotypic abnormalities, thus indicating that CYP2E1 has no critical role in mammalian development and physiology in the absence of external stimuli. When cyp2e1 knockout mice were challenged with the common analgesic acetaminophen, they were found to be considerably less sensitive to its hepatotoxic effects than wild-type animals, indicating that this P-450 is the principal enzyme responsible for the metabolic conversion of the drug to its active hepatotoxic metabolite.
Cytochromes P-450 (P-450) ()are a superfamily of
hemoproteins that carry out oxidative metabolism of many endogenous and
foreign chemicals(1) . In mammals, P-450s can be functionally
segregated into two groups, those that participate in biochemical
pathways leading to the synthesis of steroid hormones and those that
primarily metabolize foreign chemicals or xenobiotics such as drugs.
The latter enzymes are included in the CYP1, CYP2, CYP3, and CYP4
families(2) . Many of the hepatic xenobiotic-metabolizing
P-450s also metabolize endogenous compounds, but the significance of
these reactions is questionable. A clue to the lack of a critical role
for many of the P-450s, particularly those in family 2, in development,
reproduction, and longevity, is the marked species differences in their
expression and catalytic activities(3) . However, some of the
xenobiotic-metabolizing P-450s are well conserved, including those in
the CYP1 family and CYP2E1, suggesting that they may perform an
important physiological function.
CYP2E1 is the principal P-450 responsible for the metabolism of ethanol and is considered as a major component of the microsomal ethanol-oxidizing system(4, 5) . Among xenobiotics metabolized by CYP2E1 are acetaldehyde, acetaminophen, acrylamide, aniline, benzene, butanol, carbon tetrachloride, diethylether, dimethyl sulfoxide, ethyl carbamate, ethylene chloride, halothane, glycerol, ethylene glycol, N-nitrosodimethylamine, 4-nitrophenol, pyrazole, pyridine, and vinyl chloride(6) . Many of these chemicals are known toxins, established chemical carcinogens, or suspected carcinogens. CYP2E1-mediated oxidation of a variety of substrates is also believed to liberate a substantial amount of reactive oxygen that can lead to membrane lipid peroxidation and cell toxicity(7) .
CYP2E1 is also capable of metabolizing endogenous chemicals including acetone and acetol, which are key metabolites in the methylglyoxal and propanediol pathways of gluconeogenesis(8, 9) . CYP2E1 can also carry out the metabolism of arachidonic acid, resulting in the production of several hydroxyeicosatetraenoic acids(10) , some of which may have physiological and pharmacological properties(11) .
CYP2E1 is inducible by ethanol and other low molecular weight substrates(5, 12) . This induction is primarily due to a postranscriptional mechanism where presence of the substrate stabilizes the enzyme from degradation(13) . However, transcriptional mechanisms have not been ruled out(14) . This enzyme is also induced by starvation and in uncontrolled diabetes(15, 16) .
P-450s have been implicated in the hepatotoxicity of acetaminophen (also called paracetamol), an over-the-counter analgesic and antipyretic that is commonly used worldwide as a substitute for acetylsalicylic acid (aspirin®) due to its lack of gastric ulceration and general low toxicity when used within the recommended dose range (17, 18, 19) . Acetaminophen causes hepatotoxicity at a low frequency. It is metabolized to N-acetyl-p-benzoquinoneimine, a metabolite that is capable of reacting with cellular nucleophiles. The bulk of this metabolite is either reduced back to acetaminophen or conjugated with glutathione. It was postulated that toxicity results from low cellular glutathione leaving an excess of active metabolite that can cause cell toxicity(19, 20, 21, 22) .
The
P-450s responsible for acetaminophen activation have been investigated.
Ethanol was reported to increase the toxicity of acetaminophen in
mice(20, 23) , thus suggesting the involvement of
CYP2E1 in vivo. In vitro studies have also implicated
human CYP1A2 in addition to CYP2E1 in acetaminophen metabolism,
although the latter P-450 had a lower Kthan CYP1A2(24, 25) .
