0022-3565/00/2931-0296$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics JPET 293:296–303, 2000
Vol. 293, No. 1 Printed in U.S.A.
Allopurinol Prevents Early Alcohol-Induced Liver Injury in Rats1 HIROSHI KONO, IVAN RUSYN, BLAIR U. BRADFORD, HENRY D. CONNOR, RONALD P. MASON, and RONALD G. THURMAN Laboratory of Hepatobiology and Toxicology, Department of Pharmacology (H.K., I.R., B.U.B., H.D.C., R.G.T.), and Curriculum in Toxicology (I.R., R.P.M., R.G.T.), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; and Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina (H.D.C., R.P.M.) Accepted for publication January 4, 2000
This paper is available online at http://www.jpet.org
ABSTRACT Free radical formation caused by chronic ethanol administration could activate transcription factors such as nuclear factor-B (NF-B), which regulates production of inflammatory cytokines. Xanthine oxidase is one potential source of reactive oxygen species. Therefore, the purpose of this study is to determine whether allopurinol, a xanthine oxidase inhibitor and scavenger of free radicals, would affect free radical formation, NF-B activation, and early alcohol-induced liver injury in rats. Male Wistar rats were fed a high-fat diet with or without ethanol (10 –16 g/kg/day) continuously for up to 4 weeks with the Tsukamoto-French enteral protocol. Either allopurinol or saline vehicle was administered daily. Allopurinol had no effect on body weight or the cyclic pattern of ethanol in urine. Mean urine ethanol concentrations were 271 ⫾ 38 and 252 ⫾ 33 mg/dl in ethanol- and ethanol ⫹ allopurinol-treated rats, respectively. In
The establishment of a continuous intragastric in vivo enteral feeding protocol in the rat by Tsukamoto and French (Tsukamoto et al., 1984) represented a major development in alcohol research (French et al., 1986; Tsukamoto et al., 1990). With this model, not only is steatosis observed, which is characteristic of several animal models, but also inflammation and necrosis occur in ⬃2 to 4 weeks and fibrosis begins to develop in 12 to 16 weeks. Gram-negative bacterial species are a major source of endotoxin in the gut microflora (Bode et al., 1984) and blood endotoxin levels increase with alcohol (Fukui et al., 1991). Endotoxin activates Kupffer cells that produce free radicals (e.g., superoxide and nitric oxide) (Decker et al., 1989), leading to liver injury (Knecht et al., 1995). Indeed, intestinal sterilization with antibiotics (Adachi et al., 1995) and suppression of endotoxin production with lactobacillus feeding (Nanji et al., 1994) minimize alcohol-induced liver injury in the Tsukamoto-French enteral model. Moreover, GdCl3 Received for publication August 26, 1999. 1 This work was supported in part by grants from the National Institute of Alcohol Abuse and Alcoholism.
the control group, serum aspartate aminotransferase and alanine aminotransferase levels were ⬃40 I.U./l and 25 U/l, respectively. Administration of enteral ethanol for 4 weeks increased serum transaminases ⬃5-fold. Allopurinol blunted these increases significantly by ⬃50%. Ethanol treatment also caused severe fatty infiltration, mild inflammation, and necrosis. These pathological changes also were blunted significantly by allopurinol. Furthermore, enteral ethanol caused free radical adduct formation, values that were reduced by ⬃40% by allopurinol. NF-B binding was minimal in the control group but was increased significantly nearly 2.5-fold by ethanol. This increase was blunted to similar values as control by allopurinol. These results indicate that allopurinol prevents early alcoholinduced liver injury, most likely by preventing oxidant-dependent activation of NF-B.
