Development of an animal model of chronic alcohol-induced
pancreatitis in the rat
Hiroshi
Kono1,
Mikio
Nakagami1,
Ivan
Rusyn1,2,
Henry D.
Connor1,
Branko
Stefanovic3,
David A.
Brenner3,
Ronald P.
Mason2,4,
Gavin E.
Arteel1, and
Ronald G.
Thurman1,2
1 Laboratory of Hepatobiology and Toxicology, Department of
Pharmacology, 2 Curriculum in Toxicology, and
3 Department of Medicine, University of North Carolina at Chapel
Hill, Chapel Hill 27599-7365; and 4 Laboratory of
Pharmacology and Chemistry, National Institute of Environmental
Health Sciences, Research Triangle Park, North Carolina 27709
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ABSTRACT |
This study was designed to develop an animal model
of alcoholic pancreatitis and to test the hypothesis that the dose of
ethanol and the type of dietary fat affect free radical formation and pancreatic pathology. Female Wistar rats were fed liquid diets rich in
corn oil (unsaturated fat), with or without a standard or high dose of
ethanol, and medium-chain triglycerides (saturated fat) with a high
dose of ethanol for 8 wk enterally. The dose of ethanol was
increased as tolerance developed, which allowed approximately twice as
much alcohol to be delivered in the high-dose group. Serum pancreatic
enzymes and histology were normal after 4 wk of diets rich in
unsaturated fat, with or without the standard dose of ethanol. In
contrast, enzyme levels were elevated significantly by the high ethanol
dose. Increases were blunted significantly by dietary saturated fat.
Fibrosis and collagen
1(I) expression in the pancreas were not
detectable after 4 wk of enteral ethanol feeding; however, they were
enhanced significantly by the high dose after 8 wk. Furthermore,
radical adducts detected by electron spin resonance were minimal with
the standard dose; however, the high dose increased carbon-centered
radical adducts as well as 4-hydroxynonenal, an index of lipid
peroxidation, significantly. Radical adducts were also blunted by
~70% by dietary saturated fat. The animal model presented here is
the first to demonstrate chronic alcohol-induced pancreatitis in a
reproducible manner. The key factors responsible for pathology are the
amount of ethanol administered and the type of dietary fat.
medium-chain triglycerides; fibrosis; free radical; enteral
feeding
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INTRODUCTION |
EPIDEMIOLOGICAL EVIDENCE
INDICATES that chronic alcohol abuse, but not occasional alcohol
intoxication, is a major cause of chronic pancreatitis in adult
patients (26). Therapies are limited by an inadequate
understanding of the mechanisms of pathophysiology, and
progress has been slow because an appropriate animal model for
alcohol-induced pancreatitis is lacking (6). The pathology of chronic alcoholic pancreatitis consists of both intralobular fibrosis involving pancreatic acini and interlobular fibrosis with
fibrotic strictures of pancreatic ducts (8). Tsukamoto et
al. (32) demonstrated that alcohol-induced pancreatitis
can be studied in an intragastric enteral feeding model in the rat. In
that study, enteral ethanol along with a high-fat diet caused atrophy
and apoptosis in pancreatic acinar cells; however, focal necrosis and fibrosis were only present in ~30% of rats given ethanol chronically for 30-160 days. Thus modifications of this enteral feeding protocol could theoretically produce a useful model.
Free radicals are involved in alcohol-induced tissue injury
(17). Indeed,
-hydroxyethyl free radical was detected
in pancreatic secretions after alcohol exposure, but before increases
in pancreatic enzymes and pathological changes occurred
(15). The type of dietary fat is involved in free radical
formation and pathogenesis in the liver after alcohol consumption.
