Diphenyleneiodonium sulfate, an NADPH oxidase
inhibitor, prevents early alcohol-induced liver injury in the
rat
Hiroshi
Kono1,
Ivan
Rusyn1,2,
Takehiko
Uesugi1,
Shunhei
Yamashina1,
Henry D.
Connor3,
Anna
Dikalova3,
Ronald P.
Mason2,3, and
Ronald G.
Thurman1,2
1 Laboratory of Hepatobiology and Toxicology, Department of
Pharmacology and 2 Curriculum in Toxicology, University of
North Carolina, Chapel Hill 27599-7365; and 3 Laboratory of
Pharmacology and Chemistry, National Institute of Environmental Health
Sciences, Research Triangle Park, North Carolina 27709
 |
ABSTRACT |
The oxidant source in
alcohol-induced liver disease remains unclear. NADPH oxidase (mainly in
liver Kupffer cells and infiltrating neutrophils) could be a potential
free radical source. We aimed to determine if NADPH oxidase
inhibitor diphenyleneiodonium sulfate (DPI) affects nuclear factor-
B
(NF-
B) activation, liver tumor necrosis factor-
(TNF-
) mRNA
expression, and early alcohol-induced liver injury in rats. Male Wistar
rats were fed high-fat liquid diets with or without ethanol (10-16
g · kg
1 · day
1)
continuously for up to 4 wk, using the Tsukamoto-French intragastric enteral feeding protocol. DPI or saline vehicle was administered by
subcutaneous injection for 4 wk. Mean urine ethanol concentrations were
similar between the ethanol- and ethanol plus DPI-treated groups.
Enteral ethanol feeding caused severe fat accumulation, mild
inflammation, and necrosis in the liver (pathology score, 4.3 ± 0.3). In contrast, DPI significantly blunted these changes (pathology
score, 0.8 ± 0.4). Enteral ethanol administration for 4 wk also
significantly increased free radical adduct formation, NF-
B
activity, and TNF-
expression in the liver. DPI almost completely
blunted these parameters. These results indicate that DPI prevents
early alcohol-induced liver injury, most likely by inhibiting free
radical formation via NADPH oxidase, thereby preventing NF-
B
activation and TNF-
mRNA expression in the liver.
nuclear factor-
B; tumor necrosis factor-
; enteral
feeding
 |
INTRODUCTION |
CHRONIC ALCOHOL
INCREASES reactive oxygen species in the liver, which could be
involved in triggering a vicious cycle of pathology by activating
transcription factors for proinflammatory cytokines such as tumor
necrosis factor-
(TNF-
) and interleukin-1. Recent evidence from
our laboratory (17) supports the hypothesis that TNF-
plays a pivotal role in early alcoholinduced liver injury. Indeed, anti-TNF-
antibody blunted inflammation and necrosis in rats
fed enteral ethanol. Furthermore, early alcohol-induced liver injury
was prevented in TNF receptor-1 knockout mice given enteral ethanol
intragastrically (42). Thus it is likely that an
inflammatory cascade via TNF-
signaling is involved in early alcohol-induced liver injury. One key transcription factor involved in
TNF-
synthesis is nuclear factor-
B (NF-
B). Importantly, it is
activated in many cell types by oxidants (5).
The major source of reactive oxygen species in alcohol-induced liver
injury remains unclear. Xanthine oxidase and NADPH oxidase could be
sources of reactive oxygen species. Indeed, allopurinol, a xanthine
oxidase inhibitor and scavenger of free radicals, prevented free
radical formation, NF-
B activation, and alcohol-induced liver injury
in the enteral alcohol model (21). Evidence for or against
NADPH oxidase, however, is lacking. Therefore, the specific purpose of
this study was to determine if diphenyleneiodonium sulfate (DPI), an
NADPH oxidase inhibitor, would affect free radical formation, NF-
B
activation, TNF-
mRNA expression in the liver, and early
alcohol-induced liver injury in the Tsukamoto-French enteral alcohol
feeding model (39).
 |
MATERIALS AND METHODS |
Animals and treatments.
