TOXICOLOGY AND APPLIED PHARMACOLOGY 153, 102–108 (1998)

ARTICLE NO. TO988543

Role of CYP1A2 in the Hepatotoxicity of Acetaminophen:

Investigations Using Cyp1a2 Null Mice
Robert P. Tonge,*,1 Edward J. Kelly,† Sam A. Bruschi,* Tom Kalhorn,‡ David L. Eaton,† Daniel W. Nebert,§ and
Sidney D. Nelson*,2
*Department of Medicinal Chemistry, †Department of Environmental Health, ‡Department of Pharmaceutics, University of Washington, Seattle, Washington
98195–7631; and §Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, Ohio 45267– 0056

Received April 30, 1998; accepted August 6, 1998

Key Words: acetaminophen; APAP; Cyp1a2; knockout mice;

Role of CYP1A2 in the Hepatotoxicity of Acetaminophen: In-
vestigations Using Cyp1a2 Null Mice. 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.
Acetaminophen (APAP) is known to cause centrilobular he-
patic necrosis under overdose conditions. This is thought to be
mediated via the P450-generated reactive intermediate
N-acetyl-p-benzoquinone imine (NAPQI). Initially, NAPQI is
detoxified by conjugation with glutathione (GSH), but once
GSH is depleted, NAPQI reacts more extensively with hepatic
proteins leading to hepatocellular damage. The P450 isoforms
thought to be responsible for APAP hepatotoxicity in humans
are CYP2E1, CYP1A2, and CYP3A4, and thus, we have inves-
tigated the effect of murine Cyp1a2 on APAP hepatotoxicity
using Cyp1a2 knockout mice (Liang et al., Proc. Natl. Acad.
Sci. USA 93, 1671–1676, 1996). Doses of 250 mg/kg were mark-
edly hepatotoxic in these mice, and surprisingly, deaths only
occurred in the knock-out and heterozygote mice over a 24-h
period after dosing. Furthermore, there were no significant
differences among survivors of any genotype in serum ALT
concentrations, a well correlated indicator of APAP hepatotox-
icity in mice. Finally, no differences were observed in the
urinary metabolites excreted over the 24-h period, including
those derived from GSH conjugation of the major reactive
metabolite NAPQI. Consistent with the effects on hepatotoxic-
ity and metabolism, 2 h after hepatotoxic doses (500 mg/kg, ip)
of APAP no significant differences were observed in total whole
liver homogenate nonprotein thiol concentrations among the
three genotypes even though hepatic thiols were decreased
compared to control animals (>90%). In addition, when the
liver cytosol and microsome samples were examined by immu-
noblotting for the presence of APAP-protein adducts using a
specific antiserum, there were no observable differences in ei-