Indeed, medium-chain triglycerides (saturated fat) blunted increases in
endotoxin levels in portal blood, free radical formation in the liver,
and liver injury significantly in the Tsukamoto-French enteral model
(18, 23). Moreover, a high-fat liquid diet (35% of total
calories) rich in corn oil increased pancreatic injury in long-term
enteral feeding (32). Thus dietary unsaturated fat has
been associated with alcohol-induced pancreatic injury. Therefore, the
present study was designed to develop an animal model and, using the
intragastric alcohol feeding protocol, to test the hypothesis that the
dose of ethanol and the type of dietary fat affect free radical
formation and pathology in the pancreas. In the present study,
rats received progressively increasing doses of ethanol through careful
challenge as their tolerance developed over the 8 wk of the
experimental period, because pancreatic injury was minimal in the
Tsukamoto-French enteral protocol with the standard dose of ethanol
(15).
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METHODS |
Diets and animals.
A liquid diet described first by Thompson and Reitz (27),
supplemented with lipotropes as described by Morimoto et al.
(20), was used in this study. It consisted of either corn
oil (unsaturated fat) or medium-chain triglycerides (saturated fat) as
a source of fat (37% of total calories), protein (23%), carbohydrate
(5%), minerals, and vitamins plus either ethanol (35-40% of
total calories) or maltose-dextrin (control diet), as described in
detail elsewhere (29).
Female Wistar rats (200-225 g) were fed high-fat liquid diets (200 kcal · kg body wt
1 · day
1)
rich in unsaturated fat, without or with ethanol (standard dose, 8-12 g · kg
1 · day
1;
high dose, 10-18
g · kg
1 · day
1) or a diet
rich in saturated fat along with a high dose of ethanol continuously
for up to 8 wk, using the enteral protocol developed by Tsukamoto and
French (13, 30). In the standard dose protocol, ethanol was initially delivered at 8.3 g · kg
1 · day
1 (35% of
total calories) and was increased 0.6 g/kg every 2 days until the end
of the first week; then it was increased 0.6 g/kg every 4 days up to
11.5 g · kg
1 · day
1 of
ethanol (37% of total calories). This dose was continued until the end
of the experimental period. On the other hand, in the high-dose
protocol, ethanol was delivered initially at 10.2 g · kg
1 · day
1 (35% of
total calories) and was increased 0.6 g/kg every 2 days until the end
of the first week and then 0.6 g/kg every 4 days until the end of
week 4. During the second 4-wk period, ethanol delivery was
increased 0.6 0.6 g/kg each week up to 17-18
g · kg
1 · day
1 (40% of
total calories). All animals received humane care in compliance with
institutional guidelines, and alcohol intoxication was assessed
carefully to evaluate the development of tolerance to ethanol with the
use of a 0-3 scoring system (0, normal; 1, sluggish movement;
2, loss of movement but still moving if stimulated; 3, loss of
consciousness). The ethanol dose was increased progressively to
challenge animals based on this assessment, allowing ~1.4-fold more
ethanol to be delivered to adult Wistar rats than in previous studies
by this and other laboratories (15, 22, 28).
Clinical chemistry.
Ethanol concentrations in urine, which are representative of blood
ethanol levels (3), were 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 9:00 AM, the urine collection bottles were
changed and a 1-ml sample was stored at
20°C for later analysis. The ethanol concentration was determined by measuring the absorbance at
366 nm resulting from the reduction of NAD+ to NADH by
alcohol dehydrogenase (4).
After 4 or 8 wk of the enteral diet, the rats were anesthetized with
pentobarbital sodium (75 mg/kg ip) and blood was collected via the
aorta just before euthanasia. Serum was stored at
80°C until it was
assayed for amylase, lipase, and creatine kinase with analytical kits
(Sigma, St. Louis, MO).
Pathological evaluation.
Pathological changes in the pancreas were scored as described by
Tsukamoto et al. (32) as follows: steatosis (percentage of
acinar cells containing fat droplets): <25% = 1+, <50% = 2+, <75% = 3+, >75% = 4+; inflammation and necrosis:
1 focus per low-power
view = 1+; >1 focus = 2+. The type of infiltrating
inflammatory cells was also determined morphologically in hematoxylin
and eosin-stained sections. Fibrosis was scored as follows: thickened
perivenular collagen and a few thin collagen septa = 1+, thin
septa with incomplete bridging between regions = 2+, thin septa
and extensive bridging = 3+, thickened septa with complete
bridging of regions and nodular appearance = 4+.
