1 Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, and 2 Cell Biology and Anatomy, University of North Carolina, Chapel Hill 27599; 5 Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; 3 Department of Physiology and Pharmacology, Oregon Health Sciences University, Portland, Oregon 97201; and 4 Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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The continuous
intragastric enteral feeding protocol in the rat was a major
development in alcohol-induced liver injury (ALI) research. Much of
what has been learned to date involves inhibitors or nutritional
manipulations that may not be specific. Knockout technology avoids
these potential problems. Therefore, we used long-term
intragastric cannulation in mice to study early ALI. Reactive oxygen species are involved in mechanisms of early ALI; however, their key source remains unclear. Cytochrome
P-450 (CYP)2E1 is induced
predominantly in hepatocytes by ethanol and could be one source of
reactive oxygen species leading to liver injury. We aimed to determine
if CYP2E1 was involved in ALI by adapting the enteral alcohol (EA)
feeding model to CYP2E1 knockout (/
) mice. Female CYP2E1
wild-type (+/+) or
/
mice were given a high-fat liquid
diet with either ethanol or isocaloric maltose-dextrin as control
continuously for 4 wk. All mice gained weight steadily over 4 wk, and
there were no significant differences between groups. There were also
no differences in ethanol elimination rates between CYP2E1 +/+ and
/
mice after acute ethanol administration to naive mice
or mice receiving EA for 4 wk. However, EA stimulated rates 1.4-fold in
both groups. EA elevated serum aspartate aminotransferase levels
threefold to similar levels over control in both CYP2E1 +/+ and
/
mice. Liver histology was normal in control groups. In
contrast, mice given ethanol developed mild steatosis, slight inflammation, and necrosis; however, there were no differences between
the CYP2E1 +/+ and
/
groups. Chronic EA induced other CYP
families (CYP3A, CYP2A12, CYP1A, and CYP2B) to the same extent in
CYP2E1 +/+ and
/
mice. Furthermore, POBN
radical adducts were also similar in both groups. Data presented here
are consistent with the hypothesis that oxidants from CYP2E1 play only
a small role in mechanisms of early ALI in mice. Moreover, this new
mouse model illustrates the utility of knockout technology in ALI research.
CYP2E1 knockout mouse; intragastric feeding
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INTRODUCTION |
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THE ESTABLISHMENT OF A continuous intragastric enteral feeding protocol in the rat by Tsukamoto and French (32) was a major development in research in alcohol-induced liver injury. With this model, not only is steatosis observed, which is characteristic of several animal models, but inflammation and necrosis also occur in ~2-4 wk and fibrosis begins to develop in 8-10 wk. Inactivation of Kupffer cells with gadolinium chloride (GdCl3) diminished free radical formation (17) and prevented early alcohol-induced liver injury in the enteral model (1). Furthermore, intestinal sterilization with antibiotics (2) or lactobacillus feeding (24) diminished endotoxin and minimized liver injury. These results are consistent with the hypothesis that Kupffer cells activated by gut-derived endotoxin play an important role in the mechanism of early alcohol-induced liver injury.
Ethanol administration in vivo is associated with the formation of free radicals due to oxidant stress (17). Cytochrome P-450 (CYP)2E1 is induced predominantly in the hepatocyte by ethanol and could be a source of reactive oxygen species, leading to liver injury (26). French and his collaborators (3) have shown that a correlation between blood levels of alcohol and induction of CYP2E1 as alcohol cycles in the enteral model exists. Importantly, the level of CYP2E1 correlates with the degree of pathology, leading to the idea that it is involved in alcohol-induced liver disease. Moreover, inhibitors of CYP2E1 partially reduced hepatic pathology caused by enteral ethanol (3). Alternatively, NADPH oxidase in the Kupffer cell, which is activated by endotoxin, could also be a source of reactive oxygen species. Thus whether oxidant stress is from CYP2E1 in the hepatocyte or NADPH oxidase in the Kupffer cell remains unclear.