ther the intensity of staining or in the spectrum of adducts
formed between APAP-dosed mice of any genotype. The cumu-
lative data suggest that Cyp1a2 does not play a significant role
in APAP hepatotoxicity in these mice. © 1998 Academic Press
1
Present address: Proteomics Group, I4.24 CTL, Zeneca Pharmaceuticals,
Alderley Park, Macclesfield, Cheshire, SK10 7TG, England, UK.
2
To whom correspondence should be addressed. Fax: (206) 685-9297;
E-mail: sidnels@u.washington.edu.
protein-adducts; hepatotoxicity.
Acetaminophen (APAP; paracetamol) is a widely used an-
algesic and antipyretic drug that is relatively safe at normal
therapeutic dose levels but can lead to severe centrilobular
hepatic necrosis in man (Davidson and Eastham, 1966) and
experimental animals (Boyd and Bereczky, 1966) following
overdose. This toxicity is thought to be mediated via the
cytochrome P450-generated electrophilic intermediate
N-acetyl-p-benzoquinone imine (NAPQI) (Dahlin et al., 1984)
which, under normal dose conditions, is detoxified by conju-
gation with glutathione (GSH). However, in overdose situa-
tions cellular GSH is depleted to an extent where further
NAPQI production leads to hepatocellular protein adduction
and toxicity (Jollow et al., 1973; Mitchell et al., 1973).
The main P450 isoforms currently thought to be responsible
for APAP bioactivation and, thus, hepatotoxicity are CYP2E1,
CYP1A2, and CYP3A4 (Raucy et al., 1989; Patten et al., 1993;
Thummel et al., 1993; Nelson et al., 1995). An exciting new
approach is now possible to evaluate the relative roles played
by each isoform further, i.e., using specific P450 knockout
mice. The effect of mouse Cyp2e1 genotype on APAP toxicity
has already been evaluated, and the results suggested that
Cyp2e1 is the major P450 in mice responsible for hepatotox-
icity caused by APAP, although other P450 isoforms may also
contribute (Lee et al., 1996).
Here we report our investigations using Cyp1a2 knockout
mice. These animals have already been shown to metabolize
the muscle relaxant zoxazolamine in a Cyp1a2-dependent fash-
ion (Liang et al., 1996). In addition, these Cyp1a2 null mice
have been used to demonstrate the necessity of this isozyme in
the induction of murine uroporphyria (Sinclair et al., 1998) and
the unimportance of Cyp1a2 in Cyp1a1 expression (Liang et
al., 1997). Similar Cyp1a2 knockout mice (Pineau et al., 1995)
have been used to demonstrate the involvement of Cyp1a2 in
caffeine metabolism (Buters et al., 1996) and in the hepatic