Collection of pancreatic samples.
Pancreatic tissues were formalin fixed, embedded in paraffin, and
stained with hematoxylin and eosin or trichrome. Pathology was
evaluated in a blinded manner by one of the authors. Ethanol concentration in the breath was analyzed by gas chromatography to
verify that the levels were similar between the groups when collection
of the pancreatic secretions was initiated (14). The rats
were anesthetized with pentobarbital sodium (75 mg/kg), and the
pancreatic secretions were collected with a method described previously
(15), with minor modifications. Briefly, the distal bile
duct was cannulated with PE-10 tubing, and a ligature was placed on the
distal end of the pancreatic duct near the sphincter to prevent
contamination of the pancreatic juice with bile. The spin-trapping
agent
-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN; Sigma)
was administered (1 g/kg body wt) intravenously, and the pancreatic
secretions were collected into 35 µl of 0.5 mM deferoxamine mesylate
(Sigma) via retrograde flow from the ligated bile duct for
3 h to avoid ex vivo radical formation. Samples were stored at
80°C until analysis of free radical adducts by electron spin resonance (ESR) spectroscopy (17).
Samples were thawed and transferred to a quartz flat cell, and ESR
spectra were obtained with 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. Spectral data were stored on an
IBM-compatible computer and were 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 cm chart paper).
Immunohistochemical detection of 4-hydroxynonenal-modified
proteins.
Paraffin-embedded sections of pancreatic tissue were deparaffinized,
rehydrated, and stained immunohistochemically for the presence of an in
vivo marker of lipid peroxidation, 4-hydroxynonenal protein adducts, by
sequential incubation with a polyclonal antibody (Alpha Diagnostic
International, San Antonio, TX) in PBS (pH 7.4) containing 1% Tween 20 and 1% bovine serum albumin. Peroxidase-linked secondary antibody and
diaminobenzidine (Peroxidase Envision kit, DAKO, Carpinteria, CA) were
used to detect specific binding. The slides were rinsed two times with
PBS-0.1% Tween 20 between all incubations, and sections were
counterstained with hematoxylin as described elsewhere
(12). To control for nonspecific binding of the secondary
antibody, sections from the same animals were processed without the
primary antibody, followed by the procedure detailed above. No positive
staining was observed in this control experiment (data not shown).
RNase protection assays for detection of collagen I mRNA levels.
Briefly, the template for the cRNA probe for rat collagen
1(I) was
generated by subcloning the AvaI/PstI
fragment of its cDNA into the in vitro transcription plasmid pGME3
(5). The antisense collagen
1(I) probe was generated by
digestion of this plasmid with HindIII restriction
endonuclease and transcribed with the T7 RNA polymerase in the presence
of [
-32P]UTP. The antisense rat L-32 probe was
generated according to the manufacturer's instructions (Ambion,
Austin, TX). Both radiolabeled probes were simultaneously hybridized to
total RNA and digested with RNase A2 and RNase T1. Protected fragments
were resolved by electrophoresis on a 6% polyacrylamide sequencing gel
and quantified by PhosphoImage analysis (Molecular Dynamics, Sunnyvale, CA).
Statistics.
A multiple-comparison ANOVA with Bonferroni's post hoc analysis
was used for the determination of statistical significance, as
appropriate. For comparison of pathology scores, the Mann-Whitney rank
sum test was used. Before the study, a P value <0.05 was selected as the level of significance.
 |
RESULTS |
Physiological parameters.
The diets were initiated 1 wk after surgery to allow for full recovery,
and steady growth rates were observed during enteral feeding. Weight
gain in rats fed high-fat liquid diets with or without ethanol was
similar to that in rats fed the chow diet [high-fat control (without
ethanol), 2.0 ± 0.2 g/day; with standard dose of ethanol,
1.9 ± 0.1; with high dose of ethanol, 1.9 ± 0.2], indicating that the rats were adequately nourished. Including medium-chain triglycerides as the source of dietary fat did not affect
weight gain. As previously reported (1, 22, 28), for
unknown reasons, daily urine alcohol concentrations fluctuated between
0 and 600 mg/dl in all groups studied (Fig.