Recently, it was reported that CYP2E1 was induced similarly by ethanol and ethanol + GdCl3 in the enteral model; however, liver pathology was prevented by GdCl3 (19). These data suggested that oxidant stress from Kupffer cells rather than from CYP2E1 predominates in the initiation of alcohol-induced liver injury. However, much of what has been learned to date involves inhibitors or nutritional manipulations that may not be specific. Knockout technology could be useful to avoid these potential problems.
French et al. (35) delivered alcohol enterally to mice in his study
with Mallory bodies, demonstrating that successful surgery was possible
in the mouse; however, mortality was high due to drug priming.
Therefore, here we established a long-term intragastric feeding model
to study alcohol-induced liver injury in mice and adapted it to
knockout technology. The specific purpose of this study was to
determine the role of CYP2E1 in early alcohol-induced liver injury
using CYP2E1 knockout (/
) mice.
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MATERIALS AND METHODS |
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Animals.
Female CYP2E1 wild-type (+/+) or CYP2E1 /
(129/Sv ter
substrain) mice (18-20 g body wt) were obtained from a colony
maintained at the University of North Carolina at Chapel Hill. Animals
were housed in a facility approved by the American Association for Accreditation of Laboratory Animal Care. All animals received humane
care in compliance with institutional guidelines. Mice had free access
to a chow diet and water ad libitum before the study.
Operative procedures and gastric cannulation. Mice were fasted for 24 h before surgery. A 45-cm PE-90 polyethylene tube (Becton Dickinson, Sparks, MD) with a small silicon tip (1.5 mm) on one end was used for gastric cannulation using aseptic surgical techniques. Briefly, animals were anesthetized by inhalation of methoxyflurane and a vertical midline incision was made in the skin of the abdomen from the xiphoid cartilage extending to the midabdomen. A second small incision was made in the dorsal cervical area. A subcutaneous tunnel was exposed and 7-0 polypropylene sutures were passed 1-mm apart through the serosa and muscular layer of the stomach. After a small opening was made in the forestomach between sutures, the tip of the cannula was inserted 0.8 cm into the stomach. The tube was anchored to the stomach wall with Dacron felt, and the stomach was replaced in the abdominal cavity. The small incision where the cannula exits through the abdominal wall was closed with 5-0 silk and tied around the cannula, resulting in tight fixation of the cannula to the abdominal wall. The abdominal wall and skin were closed with 5-0 silk. The mouse was placed in a prone position, and an anchoring button was sutured to the muscles of the dorsal cervix. The skin was closed around the button stem with 5-0 silk. The total surgical procedure took ~30 min. Gentamicin (8 mg/kg) was administered intraperitoneally postsurgically. The cannula exited through a flanged button (Instech Laboratories, Plymouth Meeting, PA). A protective spring coil (Instech Laboratories), through which the cannula passes to the swivel (Lomir Biomedical Quebec), was connected to the anchoring button. The use of a spring coil and swivel protected the cannula and allowed free movement of animals in individual metabolic cages. The mouse was placed in a metabolic cage, and the end of the gastric cannula was connected to a swivel and infusion pump.
Diet. A liquid diet described first by Thompson and Reitz (29) supplemented with lipotropes as described by Morimoto et al. (23) was used. It contained corn oil as fat (37% of total calories), protein (23%), carbohydrate (5%), minerals, and vitamins, plus ethanol or isocaloric dextrin-maltose (control diet) as described elsewhere (31). Throughout the enteral feeding period, mice had free access to cellulose pellets as a source of fiber (Harlan Teklad, Madison, WI).
Experimental protocol.
Mice were randomly divided into two experimental groups and fed either
high-fat control or high-fat ethanol-containing diet continuously for
up to 4 wk via intragastric feeding. The diet (1.29-1.31 kcal/ml)
was infused at a rate of 0.44 ml · g body wt1 · day
1
with an infusion pump (Harvard Apparatus, Natic, MA). All animals received humane care in compliance with institutional guidelines, and
severe alcohol intoxication was assessed carefully to evaluate development of tolerance using a 0-3 scoring system (0 = normal, 1 = sluggish movement, 2 = loss of movement but still moving if stimulated, 3 = loss of consciousness). The amount of ethanol in the
diet was varied from 4.0% to 8.0% to obtain optimal delivery of
calories without compromising growth or survival. Ethanol initially was
delivered at 14 g · kg
1 · day
1
(27% of total calories) and was increased 1 g/kg for 2 days
until the end of the first week and then 1 g/kg for 4 days until the end of the experiment (final alcohol delivered = 28 g · kg
1 · day
1;
40% of total calories). Animals had free access to water.