treatment prevents free radical formation and early alcoholinduced liver injury in the enteral alcohol model (Adachi et al., 1994). Furthermore, inactivation of Kupffer cells with GdCl3 prevents the hypermetabolic state caused by acute ethanol (swift increase in alcohol metabolism, SIAM) (Bradford et al., 1993). Additionally, sterilization of the gut with antibiotics blocks SIAM (Rivera et al., 1998) and it has recently been reported that the cyclooxygenase inhibitor indomethacin blocks SIAM, supporting the hypothesis that mediators of the Kupffer cell such as prostaglandin E2 (Bradford et al., 1999) are necessary for increasing the oxygen gradient in the liver after alcohol. Collectively, these data are consistent with the hypothesis that oxidants from Kupffer cells activated by gutderived endotoxin are involved in early alcohol-induced liver injury (Thurman, 1998). It is known that if Kupffer cells are destroyed with GdCl3, generation of ␣-hydroxyethyl radicals is blocked in the enteral ethanol model in vivo (Knecht et al., 1995). Tumor necrosis factor-␣ (TNF-␣) has been shown to cause alcoholinduced liver injury based on studies with anti-TNF antibody
ABBREVIATIONS: SIAM, swift increase in alcohol metabolism; TNF-␣, tumor necrosis factor-␣; NF-B, nuclear factor-B; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ESR, electron spin resonance; POBN, ␣-(4-pyridyl-1-oxide)-N-t-butylnitrone; DPI, diphenyleneiodonium; ICAM-1, intercellular adhesion molecule. 296
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(Iimuro et al., 1997a) and in TNF receptor-1 knockout mice (Yin et al., 1999). However, activation of nuclear factor-B (NF-B), which regulates production of inflammatory cytokines (i.e., TNF-␣) (Thurman, 1998) has not been examined in relation to the generation of free radicals. Because Kupffer cells contain xanthine oxidase, which is one potential source of free radicals, the purpose of this study was to determine whether allopurinol, a compound shown to be both an inhibitor of xanthine oxidase and a free radical scavenger (Wiezorek et al., 1994) would alter free radical production, NF-B activity, and early alcohol-induced injury in rats. Allopurinol is shown herein to have a protective effect. Preliminary accounts of this work have appeared elsewhere (Kono et al., 1998).
was analyzed by gas chromatography to verify that levels were not different between the groups when experiments were initiated (Glassman et al., 1989). Rats were anesthetized with pentobarbital (75 mg/kg) and the proximal bile duct was cannulated with polyehtylene-10 tubing. After the spin trapping reagent ␣-(4-pyridyl-1-oxide)N-t-butylnitrone (POBN, 1 g/kg b.wt.; Sigma Chemical Co.) was injected slowly into the tail vain, bile samples were collected at 30-min intervals for 3 h into 35 l of 0.5 mM Desferal (deferoxamine mesylate; Sigma Chemical Co.) to prevent ex vivo radical formation. Samples were stored at ⫺80°C until analysis of free radical adducts by ESR spectroscopy as described elsewhere (Knecht et al., 1995). Specifically, samples were thawed, transferred in 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 (Duling, 1994). 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 of chart paper). Nuclear Protein Extraction and Gel Mobility Shift Assay. Binding conditions for NF-B were characterized and electrophoretic mobility shift assays were performed as described in detail elsewhere (Zabel et al., 1991). Briefly, nuclear extracts (40 g) from liver tissues were preincubated for 10 min on ice with 1 g of poly(dI-dC) and 20 g of 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, 0.05 mM EDTA, and 2 l of a 32P-labeled DNA probe (10,000 cpm/l; Cerenkov) that contained 0.4 ng of double-stranded oligonucleotide was added and mixtures were incubated for 20 min on ice and resolved on 5% polyacrylamide (29:1 cross-linking) and 0.4⫻ Tris/boric acid/EDTA gel. After electrophoresis, gels were dried and exposed to Kodak film. Specificity of NF-B binding was verified by the competition assay 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 supershift experiments, 1 g of rabbit antisera against p50 or p65 protein (a kind gift of Dr. N. R. Rice, Advanced Bioscience Laboratories, National Cancer Institute) 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⬘) (Baeuerle and Baltimore, 1989). Data were quantitated by scanning autoradiograms with GelScan XL (Pharmacia LKB, Uppsala, Sweden). Statistics. ANOVA was used for the determination of statistical significance as appropriate. For comparison of pathological scores, the Mann-Whitney rank sum test was used. A P value ⬍.05 was selected before the study as the level of significance.