The conservation across species in expression and catalytic activities of CYP2E1 and its ability to metabolize and be induced by chemicals that are generated endogenously, such as acetone and ethanol, suggests that it has an important physiological role in mammals. To investigate this possibility and to determine if this P-450 is involved in the hepatotoxicities and carcinogenesis potential of many of its substrates, mice lacking CYP2E1 expression were produced and characterized.
Figure 1: Panel A displays the restriction map of the cyp2e1 gene, the targeting vector, and the predicted homologous recombinant locus. The numbers over the horizontal double arrows are the predicted sizes of restriction fragments in kb. Panel B shows a Southern blot of the specific ES clone and wild-type ES cells, and panel C displays a Southern blot of a typical screen of tail clipping DNA from mice with different genotypes. The sizes of the fragments are in kb.
The construct was made in six cloning steps (see Fig. 1A). 1) The HindIII site in the polylinker region of pGEM-3Z (Promega) was destroyed by HindIII digestion, Klenow polymerase treatment, and religation. 2) An 8-kb SalI-SmaI cyp2e1 genomic fragment was subcloned into the same sites in the modified pGEM-3Z. 3) The plasmid made in step 2 was digested with HindIII, treated with Klenow polymerase, and ligated with XhoI linkers in order to remove the 1.8-kb fragment containing exon 2 and add a restriction site compatible with the PGK-NEO cassette. This 1.9-kb cassette was previously modified by changing the BamHI site at its 3` end to an XhoI site by use of Klenow polymerase and XhoI linkers. 4) The XhoI fragment containing the PGK-NEO cassette was subcloned into the cyp2e1 gene at the XhoI site. 5) The cyp2e1 construct, containing the PGK-NEO cassette was digested with AflII, treated with Klenow polymerase, and ligated with HindIII linkers. 6) The cyp2e1 gene was released from this construct by digestion with SalI and HindIII and inserted into the corresponding sites of pMC1TK plasmid (29) containing the herpes simplex virus thymidine kinase gene. The resulting plasmid was used as a targeting vector.
Messenger RNA was analyzed by
Northern blots using liver RNA and the rat CYP2E1 cDNA as a probe.
Total RNA was isolated from liver tissue using guanidinium thiocyanate
extraction (40) and cesium trifluoroacetic acid centrifugation
as described previously(31) . Ten µg of total RNA was
subjected to electrophoresis on 1% agarose gels containing 2.2 M formaldehyde (41) and blotted to GeneScreen Plus (DuPont)
nylon membranes using 3 M NaCl and 0.15 M sodium
citrate, pH 7.0. The CYP2E1 cDNA was labeled using random primers and
[P]dCTP. The conditions for prehybridization,
hybridization, and washing were described previously(31) .
Mice
homozygous for the disrupted allele, designated
cyp2e1, were born normally and appeared
indistinguishable from their wild-type counterparts. No differences
were found between litter size and growth rates for the
cyp2e1
animals as compared with wild-type
littermate controls. The expression of CYP2E1 was determined by
immunoblotting with anti-rat CYP2E1 antibody. As expected, a complete
absence of protein expression was found in the livers of
cyp2e1
mice (Fig. 2). The liver is the
primary site of expression of this P-450(16) . P-450s in the
CYP1A, CYP2A, CYP2B, CYP2C, and CYP3A subfamilies were expressed in the
cyp2e1
mice at similar levels to those found
in control animals, thus indicating that the loss of CYP2E1 was not
compensated by an increase in expression of other P-450s, although it
remains a possibility that a P-450 not detected with our anti-rat P-450
antibodies is overexpressed.
Figure 2:
Western
immunoblots of different P-450s in cyp2e1 mice. Each lane was loaded with 10 µg of microsomal protein
from a single mouse.