Clinical chemistry.
Ethanol concentration in urine, which was shown to be representative of
blood alcohol levels in the rat (5), was measured daily. Mice were
housed in metabolic cages that separated urine from feces, and urine
was collected over 24 h in bottles containing mineral oil to prevent
evaporation. Each day at 9 AM, urine collection bottles were changed
and a 1-ml sample was stored at 20°C in a microtube for
later ethanol analysis. Ethanol concentration was determined by
measuring absorbance at 366 nm resulting from the reduction of
NAD+ to NADH by alcohol
dehydrogenase (8).
Pathological evaluation. After 4 wk of ethanol treatment, livers were formalin-fixed, embedded in paraffin, and stained with hematoxylin-eosin to assess steatosis, inflammation, and necrosis. Liver pathology was scored as described for the rat by Nanji et al. (25) as follows: for steatosis (the percentage of liver cells containing fat), <25% = 1+, <50% = 2+, <75% = 3+, >75% = 4+; for inflammation and necrosis at one focus per low-power field = 1+ and at two or more foci = 2+. Pathology was scored in a blinded manner by one of the authors and by an expert in rodent liver pathology.
Alcohol metabolism. In naive mice, ethanol (3 g/kg ip) was given acutely before experiments. In cannulated mice, after 4 wk, ethanol-containing diet was removed immediately before experiments (13). Mice were forced to breathe into a closed, heated chamber (37°C) for 15 s, and 1 ml of breath was collected with a gas-tight syringe. The concentration of ethanol in breath was determined by gas chromatography, and rates of alcohol metabolism were calculated from linear decreases in blood alcohol concentration per unit time using Widmark's formula as described elsewhere (13).
Western blotting for CYP2E1.
Mouse livers were immediately excised after death, placed into liquid
nitrogen, and stored at 70°C. Tissue was subsequently homogenized in three volumes of 10 mM Tris chloride buffer, pH 7.4, containing 150 mM potassium chloride, 1 mM EDTA, and 0.25 M sucrose
with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) and
centrifuged at 10,000 g for 30 min,
and then 9 µg of supernatant were immunoblotted after separation of
proteins on a 9% polyacrylamide gel. Electrophoretic transfer of
protein to nitrocellulose sheets was performed as previously described (27). Nitrocellulose sheets were blocked overnight in 3% (wt/vol) nonfat dry milk in Tris-buffered saline (10 mM Tris chloride, pH 7.4, with 150 mM NaCl) at 4°C. CYP2E1 was detected using sheep anti-rabbit CYP2E1 antiserum (1:5,000), with rabbit anti-sheep horseradish peroxidase (HRP, 1:5,000), using enhanced chemiluminescence (ECL) detection. The P-450 reductase
was a rabbit anti-pig IgG (1:2,500) provided by Dr. B. S. Masters
(University of Texas at San Antonio); detection with goat anti-rabbit
HRP and ECL detection followed. Staining intensity of the complex was
determined with a Bio-Rad GS-363 molecular imager (Bio-Rad, Richmond,
CA) with a CH-imaging screen and Bio-Rad molecular analyst software.
Measurement of P-450 subfamilies. Testosterone hydroxylation was assayed as described by Brunner et al. (9) except that 400 µg of the S9 fraction from liver homogenates were used in place of microsomes. The dealkylation of ethoxyresorufin and pentoxyresorufin was determined as described by Lubet et al. (21) and adapted to microtiter plates as described by Kennedy and Jones (16). Reactions in a final volume of 250 µl contained 5 µM alkylresorufin added in 5 µl of DMSO, 100 mM sodium phosphate buffer, pH 8.0, 125 µg of S9 protein, 5 µl of dicumarol, and 250 µM NADPH to initiate the reaction. Reactions were terminated by the addition of 100 µl of acetonitrile, and resorufin was determined spectrofluorometrically using a Fluorostar microplate reader with an excitation filter of 544 nm and an emmission filter of 590 nm.