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All rights of reproduction in any form reserved.
 ACETAMINOPHEN HEPATOTOXICITY IN Cyp1a2 NULL MICE
sequestering of 2,3,7,8-tetrachlorodibenzo-p-dioxin and
2,3,4,7,8-pentachlorodibenzofuran (Diliberto et al., 1997).
Following APAP exposure, toxicity was evaluated in each
Cyp1a2 genotype by estimating nonprotein thiol depletion and
APAP-covalent binding in the liver along with serum alanine
aminotransferase (ALT) activity. APAP-covalent binding was
determined immunochemically using a specific affinity puri-
fied anti(APAP) antiserum raised in our laboratory (Tonge et
al., 1997). Analysis of the urine for all major APAP metabo-
lites, including NAPQI-derived thioether metabolites as bio-
markers of NAPQI formation (Prescott et al., 1981; Sarich et
al., 1997), was also performed. Results of these studies indicate
that Cyp1a2 does not play a significant role in APAP hepato-
toxicity in mice in contrast to other P450 isoforms known to
more efficiently oxidize APAP to its toxic metabolite. These
data are consistent with studies in humans which have shown
that induction of CYP1A2 does not significantly increase the
clearance of APAP through the formation of NAPQI (Miners et
al., 1984; Sarich et al., 1997).
MATERIALS AND METHODS
Chemicals. All chemicals were from Sigma Chemical Co. (St. Louis, MO)
unless otherwise specified. Electrophoresis and Western blotting reagents were
from BioRad (Hercules, CA).
Animal husbandry and genotyping. The Cyp1a2 knockout mice were
propagated by interbreeding heterozygotes to allow littermate comparisons of
all genotypes. Animals were weaned at 3– 4 weeks of age and a small portion
(;1 cm) of tail was excised for genotyping. The tissue was digested overnight
at 37°C in 2 ml 10 mM Tris/HCl (pH 7.4) containing 1% SDS, 5 mM EDTA,
150 mM NaCl, and 200 mg/ml Proteinase K (Boehringer-Mannheim, Mann-
heim, Germany). Total nucleic acid (TNA) was precipitated by ethanol addi-
tion, resuspended in 10 mM Tris/HCl (pH 7.4) containing 1 mM EDTA, and
heated to 100°C to inactivate the Proteinase K. The TNA was then subjected
to PCR amplification utilizing Cyp1a2-specific oligonucleotide primers de-
scribed previously (Liang et al., 1996). The PCR reaction buffer contained 10
mM Tris/HCl (pH 8.4), 1.5 mM MgCl2, 50 mM KCl, 200 mM dG/A/T/C, 1
mM of each primer, and 1 unit of Taq polymerase (Boehringer Mannheim) in
a final volume of 30 ml. The samples were overlaid with a drop of paraffin oil,
and PCR was run in a thermal cycler (M. J. Research, Watertown, MA) using
standard conditions (94°C, 30 s; 62°C, 45 s; 72°C, 60 s repeated for 35 cycles).
The products were then analyzed by agarose gel electrophoresis and visualized
using ethidium bromide staining.
APAP toxicity. Male wild-type [Cyp1a2(1/1)], heterozygous
[Cyp1a2(1/2)], and knockout [Cyp1a2(2/2)] mice (2– 4 months old) were
housed in modified rat metabolism cages (Nalgene, Naperville, IL), 2 animals
of similar genotype per cage, and fasted overnight prior to dosing, APAP (25
mg/ml in phosphate-buffered saline, pH 7.4) was given at 250 or 500 mg/kg
(ip). Animals receiving 250 mg/kg (n 5 18) were killed by cervical dislocation
24 h after dosing, and blood, liver, and urine samples were taken. Urine was
collected over 50 mg ascorbic acid into an ice-cooled container to minimize
atmospheric oxidation of urinary metabolites. Residual urine remaining in the
bladder of the mice was taken and added to that in the metabolism cage. After
the experiment the metabolism cages were washed down with deionized water
to recover urine remaining on the walls or floor of the cage. Animals receiving
500 mg/kg (n 5 22) were killed 2 h after dosing, and liver samples were
collected. Serum was prepared from blood samples for ALT measurements.
Livers were weighed and homogenized into 5 ml ice cold homogenization
buffer (10 mM Tris–HCl (pH 7.4) containing 0.25 M sucrose, 1 mM EDTA,
0.5 g/L bovine serum albumin, 2.5 mg/L aprotinin, 2.5 mg/L antipain, 2.5
103
mg/L leupeptin, and 1 mM phenylmethanesulfonyl fluoride (PMSF)), and 0.6
ml of homogenate was frozen in liquid N2 for nonprotein thiol measurements.
The remaining liver homogenate was used to prepare cytosolic and microsomal
