Department of Physiology and the Alcohol Research Center, Louisiana
State University Medical Center, New Orleans, Louisiana 70112-1393
Endotoxemia is associated with alcoholic liver
diseases; however, the effect of endotoxin on the oxidation of ethanol
is not known. We tested the hypothesis that endotoxin treatment
enhances hepatic ethanol radical production. The generation of free
radicals by the liver was studied with spin-trapping technique
utilizing the primary trap ethanol (0.8 g/kg) and the secondary
trap
-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN; 500 mg/kg). Electron paramagnetic resonance (EPR)
spectra of bile showed six-line signals, which were dependent on
ethanol, indicating the trapping of ethanol-dependent radicals.
Intravenous injections of Escherichia
coli lipopolysaccharide (0.5 mg/kg) 0.5 h before 4-POBN
plus ethanol treatment caused threefold increases of biliary radical
adducts. EPR analyses of bile from
[1-13C]ethanol-treated
endotoxic rats showed the presence of species attributable to
-hydroxyethyl adduct, carbon-centered adducts, and
ascorbate radical. The generation of endotoxin-induced increases of
ethanol-dependent radicals was suppressed by 50% on
GdCl3 (20 mg/kg iv) or
desferrioxamine mesylate (1 g/kg ip) treatment. Our data show that in
vivo endotoxin increases biliary ethanol-dependent free radical
formation and that these processes are modulated by Kupffer cell
activation and catalytic metals.
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INTRODUCTION |
ETHANOL METABOLISM in the liver results in increased
oxidative stress, and this has been implicated in the pathogenesis of alcohol-induced injury (28). With chronic ethanol feeding,
oxidation of lipids and proteins was correlated with liver pathology
(37). Similarly, erythrocyte malondialdehyde and plasma lipid
hydroperoxide contents were increased in cirrhotic patients (16). These
results emphasize the possible role of free radical-induced tissue
injury resulting from chronic alcohol consumption.
Applications of electron paramagnetic resonance (EPR) spin-trapping
technique for detection of
-hydroxyethyl radical in
response to ethanol intake have provided some additional insight into
the understanding of ethanol metabolism in the liver. In vivo
spin-trapping studies after acute ethanol treatment of chronically
alcohol-fed animals have identified some components affecting
-hydroxyethyl radical formation, including fat content in the diet
(22), the role of Kupffer cells (KC), which are the hepatic resident
macrophages (21), and cytochrome
P-4502E1 (3). These factors correlate with the extent of liver injury in chronically alcohol-fed animals (3,
21). Endotoxin (which is elevated in the plasma of these animals) has
been shown to contribute to the severity of alcohol-induced liver
injury (7, 30, 38) and that intestinal sterilization elicits protection
(2). In addition, endotoxemia is associated with alcoholic
liver diseases in humans (9).
In vivo spin-trapping technique in alcohol-treated rats has been
successful for the trapping of
-hydroxyethyl radical, which is a
result of the reaction of ethanol with primary highly reactive radical
species, such as hydroxyl, lipid alkoxyl radicals, or iron in perferryl
states. The detection of
-hydroxyethyl radical is of importance in
alcohol-induced liver injury, because its redox potential, at pH 7.0, is relatively high, being close to those of superoxide radical anion
and peroxyl radicals (E0'
CH3C ·HOH/CH3CH2OH = 0.98 V;
E0'
OO ·/H2O2 = 0.94 V; and E0'
ROO ·/ROOH = 1.0 V) (25). Because
-hydroxyethyl radical itself is a relatively strong oxidant, its
formation in high concentrations may play a role in hepatotoxicity in
the case of chronically alcohol-fed rats. However, with acute alcohol
intake,
-hydroxyethyl radical formation may be a result of
detoxification processes as ethanol reacts with highly cytotoxic
species to form the lesser reactive
-hydroxyethyl radical.
The detection of ethanol-derived or -dependent free radicals can be
used as a marker of potential changes in hepatic pathophysiology. Kupffer cell-dependent superoxide radical generation after acute alcohol (6) or in vivo endotoxin (5) treatment has been implicated in
acute hepatic inflammatory conditions. However, it is not
known if endotoxin also affects the oxidation of ethanol to form
-hydroxyethyl radical or if this event has any influence on
endotoxin-induced hepatic injury.