Male Wistar rats were fed a high-fat liquid diet with or without
ethanol (10-16
g · kg
1 · day
1,
n = 5) continuously for up to 4 wk, using the
intragastric enteral feeding protocol developed by Tsukamoto and French
(39). Either DPI (1 mg · kg
1 · day
1; Toronto
Research Chemicals, Toronto, ON, Canada) or vehicle (5% glucose, 0.2 ml/day) was administrated by subcutaneous injection for 4 wk. This dose
of DPI was shown to be well tolerated and effective in long-term
studies (8, 13). Rats were housed in a pathogen-free
facility accredited by the American Association for Accreditation of
Laboratory Animal Care, and surgical procedures used in this study were
approved by the institutional animal care and use committee.
A liquid diet described first by Thompson and Reitz (36)
supplemented with lipotropes as described by Morimoto et al.
(31) was used. It contained corn oil as fat (37% of total
calories), protein (23%), carbohydrate (5%), minerals and vitamins,
plus either ethanol (35-40% of total calories) or isocaloric
maltose-dextrin (control diet) as described previously
(38).
Urine collection and ethanol assay.
Ethanol concentration in urine, which is representative of blood
alcohol levels (3), was measured daily. Rats 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 for later
analysis. Ethanol concentration was determined by measuring absorbance
at 366 nm resulting from the reduction of NAD+ to NADH by
alcohol dehydrogenase (7).
Blood collection and transaminase determinations.
Blood was collected from the aorta after 4 wk of enteral feeding and
centrifuged. Serum was stored at
20°C until it was assayed for
alanine aminotransferase (ALT) by standard enzymatic procedures (7).
Pathological evaluation.
After 4 wk of ethanol treatment, livers were formalin fixed, embedded
in paraffin, and stained with hematoxylin and eosin to assess
steatosis, inflammation, and necrosis. Liver pathology was scored in a
blinded manner by one of the authors and by an outside expert as
described previously by Nanji et al. (32) as follows: for
steatosis (the % of liver cells containing fat), <25% = 1+, <50% = 2+, <75% = 3+, and 75% = 4+; and for inflammation and necrosis: 1 focus per low-power field = 1+; and 2 or more foci = 2+.
The number of neutrophils in the liver sections was also determined
after 4 wk by counting cells in three high-power fields (×400) per
hematoxylin and eosin- and Giemsa-stained slide. Fat accumulation
caused ballooning of hepatocytes and narrowing of the sinusoidal space.
This could affect the number of hepatocytes and sinusoidal space in
each field; therefore, the number of hepatocytes was also counted and
the number of neutrophils was expressed per 100 hepatocytes
(16).
Collection of bile and detection of free radical adducts.
Ethanol concentration in the breath was analyzed by gas chromatography
to verify that levels were similar between the groups when bile was
collected (14). Rats were anesthetized with pentobarbital sodium (75 mg/kg), and the proximal bile duct was cannulated with PE-10
tubing. After the spin-trapping reagent
-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN; 1 g/kg body wt; Sigma Chemical, St. Louis, MO) was injected slowly into the tail vein, bile samples were collected at 30-min intervals for 3 h into 35 µl of 0.5 mM Desferal (deferoxamine mesylate, Sigma Chemical) to prevent ex vivo radical adduct formation. Samples were stored at
80°C until analysis of free radical adducts by electron spin resonance (ESR) spectroscopy (18).
Samples were thawed and transferred to a quartz flat cell, and ESR
spectra were obtained using a Bruker ESP 300 ESR spectrometer.
Instrument conditions were as follows: 20-mW microwave power, 1-G
modulation amplitude, 80-G scan width, 16-min scan, and 1-s time
constant. Spectral data were stored on an IBM-compatible computer and
analyzed for ESR hyperfine coupling constants by computer simulation
(10). ESR signal intensity was determined from the
amplitude of the high-field member of the low-field doublet (second
line from the left) of the ESR spectra and expressed in arbitrary units
(1 unit = 1 mm chart paper).
Nuclear protein extraction and gel mobility shift assays.