1). In rats fed the standard dose of
ethanol, mean urine alcohol concentration was 185 ± 11 mg/dl
(Fig. 1A); however, the high dose of ethanol increased these
values significantly, by ~50% (274 ± 15 mg/dl; Fig.
1B). Furthermore, in the second 4-wk period, the mean urine alcohol concentrations were 319 mg/dl in the high-dose group and 246 mg/dl in the standard-dose group. There were no differences in mean
urine alcohol concentrations between rats fed the high dose of ethanol
with unsaturated fat and those fed the high dose with saturated fat, as
expected (271 ± 17 mg/dl).

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Fig. 1.
Representative plots of daily urine alcohol
concentrations. Typical urine alcohol concentrations in rats fed the
high-fat liquid diet with the standard dose of ethanol (A)
and the high dose of ethanol (B) and rats fed the high dose
of ethanol plus medium-chain triglycerides (MCT; C) are
shown.
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In the first 4 wk, all animals were healthy and survived during enteral
feeding; however, ~30% mortality was observed in the high-dose
ethanol group during the second 4 wk. Because analysis of these animals
was postmortem, the cause of mortality was not clear in all cases,
although it was observed that some animals died because of convulsions
during alcohol withdrawal when urine alcohol levels were low. Serum
creatine kinase values were not significantly elevated as would be
expected if circulatory shock occurred. Furthermore, there was no
effect of alcohol on average systemic blood pressure.
Pathophysiology.
In rats fed a high-fat control diet for 4 wk, the values of the serum
pancreatic enzymes
-amylase and lipase were normal (Fig.
2). Increases in these values were
minimal in rats given the standard dose of ethanol; however, values
increased significantly, approximately threefold, in rats given higher
doses of ethanol (Fig. 2). In contrast, the amylase and lipase values
were blunted significantly (~50%) by dietary saturated fat in the
rats given the higher doses of ethanol (Fig. 2). After 8 wk, the
amylase and lipase values in the animals given higher doses of ethanol were also significantly elevated compared with those in animals that
received the standard dose of ethanol (amylase, 1,877 ± 188 IU/l;
lipase, 82 ± 38 IU/l) or the high-fat control animals (amylase, 1,477 ± 173 IU/l; lipase, 136 ± 26 IU/l). However, amylase
levels at 8 wk in animals given the higher doses of ethanol were
significantly lower compared with the values at 4 wk in the same group
(Fig. 2A); this effect is similar to that observed in
humans. Specifically, it is known that serum amylase levels decrease as
pancreatic exocrine function declines during the progression of
pancreatic damage (9).

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Fig. 2.
Effect of the dose of ethanol and lipid type on levels of
serum -amylase and lipase. Blood samples were collected via the
aorta at 4 or 8 wk (W), and -amylase and lipase were measured as
described in detail in METHODS. Data are means ± SE;
n = 4-6 rats. CO, corn oil; CON, high-fat control
diet; ETH, high-fat ethanol-containing diet; STD, standard dose of
ethanol; High, high dose of ethanol. *P < 0.05 compared with rats fed a high dose of ethanol with unsaturated fat by
ANOVA and Bonferroni's post hoc test; #P < 0.05 compared with rats fed the standard ethanol dose.
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Pancreatic histology data from adult female Wistar rats on the chow
diet are shown in Fig. 3A. In
rats fed the high-fat diet with or without the standard dose of
ethanol, pathological changes were minimal after 4 wk (Fig. 3,
B and C). In contrast, the high dose of enteral
ethanol given with unsaturated fat for 4 wk caused acinar cell atrophy,
fatty infiltration in acinar and islet cells (Fig. 3D),
inflammatory cell infiltration (Fig. 3E), and focal necrosis
(Fig. 3F) in the pancreas, resulting in the total pathology score of 4.4 ± 0.5 (Table 1). The
infiltrating cells were predominantly lymphocytes. Dietary medium-chain
triglycerides prevented these pathological changes nearly completely
(total pathology score, 1.0 ± 0.4). Furthermore, after 8 wk of
the high dose of ethanol, pathological changes were greater than
after 4 wk (total pathology score, 5.0 ± 0.4; Fig.