The expression of CYP2E1 mRNA was also
analyzed in the cyp2e1 mice. Two transcripts
were detected in the liver of normal mice and mice heterozygous for the
disrupted allele (Fig. 3). In the cyp2e1
mice, neither of these two RNA transcripts were found. Instead,
two lower abundance RNAs slightly smaller than the transcripts present
in wild-type animals were detected. These may be transcripts from the
disrupted allele that should be smaller than a transcript from the
normal allele since exon 2 is deleted in the disrupted allele. The
lower abundance of these RNAs, as compared with those from the
wild-type allele, is not surprising since mRNAs that do not encode a
normal protein are usually not stable. In any case, the protein and RNA
establish with certainty that the cyp2e1 gene is not expressed
in the knockout animals. The change in size and abundance of the high
molecular weight transcript annealing with the CYP2E1 cDNA in the
cyp2e1
mice suggest that it is not due to a
cross-hybridizing mRNA derived from another gene but is most likely a
read-through transcript of the cyp2e1 gene with an alternate
polyadenylation signal.
Figure 3:
Analysis of RNA in livers of
cyp2e1 mice. Each lane was loaded with 10
µg of total liver RNA from a single mouse. The blot was exposed for
24 h with aid of an intensifying screen.
Figure 4:
Survival rate of cyp2e1-/-
() and wild-type (
) mice as a function of the dose of
acetaminophen administered. Groups of 10 mice were injected
intraperitoneally with acetaminophen in alkaline saline and survival
scored after 48 h. Two complete and independent experiments were
performed. The curves were manually fit to the data
points.
To determine the mechanism of
toxicity, levels of enzymes and other serum components, some of which
are diagnostic for liver and kidney injury, were measured in serum of
treated mice that survived in the experiments described above. At all
doses, levels of creatinine, bilirubin, and alkaline phosphatase were
within the normal range for mice and were not significantly different
between the cyp2e1 and wild-type mice. In
contrast, liver enzymes aspartate aminotransferase and alanine
aminotransferase were elevated at high doses of acetaminophen (Fig. 5). Elevation of these liver enzymes, which are considered
a measure of liver cell death, were detected at doses of 200 and 400
mg/kg in wild-type animals but were unchanged at these doses in the
cyp2e1
mice. These data indicate that liver
damage is involved in mediating the toxicity of acetaminophen. This was
confirmed by analysis of liver histology of acetaminophen-treated mice
(data not shown). At doses higher than 600 mg/kg, a significant level
of toxicity was also found in the cyp2e1
mice. These data suggest that CYP2E1 mediates the hepatotoxicity
of acetaminophen. Other P-450s such as CYP1A2 having a higher K
for acetaminophen may be responsible for the
toxicity in cyp2e1
mice at high doses of the
drug.
Figure 5:
Determinations of aspartate
aminotransferase (panel A) and alanine aminotransferase (panel B) activities in serum of cyp2e1 (
) and wild-type (
) mice as a function of the dose of
acetaminophen administered. The mean + standard deviations are
shown with n = 3 (* p < 0.05,** p < 0.01,*** p < 0.001). At the 600 mg/kg dose group
for the wild-type mice in panel B, two animals were
analyzed.
The present study using mice lacking expression of CYP2E1 establish that although this P-450 is highly conserved in mammals, it does not appear to play a significant role in development, reproductive vitality, and physiology. Under conditions of exposure to certain chemicals, CYP2E1 accentuates toxicity. Efforts are underway to use this animal model to determine whether this enzyme is responsible for the carcinogenicity of a number of its chemical substrates including N-nitrosodimethylamine and phenacetin.
CYP2E1 may also
exert a role in alcoholic liver disease. Lipid peroxidation was found
to be associated with alcoholic liver injury in humans and experiment
animals(42) . This could be the result, in part, of increased
oxygen radical production by ethanol-induced CYP2E1(7) . The
cyp2e1 mice could be used to test this
possibility.
During fasting and diabetic ketosis, serum acetone,
acetol, and 1,2-propanediol are elevated. CYP2E1 is concommitantly
induced due to protein stabilization by acetone(16) . Acetone
is primarily oxidized to acetol by CYP2E1. Acetol is further
metabolized to 1,2-propanediol by the same P-450 in a pathway of
gluconeogenesis, suggesting a physiological role for this P-450 during
pathophysiological and dietary stress(8) . The
cyp2e1 mice should be of use to determine if
CYP2E1 plays an essential role in survival under conditions of
starvation.