Collection of bile and detection of free radicals.
Ethanol concentration in the breath was analyzed by gas chromatography
to verify that levels were between 200 and 250 mg/dl when experiments
were initiated (13). Mice were anesthetized with pentobarbital (75 mg/kg), the abdomen was opened, and the common bile duct was ligated
with a 8-0 polypropylene suture near the liver. After a small opening
was made in the fundus of the gallbladder, the tip of a 20-cm length of
PE-10 polyethylene tubing was inserted 3 mm into the gallbladder. The
opening in the gallbladder was closed with a 8-0 polypropylene suture
around the tube. Spin trap
[-(4-pyridyl-1-oxide)-N-tert-butylnitrone; 1 g/kg]
was administered intraperitoneally, and bile samples were collected for
3 h into 35 µl of 0.5 mM Desferal (deferoxamine mesylate) to prevent
ex vivo radical formation. During bile collection, 4 ml/kg of saline were injected at 30-min intervals to avoid dehydration and
~200-300 µl of bile samples were collected. Samples were
stored at
80°C until analysis of free radical adducts by
electron spin resonance (ESR) spectroscopy (17). Samples were thawed
and placed in a quartz flat cell, and ESR spectra were obtained using a
Varian E-109 spectrometer equipped with a TM110 cavity. Instrument
conditions were as follows: 20-mW microwave power, 1.0-G
modulation amplitude, 80-G scan width, 16-min scan, and 1-s time
constant. Data were collected with an IBM-type computer interfaced to a
spectrometer. Simulations and double integrations of spectra to
determine amplitude were carried out with a computer program (10).
Statistics. ANOVA or Student's t-test was used for determination of statistical significance as appropriate. For comparison of pathological scores, the Mann-Whitney rank sum test was used. A P value of <0.05 was selected before the study as the level of significance.
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RESULTS |
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Body weight gain.
All mice survived surgery, and diets were initiated after 1 wk to allow
for complete recovery from surgery. Steady weight gains were observed
during 4 wk of continuous enteral feeding of liquid diets with or
without ethanol, indicating adequate nutrition (Fig.
1). Similar weight gains occurred in adult
CYP2E1 +/+ or /
mice on chow diet (data not shown). There
were no significant differences in weight gains among the groups
studied (Fig. 1).
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Ethanol concentrations in urine and ethanol metabolism.
Representative plots of daily urine alcohol concentrations in mice
given ethanol are depicted in Fig. 2. As
reported previously by several groups using the Tsukamoto-French
enteral protocol in the rat (1, 30), urine alcohol levels in mice also
fluctuated in a cyclic pattern from zero to >500 mg/dl, even though
ethanol was infused continuously. Similar patterns were observed here in CYP2E1 +/+ and CYP2E1 /
mice. The reason for this
cyclic pattern is not understood, but, because it occurred in CYP2E1
/
mice, it was concluded that CYP2E1 is not involved in
this phenomenon in the mouse. Mean urine alcohol concentrations over 4 wk were 243 ± 17 mg/dl in CYP2E1 +/+ and 235 ± 22 mg/dl in CYP2E1
/
mice. There were no significant
differences between groups.
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Serum transaminase levels.
In control groups, serum AST levels were ~40 IU/l after 4 wk. Chronic
ethanol treatment significantly increased serum AST levels about
threefold over control values in both CYP2E1 +/+ and CYP2E1
/
mice (Fig. 3); however,
there were no significant differences between groups.
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Pathological evaluation.
After high-fat control diet for 4 wk, liver-to-body weight ratio was
~4%; although enteral ethanol feeding increased this ratio
significantly, it did so to the same extent in both groups (CYP2E1 +/+
mice, 6.3 ± 0.4; CYP2E1 /
mice, 6.5 ± 0.3).