fractions by standard procedures (Fleischer and Kervina, 1974). Briefly, the
liver homogenate was centrifuged at 250g for 10 min. The pellet (unbroken
cells and connective tissue) was discarded and the supernatant was centrifuged
at 14,000g for 10 min. The resulting pellet (nuclei, plasma membrane, and
mitochondria) was discarded and the supernatant was centrifuged at 105,000g
for 60 min. The remaining supernatant (cytosol) was snap frozen in liquid N2,
and the pellet was resuspended in 10 ml homogenization buffer and centri-
fuged at 105,000g for 60 min. The supernatant was discarded, and the pellet
(microsomes) was resuspended in 1 ml homogenization buffer and snap frozen
in liquid N2. All centrifugation procedures were carried out at 4°C and all
samples were stored at 270°C prior to analysis.
Urine analysis. The urine samples, including cage washes, were lyophi-
lized to dryness and redissolved in 20 ml water. Any solid material was
removed from the sample by centrifugation (500g 10 min). Urine was hydro-
lyzed with b-glucuronidase/arylsulfatase (Helix Pomatia crude preparation) by
diluting 0.5 ml 1:1 with 200 mM sodium acetate buffer (pH 5) and incubating
overnight at 37°C with 40 ml enzyme. Both hydrolyzed and unhydrolyzed
samples were filtered and deproteinated by passage through a 10-kDa cutoff
membrane (Alltech, Deerfield, IL). Urinary metabolites were analyzed by
injection of 10 ml supernatant onto a HPLC [2 Waters 510 HPLC pumps,
Waters Automated Gradient Controller, Waters 484 Tunable Absorbance De-
tector (Waters Chromatography, Fairfax, VA), HP3393A Integrator (Hewlett
Packard, Wilmington, DE)] equipped with a C18 reversed-phase column (4.6 3
100 mm, 3 mm, 100 Å, Microsorb-MV, Rainin, Woburn, MA) and UV
absorbance monitored at 254 nm. Elution was achieved using the following
program: 0 min, 93% A, 7% B; 11 min, 93% A, 7% B; 20 min, 70% A, 30%
B; 25 min, 0% A, 100% B; 35 min, 93% A, 7% B (Buffer A, 20 mM
ammonium phosphate/0.1% acetic acid, pH 3.2; Buffer B, acetonitrile). Me-
tabolite identification was achieved by comparison of retention times with
those of authentic standards produced in our laboratory. Metabolite quantita-
tion was achieved by determining the relative UV response of each metabolite
to that of APAP and by then determining standard curves for APAP in our
HPLC method (Sarich et al., 1997). Typical chromatograms are presented in
Fig. 1.
Serum ALT estimation. ALT activity in 5 ml serum was determined using
standard commercial methods (Sigma Diagnostics, Procedure No. DG159-
UV).
Hepatic nonprotein thiol estimation. Protein was precipitated from 0.6 ml
whole liver homogenate by adding 0.6 ml 4% sulfosalicylic acid, vortexing,
and centrifuging for 10 min at 16,000g. Supernatant (0.2 ml) was mixed with
1.5 ml 0.1 mM Ellman’s reagent [5,59-dithio-bis(2-nitrobenzoic acid)] and
allowed to stand for 15 min. After this time, absorbance was read at 412 nm.
Quantitation was possible using a GSH standard curve.
Immunoblot analysis. SDS-PAGE was performed as described elsewhere
(Ausubel et al., 1993) using a discontinuous buffer system and minigels (5.5
cm, 4% stacking gel and 10% resolving gel) (Mini-Protean II, BioRad).
Sample (50 mg) was loaded into each sample well. After electrophoresis, the
proteins were transferred electrophoretically to nitrocellulose (2 h, 4°C, 100
V). APAP-modified proteins were detected by incubating the nitrocellulose at
4°C for 3 h sequentially with 1:200 diluted affinity-purified anti(APAP)
antiserum (Tonge et al., 1997) and 1:1000 diluted alkaline phosphatase-
conjugated anti-rabbit IgG (Sigma) using previously described techniques
(Kenna et al., 1987). Bound antibodies were visualized colorimetrically using
NBT/BCIP (Promega Co., Madison, WI) (Ausubel et al., 1993). Immunoblots
were digitized using a flat-bed scanner (300 d.p.i.), and densitometric analysis
was performed on a Macintosh computer using the public domain NIH Image
program (available at http://rsb.info.nih.gov/nih-image/).
Statistical analysis. Students t tests were carried out where appropriate on
a Macintosh computer with Statworks Version 1.2 (Cricket Software, Phila-
delphia, PA).
104 TONGE ET AL.