Binding conditions for NF-
B were characterized and electrophoretic
mobility shift assays were performed as described previously (43). Briefly, nuclear extracts (40 µg) from liver
tissues were preincubated for 10 min on ice with 1 µg poly(dI/dC) and
20 µg BSA (both from Pharmacia Biotech, Piscataway, NJ) in a buffer that contained 1 mM HEPES (pH 7.6), 40 mM MgCl2, 0.1 M
NaCl, 8% glycerol, 0.1 mM dithiothreitol, and 0.05 mM EDTA and 2 µl
of a 32P-labeled DNA probe (10,000 counts · min
1 · µl
1;
Cerenkov) that contained 0.4 ng of double-stranded oligonucleotide. Mixtures were incubated for 20 min on ice and resolved on 5%
polyacrylamide (29:1 cross linking) and 0.4× Tris-boric acid-EDTA
gels. After electrophoresis, gels were dried and exposed to Kodak film.
Specificity of NF-
B binding was verified by competition assays and
ability of specific antibodies to supershift protein-DNA complexes. In the competition assay, a 200-fold excess of the unlabeled
oligonucleotide was added 10 min before addition of the labeled probe.
In the supershift experiment, 1 µg of rabbit antisera against p50
protein (Santa Cruz Biotech, Santa Cruz, CA) was added to the reaction mixture after incubation with labeled probe, which was further incubated at room temperature for 30 min. Labeled and unlabeled oligonucleotides contained the consensus sequence for NF-
B (top strand: 5'-GCAGAGGGGACTTTCCGGA-3'; bottom strand:
5'-GTCTGCCAAAGTCCCCTCTG-3') (4). Data were quantitated by
scanning autoradiograms with GelScan XL (Pharmacia, Uppsala, Sweden).
RNA isolation and RT-PCR amplification.
Immediately after the rats died, liver tissues were flash frozen in
liquid nitrogen and stored at
80°C until analysis. Approximately 50 mg of liver tissue was collected, and total cellular RNA was extracted
using the Qiagen RNeasy kit (Chatsworth, CA) according to the
manufacturer's instructions. The synthesis of cDNA and PCR
amplification of TNF-
and glyceraldehyde-3-phosphate dehydrogenase was performed as described previously (24). When
appropriate, the specificity of the PCR bands was confirmed by
restriction site analysis of the amplified cDNA, which generates
restriction fragments of the expected size (data not shown). The
amplified PCR products were subjected to electrophoresis at 75 V
through 2% agarose gel for 1 h. Gels were stained with 0.5 mg/ml
ethidium bromide Tris-borate-EDTA buffer (ICN, Costa Mesa, CA) and
photographed with type 55 Polaroid positive/negative film.
Statistics.
ANOVA or Student's t-test was used for the determination of
statistical significance as appropriate. For comparison of pathological scores, the Mann-Whitney rank sum test was used. P < 0.05 was selected before the study as the level of significance.
 |
RESULTS |
Body weight.
Diets were initiated after 1 wk to allow for full recovery from
surgery. All animals survived 4 wk of the experimental period, and
animals treated with DPI exhibited no complications. In spite of
development of greater hepatic injury in ethanol groups, the rats grew
steadily, making nutritional complications an unlikely explanation for
these results. The mean body weight gains were 2.9 ± 0.4 g/day
for the ethanol group and 2.8 ± 0.2 g/day for the ethanol plus
DPI group (Fig. 1). There were no
significant differences in body weight gains between the groups.

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Fig. 1.
Effect of chronic enteral ethanol and diphenyleneiodonium
sulfate (DPI) on body weight. Body weight was measured once a week.
Values are means ± SE (n = 5). ,
High-fat control diet + vehicle; , high-fat
control diet + DPI; , high-fat ethanol-containing
diet + vehicle; , high-fat ethanol-containing
diet + DPI.
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Ethanol concentrations in urine.
As reported previously in several studies (1, 33, 37),
alcohol levels in urine fluctuate in a cyclic pattern from 0 to >500
mg/dl. The urine alcohol cycle depends on an intact
hypothalamic-pituitary-thyroid axis response to the ethanol-induced
drop in body temperature by increasing the rate of ethanol elimination
(27). DPI had no effect on cyclic patterns of ethanol.