In this study, our experimental methodology utilizes ethanol as a
primary trapping agent of highly oxidant species, forming
-hydroxyethyl radical, which is then trapped by the secondary trapping agent
-(4-pyridyl-1-oxide)-N-t-butylnitrone
(4-POBN). We aimed 1) to initially
develop the secondary spin-trapping protocol in bile using 4-POBN and
ethanol in naive animals, 2) to
investigate the effects of endotoxin on
-hydroxyethyl and
ethanol-dependent radical formation, and
3) to investigate the possible
sources of oxidants that affect radical adduct formation after acute
ethanol with or without endotoxin treatment.
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EXPERIMENTAL METHODS |
Animal preparation.
Male Sprague-Dawley rats (300-350 g; Hilltop Laboratory Animals,
Scottdale, PA) were housed in a controlled environment, exposed to a
12:12-h light-dark cycle, and provided with standard rodent chow
(Purina Mills, St. Louis, MO) and water ad libitum for at least 1 wk
before the experimental procedures were initiated. All experiments were
performed in accordance with the guidelines for care and use of
laboratory animals approved by the Louisiana State Medical Center New
Orleans Animal Care Committee.
Surgical procedure.
On the day before an experiment, animals were anesthetized with an
intramuscular injection of ketamine (90 mg/kg) and xylazine (9 mg/kg). Using aseptic surgical techniques, we placed a
catheter in the right jugular vein for intravenous injection of
spin-trap solutions, endotoxin, or
GdCl3. One day after jugular vein
catheter placement, fasted rats were anesthetized, laparotomy was
performed, and bile ducts were cannulated using 7- to 10-cm segments of
PE-50 tubing (Becton-Dickinson, Parsippany, NJ). At the end of the
surgical procedure, muscle and skin layers were closed with wound clips and the end of the catheter was exteriorized for bile collection. Bile
cannulation typically took ~10 min. Rats were placed on a warm
heating pad, and anesthesia was maintained during bile collection.
Treatment protocols.
To minimize free radical formation in spin-trap solutions before
intravenous administration in rats, the solvent saline was treated with
Chelex (11) [by stirring 10 g washed Chelex 100 (Bio-Rad,
Hercules, CA) in 100 ml saline overnight] and added with
desferrioxamine mesylate (DF). EPR spectra of 4-POBN (150 mg/ml) in
chelexed saline solution containing 0.3 mM DF showed weak six-line
signals, which were not increased by addition of ethanol (30%, final
concn), indicating that ethanol-dependent radical was not present in
spin-trap cocktail before intravenous injection. If such
treatment was ignored (by injecting 4-POBN plus ethanol dissolved in
untreated saline), free radical adduct formation in bile was 70%
higher [EPR intensities of saline group and saline plus DF group
were 2.28 ± 0.07 and 1.33 ± 0.12 U
(n = 3), respectively]. With the
intravenous injection protocol in our study, we found the presence of
three-line nitroxide signals [nitrogen hyperfine coupling constant
(aN) = 16.7 G]
from plastic additives of syringes (12) in plasma but not in
bile.
Figure 1 shows the experimental protocol
used in this study. Mixture of 4-POBN (350 or 500 mg/kg) and ethanol
(0.8 g/kg) in 1.0 ml chelexed saline containing 0.3 mM DF was used as
spin-trap cocktail for intravenous injection (over ~3 min). In some
experiments, gavaging rats with either saline or ethanol (10 ml of 40%
ethanol in saline per kg) was performed 30 min before intravenous
injection of 4-POBN (500 mg/kg) prepared in 1.0 ml chelexed saline
containing 0.3 mM DF. After bile cannulation, 4-POBN or 4-POBN plus
ethanol mixture was injected and bile collection commenced. Up to six bile samples (0.5 ml each) were collected into plastic Eppendorf tubes
during successive ~15- to 20-min intervals. Iron and copper are
normally released into the bile and thus are capable of reacting with
biliary lipid hydroperoxides and/or thiols to form free
radicals in the collection tubes. To prevent this ex vivo free radical formation, a 25-µl aliquot (prepared in chelexed saline) of iron chelator 2,2'-dipyridyl (1 mM, final concn) and copper
chelator bathocuproinedisulfonic acid (0.5 mM, final concn) was added
into each collection tube (20). Metal chelators were not added into tubes for experiments designed for biliary iron determination. Blood
was collected from the inferior vena cava, and prepared plasma samples
were subsequently stored at
20°C. Bile samples were frozen
immediately on dry ice and stored at
70°C.