FIG. 2. Effect of mouse Cyp1a2 genotype on the urinary excretion of

APAP. Mice were dosed with 250 mg/kg APAP (ip) (n 5 18), and their urine
was collected for 24 h. Three urine samples (n 5 3) from each genotype were
analyzed by HPLC. Each urine sample was pooled from 2 animals.

dosing of the animals), there were no statistically significant

differences in serum ALT levels among genotypes. The mean
FIG. 1. Typical HPLC chromatograms of urinary metabolites of APAP. ALT levels were 15,804 6 5345 U/L (mean 6 SD, n 5 5),
Urine was analyzed undiluted (unhydrolyzed) or diluted (1:1) before analysis 14,485 6 1851 U/L (mean 6 SD, n 5 5), and 13,712 6 5254
(hydrolyzed). A, APAP glucuronide (4 min); B, 3-hydroxy APAP (7.8 min); C, U/L (mean 6 SD, n 5 4) for (1/1), (1/2), and (2/2) mice,
APAP sulfate (9.5 min); D, 3-(cystein-S-yl) APAP (APAP-Cys) (11.3 min); E, respectively. Serum obtained from 2 mice at the point of death
APAP (12.1 min); F, 3-methoxy APAP (16.7 min); G, 3-(N-acetyl cystein-S-
contained ALT activities among the highest measured (Fig. 3).
yl) APAP (APAP-NAC) (22.6 min).
Serum obtained from control mice contained no measurable
ALT activity.
RESULTS Hepatic nonprotein thiols. Following exposure of each
genotype to 500 mg/kg APAP for 2 h, there was a massive
Urine analysis. Mice were exposed to 250 mg/kg APAP decrease in hepatic nonprotein thiols (.90.1%) compared to
for 24 h because this was the maximum tolerated dose for this
duration in the pilot study. Despite this, 4 animals [2 (1/2)
and 2 (2/2)] died during the experiment between 6 and 11 h.
There were no statistically significant differences in any
APAP-derived urinary metabolite assayed between genotypes
(Fig. 2). The major APAP-derived urinary metabolite in each
genotype was APAP-glucuronide (53.1–59.1% total metabo-
lites), and the APAP-GSH derived metabolites (APAP-Cys and
APAP-NAC) varied between 19.2% and 21.4%. Unmetabo-
lized urinary APAP was between 7.8% and 10.8% of total
administered dose. Metabolite recovery for (1/1) mice was
16.2 6 3.4 g (95.3 6 19.8%), for (1/2) mice was 16.4 6 2.2 g
(88.2 6 11.8%), and for (2/2) mice was 19.1 6 2.9 g (101 6
15.5%) (mean 6 SD, n 5 3).
Serum ALT. The majority of mice exposed to APAP at 250
mg/kg for 24 h showed elevated serum ALT activities (Fig. 3) FIG. 3. Effect of mouse Cyp1a2 genotype on serum ALT activity follow-
although this effect was variable. Activities ranged from 453 to ing APAP exposure. Mice were dosed with 250 mg/kg APAP ip (n 5 18), and
a blood sample was taken after 24 h for ALT estimation. Figure shows ALT
22,034 U/L, 12,218 to 17,364 U/L, and 0 to 18,656 U/L for levels for individual animals. Black crosses represent animals that died during
(1/1), (1/2), and (2/2) mice, respectively. If the outlying the dose period from which serum could be obtained (n 5 2). Control animals
points at 0 and 453 U/L were disregarded (possibly due to low showed zero serum ALT activities (data not shown).
 ACETAMINOPHEN HEPATOTOXICITY IN Cyp1a2 NULL MICE 105

Cyp1a2 genotype had a negligible effect on the quantity of

either microsomal or cytosolic adducts formed (Fig. 4B). Fur-
ther, there were no observable differences in the spectrum of
proteins modified by APAP in any genotype (Fig. 5). The main
APAP-protein adducts had molecular weights of 108, 77, 50,
47, 39, and 34 kDa in cytosol and 142, 117, 105, 78, 73, 70,
and 38 kDa in microsomes. There were no antibody detectable
proteins in the control microsomal samples, but a minor, non-
specific, 34-kDa protein was detected in control cytosolic
samples (Fig. 5).

DISCUSSION

Previous investigations have suggested that the human P450

isoforms responsible for APAP bioactivation are CYP2E1,
CYP1A2, and CYP3A4 (Raucy et al., 1989; Patten et al., 1993;
Thummel et al., 1993; Nelson et al., 1995). However, this
investigation has demonstrated that Cyp1a2 plays a negligible
role in the hepatotoxicity of acetaminophen in the mouse under
the conditions used in this study.

FIG. 4. Effect of mouse Cyp1a2 genotype on hepatic nonprotein thiol

depletion (A) and on the formation of APAP-hepatic protein adducts (B)
following APAP exposure. Mice were dosed with 500 mg/kg APAP ip (n 5
22) and were killed 2 h later. Livers were removed, homogenized, and
analyzed for the presence of GSH. Cytosolic and microsomal fractions were
prepared from the liver homogenate and analyzed immunochemically for the
presence of APAP-protein adducts. The quantity of adducts was assessed by
the relative density of staining on immunoblots. Numbers in parentheses in A
represent numbers of animals in each group. *Significantly different than
(1/2) microsomes ( p , 0.05).