There were no significant differences in mean urine alcohol
concentrations between rats given ethanol (Fig.
2A; 251 ± 38 mg/dl) and
rats given ethanol plus DPI (Fig. 2B; 262 ± 33 mg/dl).

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Fig. 2.
Representative plots of daily urine alcohol
concentrations. Urine alcohol concentrations were measured daily as
described in MATERIALS AND METHODS. Typical urine alcohol
concentrations in rats fed high-fat ethanol-containing diet + vehicle (A) and high-fat ethanol-containing diet + DPI
(B) are shown.
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Serum transaminase levels.
Serum ALT levels were ~40 IU/l after 4 wk of high-fat control diet
(Table 1). However, administration of
enteral ethanol for 4 wk increased serum ALT levels significantly by
about fourfold. DPI significantly blunted these values by ~70%.
Pathological evaluation.
In control groups, there were no pathological changes after 4 wk (Fig.
3, A and B).
Administration of enteral ethanol for 4 wk caused severe fatty
infiltration, mild inflammation, and necrosis (Fig. 3C),
resulting in a total pathology score of 4.3 ± 0.3 (Table 1).
Increases in pathology scores were blunted almost completely by DPI
treatment (Fig. 3D; total pathology score: 0.8 ± 0.4).

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Fig. 3.
Photomicrographs of livers after ethanol treatment. Livers from
rats given high-fat control and high-fat ethanol-containing diets are
shown. Original magnification, ×100. Representative photomicrographs
of hematoxylin and eosin-stained liver sections. A: high-fat
control diet + vehicle. B: high-fat control diet + DPI. C: high-fat ethanol-containing diet + vehicle.
D: high-fat ethanol-containing diet + DPI.
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The number of infiltrating neutrophils in the liver was minimal and not
different between the groups in the absence of ethanol. However, a
significant increase was caused by 4 wk of enteral ethanol feeding
(Table 1). This increase was prevented totally by DPI.
Effect of chronic ethanol and DPI on free radical adduct formation.
Radical adducts were barely detectable in bile from rats fed an
ethanol-free, high-fat control diet in both groups (data not shown).
However, administration of enteral ethanol for 4 wk caused a
significant increase in free radical adduct formation (Fig. 4). This increase was blunted
significantly by DPI. ESR hyperfine coupling constants were
aN = 15.70 G and
a
= 2.72 G, characteristic of both
the
-hydroxyethyl and lipid-derived POBN radical adduct. Studies
using [13C]ethanol have demonstrated that both radical
adducts are present in approximately equal amounts (18).
ESR signal intensity was determined from the amplitude of the
high-field member of the low-field doublet (second line from the left)
of the ESR spectra (Fig. 5). The
intensity of these signals was increased significantly by enteral
ethanol but was blunted ~90% by DPI (Fig. 5).

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Fig. 4.
Effect of chronic enteral ethanol and DPI on electron
spin resonance (ESR) spectra. Rats were fed enteral liquid diets for 4 wk intragastrically. After injection of the spin-trapping reagent
-(4-pyridyl-1-oxide)-N-t-butylnitrone (1 g/kg
iv, Sigma Chemical), bile was collected into Desferal (deferoxamine
mesylate, 0.5 mM) and analysis of ESR spectra was performed as
described in MATERIALS AND METHODS. Representative ESR
spectra are shown.
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Fig. 5.
Effect of chronic enteral ethanol and DPI on average
radical adduct signal intensity. Conditions were the same as those
given in Fig. 4 legend. ESR signal intensity was determined from the
amplitude of the high-field member of the low-field doublet (second
line from the left) of the ESR spectra and was averaged for rats
treated as described in MATERIALS AND METHODS. Values are
means ± SE (n = 4). * P < 0.05 compared with rats fed high-fat ethanol-containing diet + vehicle (VEH) (ANOVA and Bonferroni's post hoc test).
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Effect of chronic ethanol and DPI on hepatic NF-
B activity.
NF-
B binding activity in hepatic nuclear extracts was minimal after
4 wk of high-fat control diet (Fig.
6A). In contrast, enteral
ethanol significantly increased NF-
B binding by nearly threefold.