3E).

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Fig. 3.
Representative photomicrographs of pancreata from rats fed
different diets after ethanol treatment. A: chow.
B: 4-wk high-fat control diet. C: 4-wk high-fat
standard dose ethanol-containing diet. D: 4-wk high-fat,
high ethanol-containing diet. E: 8-wk high-fat, high
ethanol-containing diet. Original magnification, ×200. F:
higher magnification (×400) view of inflammation and necrosis in rats
fed the high-fat liquid diet with the high dose of ethanol for 4 wk.
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Fibrogenesis in the pancreas after chronic enteral ethanol feeding.
Trichrome staining of the pancreas from adult female Wistar rats on the
chow diet is shown in Fig. 4A.
No significant fibrotic changes were detected after 4 wk of enteral
feeding as reflected by the fibrosis score (Table
2), confirming a previous study from this
laboratory (15). However, 8 wk of enteral feeding of the
high dose of ethanol significantly increased the fibrotic score
compared with the fibrotic score of animals receiving the standard dose
of alcohol or the high-fat control diet (Fig. 4E, Table 2).
Under conditions in which fibrosis was detected, these changes tended
to be focal. In addition to the scoring of pancreata for fibrotic
changes, levels of collagen
1(I) mRNA expression in the pancreas
were also determined (Fig. 5). There were
no detectable signals in animals fed the chow or high-fat diets,
regardless of the length of treatment (Fig. 5). Enteral feeding with
ethanol for 4 wk also did not affect collagen
1(I) mRNA expression
(Fig. 5), as observed previously (15). However, high-dose
ethanol given for 8 wk caused extensive increases in collagen
1(I)
mRNA expression.

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Fig. 4.
Trichrome staining in the pancreas after chronic enteral ethanol
feeding. Original magnification, ×200. Representative
photomicrographs show histology of the pancreas from rats given
high-fat control (chow; A) and high-fat ethanol-containing
(B-E) diets.
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Fig. 5.
Effect of chronic ethanol and lipid type on collagen
1(I) mRNA expression in pancreatic tissue. Pancreatic tissues were
assayed for fibrillar collagen 1(I) mRNA using an RNase protection
assay with L32 as the housekeeping gene. Representative gels are shown.
EtOH, ethanol.
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Radical adducts in pancreatic secretions.
Radical adducts were barely detectable in pancreatic secretions from
rats fed either the chow or the high-fat control diet without ethanol
(data not shown). Moreover, POBN radical adducts in pancreatic
secretions from rats given the standard dose of ethanol were minimal
after 4 wk (Fig. 6). In
contrast, the high dose of ethanol increased radical adducts
approximately twofold. Furthermore, dietary saturated fat in the form
of medium-chain triglycerides blunted this increase significantly.
Computer simulation of these spectra identified the
POBN-
-hydroxyethyl radical adduct (17). The average
radical intensity was approximately fourfold greater in pancreatic
secretions from rats fed the high dose than those fed the standard dose
of ethanol (Fig. 7). Furthermore, this
increase was blunted significantly (~70%) by dietary saturated fat.

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Fig. 6.
Effect of the dose of ethanol and lipid type on electron
spin resonance (ESR) spectra in pancreatic secretions. After 4 wk of
enteral feeding, the diet was discontinued immediately before
experiments. After injection of the spin-trapping agent
-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN; 1 g/kg body
wt iv), pancreatic secretions were collected as described in detail in
METHODS. Computer simulation identified the species as the
-hydroxyethyl radical adduct (coupling constants:
aN = 15.8 G,
= 2.3 G). Representative ESR
spectra are shown.
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Fig. 7.
Effect of the dose of ethanol and lipid type on average
radical adduct signal intensity from pancreatic secretions. Conditions
were the same as those depicted in Fig. 6. ESR signal intensity was
quantitated as the double integral of peaks from pancreatic secretions.