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Fig. 1.
Experimental protocol for spin-trapping in the bile of naive and
lipopolysaccharide (LPS)-treated rats. 4-POBN,
-(4-pyridyl-1-oxide)-N-t-butylnitrone.
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In experiments with lipopolysaccharide (LPS) treatment, the rats
received an intravenous injection of LPS (0.5 mg/kg) or an equal volume
of saline 0.5 or 1.5 h before bile duct cannulation. The lyophilized
LPS was prepared by sonication in isotonic nonpyrogenic saline and
filtered through a 0.45-mm sterile filter (Millex). Aliquots of 1 mg/ml
LPS were kept at
70°C until use. Injection schedules for the
other treatment groups were as follows:
GdCl3 (20 mg/kg) dissolved in
acidic saline was injected intravenously 24 h before bile cannulation
(jugular vein catheters were placed 1 day before
GdCl3 injection), and DF (1 g/kg)
was administered intraperitoneally 1 h before bile cannulation.
Experiments with either GdCl3 or
DF treatment were performed with a paired control. Each pair of bile
samples was treated identically throughout the experiments. From EPR
data, we determined the inhibitory effects of
GdCl3 or DF from paired samples.
EPR measurements.
Radical adducts as nitroxides when formed in vivo are reduced by
endogenous reductants to form hydroxylamines that are not detectable by
EPR. To retrieve EPR signals of 4-POBN radical adducts, an aliquot of
potassium ferricyanide was used to oxidize hydroxylamines back to
nitroxides (39). The final concentration of potassium ferricyanide
giving optimum EPR signals was 0.5 mM, which was used in all the bile
samples. To test whether the added potassium ferricyanide is capable of
oxidizing ethanol (that might have been present in bile samples),
potassium ferricyanide (0.5 mM) was added to a mixture of
[1-13C]ethanol (50 mM)
and 4-POBN (20 mM) in 0.1 M phosphate buffer, pH 7.4. No 4-POBN radical
adducts were detected, thus excluding such a possibility.
We analyzed more than one bile sample from each rat, typically bile
samples
4-6
(which produced the strongest radical adduct signals), by EPR
spectroscopy. The EPR spectra were recorded at room temperature on an
ER 200D spectrometer operated at 9.72 GHz with a 100-kHz modulation
frequency. After addition of potassium ferricyanide, the samples were
pipetted to a quartz flat cell, which was then centered in a
TE011 cavity. The data were
transferred to an IBM personal computer where multiple spectra from a
sample were digitally accumulated to achieve an acceptable
signal-to-noise spectrum. For some spectra, computer-simulation
analyses were performed using the software described by Duling (17) to
determine hyperfine coupling constants of 4-POBN radical adducts.
Biochemical analyses.
Total glutathione content in bile samples that had been collected into
the iron and copper chelator mixture was determined using
5,5'-dithio-bis(2-nitrobenzoic acid), where glutathione reductase
was used to convert oxidized glutathione to glutathione, and total
glutathione was determined (18).
To prevent interference from 4-POBN, experiments without 4-POBN
injections and without the iron and copper chelator mixture in the bile
collection tubes were performed to investigate LPS effects on biliary
iron contents. Iron(II) concentrations were determined by adding 100 µl of bile into 900 µl of 0.5 mg/ml 2,2'-dipyridyl in glacial
acetic acid. The absorbances were measured at 522 nm using a Hitachi
U-2000 spectrophotometer (20). Plasma from these rats was subjected to
aspartate aminotransferase, alanine aminotransferase, and ethanol
assays (diagnostic kits were from Sigma Chemical, St. Louis, MO).
Materials.
DF, 2,2'-dipyridyl, bathocuproinedisulfonic acid, glutathione,
sulfosalicylic acid, 5,5'-dithio-bis(2-nitrobenzoic acid), glutathione reductase, and
ethyl-1-13C alcohol (98% atom
13C) were purchased from Sigma
Chemical. 4-POBN was obtained from Aldrich (Milwaukee, WI). Ethyl
alcohol (200 proof) was obtained from Quantum Chemical (Tuscola, IL).
Escherichia coli (0111:B4) LPS was
obtained from Difco.