Figure 4 shows representative
photomicrographs of livers from CYP2E1 +/+ and CYP2E1
/
mice after 4 wk of enteral alcohol feeding. In control groups,
histology was normal (Fig. 4, A and
B). CYP2E1 +/+ and CYP2E1
/
mice given ethanol developed mild steatosis in
pericentral to midzonal regions (Fig. 4,
C and
D), inflammation (Fig. 4,
E and
G), and necrosis (Fig. 4,
F and
H) after 4 wk, with total pathology
scores of 4.3 ± 0.8 and 4.2 ± 0.7, respectively. Chronic
enteral ethanol induced liver injury in both groups, and there were no
significant differences in hepatic pathology scores (Fig.
5) between the CYP2E1 +/+ and
/
groups. A macrovesicular pattern of fat accumulation
was observed mainly in the pericentral to midzonal regions
in both groups (Fig. 4), except for one to three layers of hepatocytes
around central veins. This phenomenon appears to be specific to the
mouse. Furthermore, infiltrating leukocytes observed in this study were
largely lymphocytes with macrophages. In addition, inflammatory foci
predominantly containing neutrophils with residual eosinophils were
also occasionally observed.
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Western blotting for CYP2E1.
P-450 reductase expression was
increased significantly by enteral ethanol in both CYP2E1 +/+ and
CYP2E1 /
mice (Fig. 6). CYP2E1 expression was minimal in the liver from CYP2E1 +/+ mice given
high-fat control diet without ethanol after 4 wk (Fig. 6). In contrast,
enteral ethanol increased CYP2E1 expression significantly. However,
CYP2E1 expression was not detected in livers from CYP2E1
/
mice fed high-fat liquid diets with or without ethanol
as expected (20).
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Effects of enteral ethanol on induction of
P-450 subfamilies.
The effect of ethanol treatment on several
P-450 subfamilies was assessed by
monitoring the hydroxylation of testosterone and the dealkylation of
7-ethoxyresorufin and 7-pentoxyresorufin (Table
1). For all of activities measured, there
were no significant differences between the CYP2E1 control +/+ and
control CYP2E1 /
mice, suggesting that the constitutive
loss of CYP2E1 did not significantly alter the expression of other
isoforms such as the CYP3A (testosterone 6
activity), CYP2A12
(testosterone 7
hydroxylase activity), CYP1A (ethoxyresorufin
dealkylation), or the CYP2B family (pentoxyresorufin dealkylation)
(14a). There was a great deal of variation in the expression levels of
the enzymes responsible for these activities, which was confirmed for
the CYP3A family by immunoblot analysis. In both strains, ethanol
treatment resulted in an induction of the CYP3A family. Testosterone
7
hydroxylation was also induced, as was the dealkylation of
pentoxyresorufin. In contrast, there was no induction of CYP1A in
either CYP2E1 +/+ or
/
animals with ethanol. Although the results of the current study clearly demonstrate that CYP2E1 is not
present or induced in the knockout mice, there is normal constitutive expression of other isoforms and ethanol treatment causes a variable induction of the CYP3A and CYP2B families.
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Effects of chronic enteral ethanol on free radical formation in
bile.
After 4 wk of high-fat control diet, free radical formation in bile was
minimal in both CYP2E1 +/+ and /
mice (data not shown).
In contrast, enteral ethanol for 4 wk increased free radical formation
about threefold but to the same extent in both groups (Fig.
7). There were no differences in radical
intensities between the groups. Thus CYP2E1 deficiency did not affect
radical formation caused by alcohol feeding in mice.
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DISCUSSION |
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Advantages of knockout technology for the study of alcohol-induced liver disease. Knockout technology, developed by Smithies (28), has distinct advantages. The advantage of knockout technology is that deletions or disruptions in DNA result in alterations that are specific. Specific genetic manipulation allows deletion of key enzymes or receptors so that potentially nonspecific agents can be avoided. Application of chronic enteral ethanol feeding to knockout mice provides a powerful tool for mechanistic studies in alcohol research in which interpretations in vivo may be complicated.