DPI treatment significantly blunted this increase by ~50%.

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Fig. 6.
Effect of chronic enteral ethanol and DPI on hepatic
nuclear factor- B (NF- B) activity. A: nuclear extracts
(40 µg of total protein in each line) were prepared from frozen
livers and used for gel shift assays as described in MATERIALS
AND METHODS. Data shown are results of densitometric analysis of
the NF- B/DNA complex images. Density of the image in livers of rats
fed high-fat control diet was set to 100%. Values are means ± SE
(n = 5). * P < 0.05 compared with
rats fed high-fat control diet. # P < 0.05 compared
with rats fed high-fat ethanol-containing diet (ANOVA with
Bonferroni's post hoc test). B: protein binding to labeled
oligonucleotide probe is specific for the active form of NF- B.
Lane 1, labeled probe with no nuclear extract added.
Lane 2, nuclear extract from livers of rats fed high-fat
ethanol-containing diet was incubated with 32P-labeled
double-stranded oligonucleotide encompassing the B motif to detect
NF- B DNA binding activity. Lane 3, a 200-fold excess of
the unlabeled oligonucleotide was used in competition assays.
Lane 4, p50 antibodies were used in supershift experiments
as described in MATERIALS AND METHODS. Open arrow, native
NF- B/DNA complex; filled arrow, supershifted complex.
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To confirm that protein binding in nuclear extracts to labeled
oligonucleotide probe was specific for the active form of NF-
B, gel
shift assays were carried out either in the presence of an excess of
unlabeled double-stranded oligonucleotide with a consensus sequence for
NF-
B binding or with an antibody specific for the NF-
B p50
subunit (Fig. 6B). In the absence of nuclear proteins, no
protein-DNA complex was detected (Fig. 6B, lane
1). Furthermore, unlabeled oligonucleotide that contained the
NF-
B binding site could effectively compete for DNA binding with
32P-labeled probe (Fig. 6B, lane 3).
Moreover, addition of anti-p50 serum reduced the intensity of the
complex and supershifted the band to a higher molecular mass (Fig.
6B, lane 4).
Effect of chronic enteral ethanol and DPI on TNF-
mRNA
expression in liver.
TNF-
mRNA expression in the liver was minimal after 4 wk of high-fat
control diet with or without DPI treatment (Fig.
7). In contrast, TNF-
mRNA expression
was increased by enteral ethanol after 4 wk. This increase was blunted
by DPI treatment.

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Fig. 7.
Effect of chronic enteral ethanol and DPI on tumor
necrosis factor- (TNF- ) mRNA expression in the liver. TNF- and
the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
mRNA were determined using RT-PCR as described in MATERIALS AND
METHODS. Data shown are representative of 4 samples/group.
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 |
DISCUSSION |
Possible sources of oxidants in early alcohol-induced liver injury.
Chronic enteral ethanol enhances reactive oxygen species in the liver.
ESR spectroscopy of bile from animals treated with xenobiotics and
given spin-trapping agents has been demonstrated (19) to
be useful in monitoring hepatic free radical-adduct formation in vivo.
Radical adducts detected in bile may be derived from both parenchymal
and nonparenchymal cells. In the Tsukamoto-French intragastric ethanol
infusion model,
-hydroxyethyl and lipid-derived free radicals have
been detected (18). These radicals are most likely
involved in alcohol-induced liver injury; however, the source of
oxidants has remained unclear.
Two possible sources of radical adducts in alcoholic liver injury have
been suggested. One possibility is that cytochrome P-450
(CYP)2E1, induced predominantly in the hepatocyte by ethanol, is the
source of reactive oxygen species (2). In support of this
hypothesis, Ronis and co-workers (35) have shown that a correlation exists between blood levels of alcohol and induction of
CYP2E1 as alcohol cycles in the enteral model. Also, the level of CYP2E1 correlates with the degree of pathology, and inhibitors of
CYP2E1 partially reduced hepatic pathology due to enteral ethanol (2). These results, while correlative, support the idea
that oxidants from CYP2E1 may play a role in early alcohol-induced liver injury. On the other hand, it was reported (25) that
CYP2E1 was induced to the same extent by ethanol or ethanol plus the Kupffer cell toxicant gadolinium chloride (GdCl3) treatment
in the rat enteral model; however, liver pathology was prevented by
GdCl3. Importantly, it was recently reported from our
laboratory (20) that there were no differences in free
radical formation and early alcohol-induced liver injury between
wild-type and CYP2E1 knockout mice fed enteral ethanol chronically.
