In vivo endotoxin enhances biliary ethanol-dependent free radical generation

Walee Chamulitrat, Jean Carnal, Nicole M. Reed, and John J. Spitzer

Department of Physiology and the Alcohol Research Center, Louisiana State University Medical Center, New Orleans, Louisiana 70112-1393

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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 alpha -(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 alpha -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.

spin-trapping; Kupffer cells; alpha -hydroxyethyl radical; lipopolysaccharide; oxidants

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -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, alpha -hydroxyethyl radical formation may be a result of detoxification processes as ethanol reacts with highly cytotoxic species to form the lesser reactive alpha -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 alpha -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 alpha -hydroxyethyl radical, which is then trapped by the secondary trapping agent alpha -(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 alpha -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.

    EXPERIMENTAL METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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, alpha -(4-pyridyl-1-oxide)-N-t-butylnitrone.

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.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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.

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 <IT>a</IT><SUP>H</SUP><SUB>&bgr;</SUB> = 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.

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; ddager  P < 0.0001, 4-POBN + ethanol vs. 4-POBN + ethanol + LPS). Ethanol treatment increased radical adduct formation in bile of rats pretreated with LPS (dagger  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.

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.

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 alpha -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).



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