control mice (Fig. 4). Concentrations of nonprotein thiols per

gram wet wt liver in control tissues were 0.55 6 0.05 mM/g,
0.59 6 0.10 mM/g, and 0.54 6 0.15 mM/g for (1/1), (1/2),
and (2/2) mice, respectively. In comparison, those in APAP-
treated animals were 0.04 6 0.01 mM/g, 0.05 6 0.01 mM/g,
and 0.05 6 0.01 mM/g, respectively (mean 6 SD, see Fig. 4A
for number of replicates). There were no statistical differences
in nonprotein thiol levels between mice of any genotype either FIG. 5. Immunoblots showing APAP-protein adducts in cytosol and mi-
before or after APAP treatment (Fig. 4A). crosomes of control (C) and APAP-exposed mice of different Cyp1a2 geno-
type. The migration of prestained molecular weight standards are indicated on
Hepatic APAP-protein adducts. Immunochemical analy- the left of each panel, and the estimated molecular weight (kDa) of APAP-
ses for the presence of APAP-hepatic protein adducts in control modified immunoreactive proteins for all genotypes are shown on the right of
and APAP-treated mice (500 mg/kg 2 h) revealed that the each panel.
106 TONGE ET AL.
APAP hepatotoxicity has often been estimated by measuring
the levels of hepatic enzymes such as transaminases, and it is
generally accepted that such measurements are a reliable indi-
cator of the tissue damage (Placke et al., 1987; Pumford et al.,
1989; Roberts et al., 1991). The high serum ALT levels in mice
24 h following APAP exposure (250 mg/kg) clearly indicate
APAP-mediated hepatotoxicity in these animals, but this effect
was not dependent on Cyp1a2 genotype. This finding was
further substantiated by the similarity in urinary excretion of
the NAPQI-derived metabolites APAP-Cys and APAP-NAC
between different genotypes. Generally, APAP is primarily
excreted as the glucuronide and sulfate conjugates (Brodie and
Axelrod, 1948), but a proportion is oxidized to NAPQI in the
liver by cytochromes P450 (Mitchell et al., 1973a; Potter et al.,
1973; Dahlin et al., 1984). NAPQI is detoxified by conjugation
with GSH but can react extensively with cellular macromole-
cules when GSH is depleted. This leads to the characteristic
centrilobular necrosis seen in APAP hepatotoxicity (Jollow et
al., 1973; Mitchell et al., 1973b). APAP-GSH is subsequently
excreted in the bile (Hinson et al., 1982) and forms APAP-Cys
by the action of g-glutamyltranspeptidase and aminopeptidase.
APAP-Cys can be acetylated in the kidney by N-acetyl trans-
ferase to give the mercapturate, APAP-NAC. APAP-Cys and
APAP-NAC are detected in the urine following APAP expo-
sure (Jollow et al., 1974; Wilson et al., 1982) and are good
indicators of NAPQI formation (Prescott et al., 1981, 1983;
Sarich et al., 1997).
When administered at 500 mg/kg, APAP is a well docu-
mented hepatotoxin in mice (Jollow et al., 1973), and APAP-
covalent binding and GSH depletion are classical biomarkers
of APAP hepatotoxicity (Jollow et al., 1973; Mitchell et al.,
1973). For example, covalent binding and GSH depletion is
highest in species most susceptible to APAP hepatotoxicity
(Davis et al., 1974). In these studies Cyp1a2 genotype had no
effect on either of these parameters following APAP adminis-
tration, and thus, these findings also suggest that Cyp1a2 has
only a minor role in the bioactivation of APAP at concentra-
tions likely to be hepatotoxic.
Several groups have investigated and identified the forma-
tion of APAP-protein adducts in mice by immunochemical,
radiochemical, and fluorescent means. After doses of APAP in
vivo, the adducted proteins so far identified include a 56-kDa
selenium binding protein of unknown function (Bartolone et
al., 1992; Pumford et al., 1992), a 74-kDa nuclear lamin A
(Hong et al., 1994), a 44-kDa glutamine synthetase (Bulera et