Statistical analysis.
Analysis of variance statistical methods were applied in Fig. 4 where
pairwise comparisons were made using Fisher's protected least
significant difference test. Single-sample
t-test analyses were applied in Fig.
8. Data were considered statistically significant at
P < 0.05.
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RESULTS |
Use of low-dose ethanol as a marker of oxidant formation in vivo.
Initial experiments were performed to develop a spin-trapping protocol
using ethanol and 4-POBN as outlined in Fig. 1. When a relatively low
dose of ethanol (0.8 g/kg) was injected intravenously together with
4-POBN (500 mg/kg), an EPR spectrum of the bile collected 2 h later
showed very weak six-line signals indicative of 4-POBN radical adducts.
This radical adduct formation was consistent with the study by Moore et
al. (29) in which ethanol and 4-POBN were injected into the inferior
vena cava. After signal averaging by digitally accumulating three EPR
spectra, EPR signals were improved to an acceptable signal-to-noise
spectrum (Fig.
2A). Experiments with ethanol injection after 4-POBN with the same dosages
reproduced a similar EPR spectrum (Fig.
2B). 4-POBN radical adduct formation
increased over time from the measured bile samples 1-6
(not shown). Intravenous injection of 4-POBN alone resulted in almost
nondetectable signals (Fig. 2C).
Data in Fig. 2 are representative spectra from four experiments.

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Fig. 2.
Electron paramagnetic resonance (EPR) spectra of radical adducts
detected in bile samples from rats treated with 4-POBN (500 mg/kg) and
ethanol (0.8 g/kg). A: rat was
administered 4-POBN + ethanol mixture prepared in chelexed saline
containing 0.3 mM desferrioxamine mesylate (DF).
B: rat was administered 4-POBN
followed by an injection with 25% ethanol in chelexed saline
containing 0.3 mM DF. C: same as
A, except that ethanol was replaced
with saline. Spectrometer conditions were as follows: modulation
amplitude, 0.63 G; microwave power, 20 mW; time constant, 0.5 s; scan
range, 80 G; scan time, 410 s, accumulated over 3 scans.
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Another set of experiments was performed using intragastric ethanol
administration 30 min before bile cannulation and subsequent intravenous injection of 4-POBN. Results (including the control experiments) similar to those shown in Fig. 2 were obtained and shown
to be reproducible from four experiments (not shown). Data from these
initial bile experiments demonstrated that, in naive rats, ethanol
could be used as a primary spin-trap to form radical species that were
trapped by a secondary spin-trap 4-POBN. The EPR spectrum of radical
adduct in Fig. 2A was computer
simulated to extract hyperfine coupling constants of
aN = 15.7 G and
= 2.76 G, which are similar to reported parameters for 4-POBN adducts of carbon-centered radicals (27). As we did not know the chemical structures of these
4-POBN radical adducts, we tentatively designated these ethanol-dependent free radicals as carbon-centered radicals.
Effects of in vivo endotoxin on ethanol-dependent free radical
formation.
With the convenience of intravenous injection using the jugular vein
catheter, we used the spin-trapping protocol outlined in Fig. 1 in
these studies. To investigate the effects of endotoxin, LPS (0.5 mg/kg)
was injected 0.5 h before bile cannulation. With this protocol (Fig.
1), LPS treatment for ~2.5 h did not consistently increase plasma
aspartate and alanine transaminase activities. LPS treatment increased
the formation of biliary radical adducts (Fig.
3B).
Spin-trapping experiments using 4-POBN alone in LPS-treated rats
resulted in much weaker radical adducts than those obtained from
experiments using 4-POBN plus ethanol (Fig.
3C).

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Fig. 3.
Endotoxin effects on biliary 4-POBN radical adducts in rats injected
with 500 mg/kg 4-POBN and 0.8 g/kg ethanol (as outlined in Fig. 1).
A: rat was injected with 4-POBN + ethanol solution. B: rat was
pretreated with LPS (0.5 mg/kg) before 4-POBN + ethanol injection.
C: rat was pretreated with LPS (0.5 mg/kg) before 4-POBN injection. Spectrometer conditions were the same
as in Fig. 2 legend.
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EPR spectra from five bile samples after intravenous injection of
4-POBN in naive rats showed almost nondetectable signals (e.g., Fig.