Data are means ± SE; n = 6 rats.
*P < 0.01 compared with rats fed CO with EtOH,
#P < 0.05 compared with the high-dose group by ANOVA and
Bonferroni's post hoc test.
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Immunohistochemical detection of 4-hydroxynonenal modified
proteins.
To test whether ethanol administration causes lipid peroxidation in the
pancreas, 4-hydroxynonenal-modified proteins were detected by
immunohistochemistry (24). Despite increases in free
radical formation (Fig. 7), no detectable increases in 4-hydroxynonenal protein adducts were detected after 4 wk of enteral feeding in any
group (data not shown). After 8 wk of enteral feeding, significant accumulation of 4-hydroxynonenal (brown staining) was observed in the
pancreas of rats fed high-dose ethanol but not in rats fed the chow or
high-fat control diets (Fig. 8) or in
rats given the standard dose of ethanol (data not shown).

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Fig. 8.
Chronic ethanol treatment increases 4-hydroxynonenal in
the pancreas. Sections of pancreatic tissue from untreated rats
(A) and rats treated for 8 wk with high-fat (B)
or high-dose ethanol-containing (C) liquid diets were
stained immunohistochemically for 4-hydroxynonenal-modified proteins
(brown staining) as detailed in METHODS. C:
pathology caused by alcohol, including acinar atrophy, necrotic foci,
and increased stroma around acini. Original magnification, ×200.
Representative photomicrographs from 4 animals/group.
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 |
DISCUSSION |
Development of a new animal model to study alcohol-induced
pancreatic injury.
The mechanism of chronic alcohol-induced pancreatitis remains unclear,
and a major problem has been the lack of an appropriate animal model
for study. Tsukamoto et al. (32) previously reported that
feeding a high-fat diet with the standard dose of ethanol caused
atrophy and apoptosis in pancreatic acinar cells; however, focal necrosis and fibrosis were only present in ~30% of the rats fed enteral ethanol for 30-160 days. Because the severity of
clinical alcohol-induced pancreatitis and fibrosis is related to dose- and time-dependent alcohol consumption in humans
(11), here, the chronic intragastric enteral
feeding protocol was modified to increase ethanol delivery. This goal
was achieved by increasing the alcohol levels in the diet based on
behavioral assessment of the development of tolerance to ethanol with
the use of the 0-3 score system. In this manner, alcohol delivery
in female Wistar rats in the 200- to 225-g weight range could be
increased to 17-18 g · kg
1 · day
1. Alcohol
delivery was increased ~1.4-fold over previous studies from this and
other laboratories with rats in the same weight range (Refs.
13, 15, 31; Fig. 2). As a
result, pancreatic enzymes (amylase and lipase) and pathology scores
were increased after 8 wk of the high dose of ethanol (Table 1, Figs. 2
and 3). Furthermore, fibrosis and collagen
1(I) mRNA expression
increased significantly after 8 wk of the high dose of ethanol in
nearly all animals (Table 2, Figs. 4 and 5). Thus it is concluded that the total amount of alcohol consumed is a critical factor in producing chronic alcohol-induced pancreatic injury. Furthermore, the animal model presented here is the first reproducible demonstration of chronic
alcohol-induced pancreatitis in the rat.
Gender is also an important factor in alcohol-induced tissue injury.
Indeed, alcohol-induced liver injury is greater in females than in
males in the enteral alcohol model (16). The present study
differs from previous work (32) in that female
rats were used, and gender may therefore play a role in the observed
pancreatic damage; however, the damage resulting from the use of the
alcohol dosing regimen described in the METHODS section
also occurs in males (Kono et al., unpublished observations),
consistent with the idea that gender cannot completely explain the
greater pathology observed in this study.
Dietary fat and alcohol-induced pancreatic injury.
Dietary fat is also an important factor in alcohol-induced tissue
injury (21). Indeed, dietary medium-chain triglycerides reduced lipid peroxidation in the enteral model (23).