al., 1995), a 50-kDa glutamate dehydrogenase (Halmes et al.,
1996), a 54-kDa aldehyde dehydrogenase (Landin et al., 1996),
a 100-kDa N10-formyl tetrahydrofolate dehydrogenase (Pum-
ford et al., 1997), a 38-kDa glyceraldehyde-3-phosphate dehy-
drogenase (Dietze et al., 1997), and a 130-kDa carbamyl
phosphate synthetase I (Gupta et al., 1997). Other workers
have demonstrated liver microsomal APAP-protein adducts of
25, 44, 52, 72, 76, and 105 kDa in CD1 mice (Bulera et al.,
1996) and went on to identify the 44-kDa adduct as glutamine
synthetase (Bulera et al., 1995). It is possible that our major
38-kDa adduct could be 44-kDa glutamine synthetase and that
the unidentified adducts that we observed at 70.5, 78, and 105
kDa could be similar to those seen at 72, 76, and 105 kDa in
CD1 mice.
The major APAP adducts seen in our cytosolic fractions
were at 39, 47, 50, 77, and 108 kDa. It is possible that the
observed cytosolic 39-kDa adduct is glyceraldehyde-3-phos-
phate dehydrogenase but the others remain unidentified at this
time. The cytosolic adduct pattern observed in these studies is,
however, very similar to that seen following the exposure of
male B6C3/F1 mice to [14C] APAP (400 mg/kg ip 2 h) by
phosphorimaging in our laboratory (Dietze et al., 1997). This
observation lends additional evidence in support of the speci-
ficity of our affinity-purified anti(APAP) antiserum (Tonge et
al., 1997).
Cyp2e1 knockout mice have been used to examine the role
played by Cyp2e1 in APAP activation (Lee et al., 1996), and
it was demonstrated that these animals were considerably less
sensitive to APAP-induced hepatotoxicity than wild-type ani-
mals. Minimal lethality was observed after 48 h to a dose of
400 mg/kg APAP in Cyp2e1(2/2) animals, whereas .50% of
the wild-type Cyp2e1 animals died at this dose level. When the
APAP dose was increased to .600 mg/kg, toxicity was also
observed in the Cyp2e1(2/2) mice. This suggests that al-
though Cyp2e1 primarily mediates APAP hepatotoxicity, other
P450s such as Cyp1a2, with a higher Km for APAP than
Cyp2e1, may mediate APAP toxicity at very high doses. Since
our animals exhibited marked hepatotoxicity and death at doses
well below 600 mg/kg, it was impossible to evaluate the effect
of Cyp1a2 genotype at such high doses. Such an evaluation
would be relevant only in a situation where Cyp2e1 was either
absent or inhibited.
The relevance of the information obtained at hepatotoxic
doses of APAP in animal model systems to humans will have
to await studies with human hepatocytes. Studies with human
liver microsomes (Raucy et al., 1989) and with transfected
HepG2 cells (Patten et al., 1993) suggest that both human
CYP2E1 and CYP1A2 are important for APAP oxidation at
high concentrations. In addition, studies with purified
CYP3A4, CYP2E1, and CYP2A6 (Thummel et al., 1993; Chen
et al., 1998) suggest that CYP3A4 is the major isoform cata-
lyzing APAP oxidation to NAPQI at therapeutic concentrations
(;0.1 mM). This is in contrast to the relative isozyme contri-
butions at higher APAP concentrations (1–10 mM), i.e.,
CYP2E1. CYP2A6. CYP1A2. Interestingly, induction of
CYP1A2 in humans by either cigarette smoking (Miners et al.,
1984) or by the widely used drug omeprazole (Sarich et al.,
1997) did not increase the formation of NAPQI after therapeu-
tic doses of APAP.
In conclusion, it would appear that Cyp1a2 does not play a
significant role in APAP hepatotoxicity in mice as long as other
P450s are present and that this is likely to be the case in
humans.
 ACETAMINOPHEN HEPATOTOXICITY IN Cyp1a2 NULL MICE
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health Grants GM25418
and GM32165 (S. D. Nelson), ES07033 (S. D. Nelson and D. L. Eaton), and
ESO6321 (D. W. Nebert).
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