These data suggest that oxidants from CYP2E1 either are not involved or
play only a minor role in early alcohol-induced liver injury.
Alternatively, oxidant-generating enzymes in Kupffer cells may
contribute significantly to a dramatic increase in release of reactive
oxygen species after ethanol administration. Indeed, destruction of
Kupffer cells with GdCl3 diminishes free radical formation
significantly (18) and prevents liver injury in the enteral alcohol model (1). Kupffer cells contain xanthine
oxidase, NADPH oxidase, and other oxidant-generating enzymes. It has
been shown recently (21) that allopurinol, a xanthine
oxidase inhibitor and a free radical scavenger, prevented
alcohol-induced liver injury in the enteral model. Moreover, DPI, an
NADPH oxidase inhibitor, prevented free radical formation and early
alcohol-induced liver injury almost completely in this study (Figs.
3-5). Thus oxidants from Kupffer cells could play a major role in
early alcohol-induced liver injury (see Fig.
8). Indeed, release of superoxide anion leads to formation of both
-hydroxyethyl and lipid-derived radicals, which could then be used as secondary or "marker" radicals because oxygen-derived radical adducts are generally too unstable to be detected in vivo (18). Importantly, the spin-trap POBN is
rapidly absorbed and distributed throughout the body after
intraperitoneal administration, and POBN radical adducts are relatively
stable in vivo (29). Thus both
-hydroxyethyl and
lipid-derived adducts of POBN formed in the liver are excreted into the
bile and detected by ESR.

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Fig. 8.
Working hypothesis showing that Kupffer cells activated
by gut-derived endotoxin produce free radicals during enteral ethanol
administration. In this study, DPI, an NADPH oxidase inhibitor,
prevented early alcohol-induced liver injury almost completely. These
data are consistent with the hypothesis that oxidants from NADPH
oxidase play an important role in early alcohol-induced liver injury.
Kupffer cells then increase inflammatory cytokines such as TNF- ,
because Kupffer cells are a major source of NADPH oxidase in the early
phases of alcohol-induced liver injury.
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Possible mechanisms of effect of DPI in early alcohol-induced liver
injury.
A group of iodonium compounds, including DPI, represent a class
of inhibitors of flavin enzymes that reduce activities of NADPH-dependent oxidase in the neutrophil and macrophage
(34). DPI inhibited superoxide generation by macrophages
(15) and has been used to inhibit production of oxidants
in several studies (9). Because it has been suggested that
Kupffer cell NADPH oxidase plays a pivotal role in ethanol-induced
superoxide production in the liver (6), this study was
undertaken to determine if DPI could prevent liver injury by inhibition
of free radical formation in the enteral feeding model. Indeed, DPI
blunted increases in free radical formation (Figs. 4 and 5), NF-
B
activity (Fig. 6), and TNF-
mRNA expression (Fig. 7) in the liver
and prevented early alcohol-induced liver injury almost completely
(Fig. 3 and Table 1).
DPI may also inhibit several other hepatic radical-generating enzymes,
such as microsomal NADPH-CYP reductase, mitochondrial oxidases, and
nitric oxide synthase (26, 30, 41). However, it was
reported recently (22) that the free radical formation and
liver injury observed in wild-type mice fed enteral ethanol were almost
completely blunted in NADPH oxidase-deficient mice. These data are
consistent with the hypothesis that NADPH oxidase is the predominant
source of oxidants in alcohol-induced liver injury.
Infiltrating neutrophils are also a source of oxidants via NADPH
oxidase. However, the number of infiltrating neutrophils increased
significantly only after 3-4 wk of enteral feeding in this study
(Table 1), suggesting that Kupffer cells are most likely a predominant
source of oxidants, at least during the early phases of the
pathological process. Therefore, it is concluded that DPI prevents
liver injury, most likely by inhibiting free radical formation via
Kupffer cell NADPH oxidase, preventing NF-
B activation and
inflammatory cytokine production (see Fig. 8).