2C). The mean value of these signal
intensities was used as a background (or noise) level to subtract from
those values from all other experiments using 4-POBN. The absolute EPR
intensities of radical adducts, associated with LPS and/or
ethanol treatments, are shown in Fig.
4. Coadministration of ethanol
with 4-POBN or LPS treatment significantly increased biliary formation
of 4-POBN radical adducts (P < 0.05 vs. 4-POBN). LPS treatment of rats markedly increased
radical adduct formation by threefold in 4-POBN plus ethanol-injected
rats (P < 0.0001 vs. 4-POBN plus
ethanol) and by twofold in 4-POBN-injected rats
(P < 0.05 vs. 4-POBN). Among the
LPS-treated groups, coadministration of ethanol with 4-POBN increased biliary radical adduct formation by fivefold
(P < 0.0001 vs. 4-POBN plus LPS).
The effects of LPS on ethanol-dependent radical adduct formation were
not additive but rather synergistic [i.e., (LPS plus ethanol) > (ethanol) plus (LPS)]. This synergistic effect implies that the
potentiation of biliary ethanol-dependent radical formation due to LPS
treatment was likely to have resulted from free radical chain reactions
initiated by ethanol radical.

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Fig. 4.
Relative EPR signal intensities of bile samples from rats injected with
4-POBN alone, and 4-POBN + ethanol (as outlined in Fig. 1). Dosage of
4-POBN used in these experiments was 350 mg/kg. The mean value of EPR
signals from bile from rats treated with 4-POBN alone (considered noise
levels) was subtracted from those signals from bile samples from other
experiments. Ethanol treatment with or without LPS significantly
increased biliary radical adduct formation
(* P < 0.005, 4-POBN vs.
4-POBN + ethanol or 4-POBN + ethanol + LPS). LPS treatment
significantly increased biliary radical adduct
(* P < 0.05, 4-POBN vs. 4-POBN + LPS; P < 0.0001, 4-POBN + ethanol vs. 4-POBN + ethanol + LPS). Ethanol treatment
increased radical adduct formation in bile of rats pretreated with LPS
( P < 0.0001, 4-POBN + LPS vs. 4-POBN + ethanol + LPS). Data are means ± SE;
n = 4-7. Spectrometer conditions
were the same as in Fig. 2 legend, except that 4 scans were
accumulated.
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Characterization of LPS-induced ethanol-dependent radical adducts.
Further spin-trapping experiments were performed to investigate the
time course of LPS effects. Radical adduct formation in rats pretreated
with LPS for 0.5 h was greater than in rats pretreated with LPS for 1 h
(Fig. 5). These experiments with paired
timed controls were reproducible in three experiments. It is noted that the signal intensity of Fig. 5B was
comparable with that of that spectra from non-LPS-treated rats (see
Fig. 2A), indicating that ethanol-dependent free radical production induced by LPS occurred at an
early time.

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Fig. 5.
Time-dependent LPS-induced 4-POBN radical adduct formation in bile.
A: an EPR spectrum of radical adducts
detected in bile from rat injected with LPS (0.5 mg/kg) 30 min before
bile cannulation. B: same as
A, except that rat was injected with
LPS (0.5 mg/kg) 1 h before bile cannulation. Spectrometer conditions
were the same as in Fig. 2 legend.
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An EPR spectrum accumulated over eight scans of a bile sample from a
4-POBN plus
[1-13C]ethanol-injected
rat that had been treated with LPS exhibited a component showing a
twelve-line hyperfine structure indicative of
13C (which has nuclear spin equal
to 1/2) from
[13C]ethanol (Fig.
6A). The
composite simulation in Fig. 6B
exhibited a composite of three species attributable to a
[13C]ethanol radical
adduct
[4-POBN/13· CH (OH)CH3,
Fig. 6C], a carbon-centered
radical adduct (4-POBN/· R, Fig.
6D), and the ascorbate semidione
radical (Fig. 6E).
Mole ratio calculations of individual species indicated that at least 38% of the total free radicals were an adduct of
-hydroxyethyl radical from the administered ethanol. Ascorbate radical contributed 7% of the total free radicals, and the remaining 55% were
4-POBN/· R. The latter radical adduct
species resulted from the trapping of free radicals that did not
originate from ethanol moiety and are assumed to be carbon-centered
radical adducts (27).