Furthermore, dietary medium-chain triglycerides prevented free
radical formation and early alcohol-induced liver injury in the
Tsukamoto-French enteral model (18). In the present study,
dietary medium-chain triglycerides also blunted free radical
formation and tissue injury in the pancreas (Figs. 6 and 7). Dietary
medium-chain triglycerides significantly blunted the increase in plasma
endotoxin levels after enteral ethanol, the response of Kupffer cells
to endotoxin, and liver injury (18). Therefore,
medium-chain triglycerides could affect activation of tissue
macrophages or other critical cell types in the pancreas. Thus dietary
medium-chain triglycerides prevented pancreatic injury, most likely by
inhibition of free radical formation. Together, these data indicate
that dietary fat is also an important factor in the pathogenesis of
chronic alcohol-induced pancreatic injury.
What is the mechanism of damage caused by alcohol in the pancreas
? Figure 9 is a schematic depiction of
our working hypothesis on the mechanism by which alcohol damages the
pancreas. In the present study, enteral feeding with a higher delivery
of ethanol increased formation of both carbon-centered free radicals
and 4-hydroxynonenal-modified proteins in the pancreas (Figs.
6-8). These data are consistent with the hypothesis that oxidative
stress is involved in the pathogenesis of early alcohol-induced
pancreatic injury (see Fig. 9).

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Fig. 9.
Working hypothesis. Ethanol changes gut permeability and
microflora during enteral feeding, and the high dose of ethanol could
increase endotoxin levels. Increased endotoxin most likely increases
infiltration of leukocytes such as macrophages and neutrophils, which
produce free radicals in the pancreas. One key factor in causing
pathology seems to be the total amount of ethanol consumed
chronically.
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It is also proposed that the infiltration of inflammatory cells plays a
key role in the progression of pancreatic damage (Fig. 9). Infiltrating
leukocytes such as lymphocytes and macrophages can be activated by
gut-derived endotoxin and/or other stimuli to produce oxidants after
alcohol consumption (Fig. 9). Indeed, 4 wk of high-dose ethanol
increased endotoxin levels in portal blood (e.g., 149 ± 17 pg/ml), and free radical formation (Figs. 6 and 7) was ~2.5-fold
higher than in a previous study (16) performed with the
standard ethanol protocol. Furthermore, inflammatory foci increased
significantly in the high-dose group compared with those in the
standard group in this study (Table 1). There are already links in the
literature between acute pancreatitis, endotoxins, and inflammatory
cells. For example, endotoxemia predicts outcome in acute pancreatitis
in humans (34), and gut sterilization with antibiotics
protects against acute pancreatitis in rats (19). Inflammatory cell infiltration is also known to correlate well with the
prognosis of pancreatitis in humans and in animal models (2, 7,
25, 33). The question remains whether these mechanisms are also
important during the chronic pancreatitis caused by alcohol, and this
question will be the focus of future research.
Clinical implications.
The Tsukamoto-French enteral alcohol model has a strong nutritional
component, with liver injury dependent on unsaturated fat in the diet
(21). In this study, pancreatic injury caused by the high
dose of ethanol was prevented by medium-chain triglycerides (Table 1).
Importantly, in this study, increases in fibrosis were observed only
after 8 wk (Figs. 4 and 5), leading to the conclusion that chronic
alcohol-induced pancreatic injury is dependent on the total amount of
alcohol consumed and the type of dietary fat. Furthermore, this is the
first demonstration of reproducible chronic alcohol-induced
pancreatitis in a relatively short experimental period. Thus this
animal model may be useful for the study of mechanisms of chronic
alcohol-induced pancreatitis and for developing useful therapeutic strategies.
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ACKNOWLEDGEMENTS |
This study was supported, in part, by grants from the National
Institute on Alcohol Abuse and Alcoholism.
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FOOTNOTES |
Address for reprint requests and other correspondence: R. G. Thurman, Dept. of Pharmacology, CB#7365, Mary Ellen Jones Bldg., Univ. of North Carolina, Chapel Hill, NC 27599-7365 (E-mail:
thurman{at}med.unc.edu).
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 13 September 2000; accepted in final form 7 January 2001.
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