Role of NF-
B in early alcohol-induced liver injury.
NF-
B is normally present in an inactive form bound to its
inhibitor, I
B, in the cytoplasm (5). Typically, NF-
B
is rapidly activated in response to immunologic stimuli such as
lipopolysaccharide, cytokines such as TNF-
, and oxidants
(5). Activation of NF-
B involves rapid phosphorylation
and proteolytic cleavage of I
B from NF-
B, leading to
translocation of NF-
B to the nucleus (5). Binding sites
for NF-
B have been identified within the regulatory elements of
genes for several proinflammatory cytokines such as TNF-
and
interleukin-1. Thus NF-
B plays an important role in regulation of
the innate immune system that participates in inflammation. Increases
in inflammatory cytokines and adhesion molecules by activation of
NF-
B could be one explanation for pathogenesis of early
alcohol-induced liver injury. Indeed, NF-
B activity in the liver was
increased significantly by about three- to fivefold over control values
by alcohol (Fig. 6) (28). Moreover, DPI blunted this
increase and prevented liver injury significantly in this study (Table
1 and Fig. 6). These results indicate that NF-
B activation plays an
important role in early alcohol-induced liver injury (see Fig. 8).
TNF-
plays an important role in early alcohol-induced liver
injury.
Activation of NF-
B could increase TNF-
expression in the liver
(40). TNF-
is a cytokine produced mainly by activated macrophages that stimulates endothelial cells to synthesize adhesion molecules [i.e., intercellular adhesion molecule-1 (ICAM-1)], leading
to accumulation of leukocytes in the liver. Recent evidence from our
laboratory (42) supports the hypothesis that TNF-
plays
a pivotal role in early alcohol-induced liver injury. This conclusion
is based on the observation that alcohol-induced liver injury is
present in wild-type mice fed enteral ethanol chronically but is
prevented in TNF receptor-1 knockout mice (42). Indeed, ethanol increases TNF-
mRNA and ICAM-1 expression in the liver in
the enteral alcohol model (Fig. 7) (16, 17). Furthermore, anti-TNF-
antibody reduces inflammatory cell infiltration and necrosis in the Tsukamoto-French model (17). Moreover,
TNF-
mRNA expression was blunted significantly by DPI in this study (Fig. 7).
The predominant pathological change observed in this study was
steatosis (Table 1 and Fig. 3) (20, 42). Feingold and Grunfeld (12) reported that the synthesis of fatty acids
in the liver is increased after TNF-
administration. TNF-
also stimulates peripheral lipolysis, leading to an increase in circulating free fatty acids (11). Importantly, TNF-
is also
involved in fat accumulation caused by enteral ethanol in the liver.
Indeed, steatosis was observed in wild-type mice fed enteral ethanol
but was significantly blocked in TNF receptor-1 knockout mice
(42). In this study, DPI blunted increases in hepatic fat
accumulation and TNF-
mRNA expression significantly (Figs. 3 and 7).
Thus it is concluded that TNF-
plays an important role in early
alcohol-induced liver injury (Fig. 8).
DPI prevented early alcohol-induced liver injury almost completely by
inhibiting free radical formation in this study. These data are
consistent with the hypothesis that oxidants from NADPH oxidase play a
major role in early alcohol-induced liver injury (see Fig. 8). Kupffer
cells then increase production of inflammatory cytokines such as
TNF-
, because Kupffer cells are the predominant source of NADPH
oxidase in liver during the early phases of alcohol-induced liver injury.
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ACKNOWLEDGEMENTS |
Portions of this work have been previously published in abstract
form (23).
 |
FOOTNOTES |
Address for reprint requests and other correspondence: I. Rusyn, Laboratory of Hepatobiology and Toxicology, Dept. of
Pharmacology, CB#7365, Mary Ellen Jones Bldg., Univ. of North Carolina,
Chapel Hill, NC 27599-7365.
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.
Received 18 August 2000; accepted in final form 18 December 2000.
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