Application of a new enteral feeding model to alcohol-induced liver injury studies in mice. The new model described in this study was designed to provide enteral nutrition for long-term studies in mice. Surgery in the mouse is more difficult than the rat due to the smaller body size; however, postoperative survival rates of over 90% were achieved with care. Administration of antibiotics postsurgically and fasting for 24 h before surgery were important factors for success. Furthermore, it was also important that diets were initiated 1 wk after surgery to allow for full recovery from surgery for optimal survival.
The continuous intragastric infusion of a high-fat liquid diet with or without ethanol has been successfully achieved for periods of 28 days and could be easily extended. During continuous intragastric feeding, careful observations for intoxication and nursing care were required at least every 12 h for maximal success. Accordingly, animals given ethanol were observed frequently for signs of severe alcohol intoxication, i.e., sluggish movement, relaxation of legs, and loss of consciousness, using the 0-3 scoring system detailed in MATERIALS AND METHODS. With this system, it was possible to detect tolerance to alcohol during the first 1-2 wk, thus allowing alcohol delivery to be increased gradually. Because histopathological findings were similar in livers from wild-type mice (Fig. 4) and rats, it is concluded that long-term enteral feeding in the mouse is practical. Nutrition and dietary factors are important in the pathogenesis of alcoholic liver injury (11). Therefore, it is important that weight gain be demonstrated to minimize possible nutritional complications. In the present study, adult mice achieved steady weight gains, indicating that nutrition was adequate (Fig. 1). Furthermore, early alcohol-induced liver injury was prevented in tumor necrosis factor receptor-1 knockout mice given enteral ethanol using the same protocol (34). Both controls and knockout mice received the same diet, but only wild-type mice exhibited pathology. Thus it is concluded that nutritional complications cannot explain these results. The main pathological change in early alcohol-induced liver injury in the mouse was steatosis; however, inflammation and necrosis were also observed. Fat accumulation in livers from CYP2E1 +/+ mice given ethanol was observed mainly in pericentral to midzonal regions, except for one to three layers of hepatocytes around central veins, which may be hypoxic. Enteral ethanol causes hypoxia in pericentral regions of the liver in rats (4). Because ATP levels decline in anoxia, decreased fat accumulation in the last cell layers may be due to a lack of energy for triglyceride synthesis.Is CYP2E1 involved in early alcohol-induced liver injury?
Reactive oxygen species are involved in mechanisms of alcohol-induced
liver injury in the rat (17). CYP2E1 induced predominantly in the
hepatocyte after alcohol could be a source of reactive oxygen species
(15, 26). Indeed, French et al. (12) correlated the level of CYP2E1
with liver pathology, lipid peroxidation, and formation of the
-hydroxyethyl radical adduct produced in vivo and in vitro.
Furthermore, inhibitors of CYP2E1 partially decreased alcohol-induced
liver injury (6). Moreover, Wong et al. (33) reported that CYP2E1 was
the major factor in carbon tetrachloride-induced hepatotoxicity in
mice. These data are consistent with the hypothesis that mechanisms of
alcohol-induced liver injury involve oxidant stress from CYP2E1. On the
other hand, reactive oxygen species are also produced by various white
blood cells such as monocytes and macrophages. Kupffer cells, the
resident macrophage in the liver, could be a rich source of reactive
oxygen species via NADPH oxidase. Indeed, destruction of Kupffer cells by GdCl3 blunted free radical
formation in the Tsukamoto-French model and prevented early
alcohol-induced liver injury (17, 19). Thus whether oxidant stress is
from CYP2E1 predominantly in hepatocytes or NADPH oxidase in Kupffer
cells has remained unclear.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the National Institute on Alcohol Abuse and Alcoholism.
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FOOTNOTES |
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Preliminary accounts of this work have appeared elsewhere (18).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. G. Thurman, Laboratory of Hepatobiology and Toxicology, Dept. of Pharmacology, CB#7365, Mary Ellen Jones Bldg., Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365 (E-mail: thurman{at}med.unc.edu).
Received 2 April 1999; accepted in final form 8 August 1999.
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