Materials and Methods Animals and Diets. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10 –16 g/kg/day) continuously for up to 4 weeks with the intragastric enteral feeding protocol developed by Tsukamoto and French (Tsukamoto et al., 1984; French et al., 1986). Either allopurinol (100 mg/kg/day as a bolus and 100 mg/kg/day in diet; Sigma Chemical Co., St. Louis, MO) or vehicle (saline, 0.5 ml/day) was administered daily based on protocols developed by Cohen (1992) and Karwinski et al. (1991). All animals received humane care in compliance with institutional guidelines. A liquid diet described by Thompson and Reitz (1978) supplemented with lipotropes as described by Morimoto et al. (1994) was used. It contained corn oil 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 isocaloric maltose-dextrin (control diet) as described elsewhere (Tsukamoto et al., 1990). Urine Collection and Ethanol Assay. Ethanol concentration in urine, which is representative of blood alcohol levels (Badger et al., 1993), 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 9:00 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 as described elsewhere (Bergmeyer, 1988). Blood Collection and Transaminase Determinations. Blood was collected via the tail vein once a week and centrifuged. Serum was stored at ⫺20°C until assayed for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) by standard enzymatic procedures (Bergmeyer, 1988). Pathological Evaluation. After 4 weeks of ethanol treatment, animals were sacrificed; livers were removed, sectioned, and fixed in formalin. Paraffin-embedded sections were stained with hematoxylin-eosin for histological evaluation of steatosis, inflammation, and necrosis. Liver pathology was scored as described by Nanji et al. (1989) as follows: steatosis (the percentage of liver cells containing fat): ⬍25% ⫽ 1⫹, ⬍50% ⫽ 2⫹, ⬍75% ⫽ 3⫹, 75%⬎ ⫽ 4⫹; inflammation and necrosis: 1 focus per low-power field ⫽ 1⫹; 2 or more foci ⫽ 2⫹. Pathology was scored in a blinded manner by one of the authors and by an outside expert in rodent liver pathology. The number of neutrophils in liver sections was determined after 4 weeks by counting cells in three high-power fields (400⫻) per slide. Fat accumulation causes 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 also was counted and the number of neutrophils was expressed per 100 hepatocytes. The mean values were used for statistical analysis. Collection of Bile and Detection of Free Radical by Electron Spin Resonance (ESR). Ethanol concentration in the breath
Results Body Weight. To allow for full recovery from surgery, liquid diets were initiated after 1 week. In spite of development of greater hepatic injury in ethanol-treated groups, all rats grew steadily, making nutritional complications an unlikely explanation for these results. Animals treated with allopurinol had no complications during the experimental period. The mean body weight gains were 3.9 ⫾ 0.4 g/day for the ethanol group and 3.8 ⫾ 0.2 g/day for the ethanol ⫹ allopurinol 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 allopurinol on body weight of rats. Male Wistar rats (275–300 g) were used in this study. Body weight was measured once a week. Data represent means ⫾ S.E. (n ⫽ 6). E, high-fat control diet; 䡺, high-fat control diet with allopurinol; F, high-fat ethanol-containing diet; and f, high-fat ethanol-containing diet with allopurinol.
Ethanol Concentrations in Urine. As it was reported previously by several groups (Tsukamoto et al., 1985; Adachi et al., 1994; Nanji et al., 1997), alcohol levels fluctuate in a cyclic pattern from zero to ⬎500 mg/dl for unknown reasons. Allopurinol had no effect on this cyclic pattern of ethanol in urine. There were no significant differences in mean urine alcohol concentrations between rats given ethanol (Fig. 2, top; 271 ⫾ 38 mg/dl) and ethanol ⫹ allopurinol (bottom; 252 ⫾ 33 mg/dl). Serum Transaminase Levels. In control groups, serum AST and ALT levels were ⬃60 and 25 U/l, respectively, after 4 weeks of high-fat control diet (Fig. 3). Administration of enteral ethanol for 4 weeks caused a 5-fold increase in transaminase levels. Allopurinol blunted this increase significantly by ⬃50%. Pathological Evaluation. In control groups, there were no pathological changes in the liver after 4 weeks on a highfat diet (Fig. 4). As expected, severe fat accumulation, mild inflammation, and necrosis were observed after 4 weeks of enteral ethanol feeding, resulting in a total pathology score of 5.3 ⫾ 0.3 (Fig. 5). Increases in the pathology scores were blunted significantly by ⬃60% by allopurinol (total pathology score 2.5 ⫾ 0.4). The number of infiltrating neutrophils in the liver was minimal and not different between the groups in the absence of ethanol; however, enteral ethanol for 4 weeks increased this number ⬃3-fold over control values (Fig. 6). This increase was significantly blunted by ⬃50% by allopurinol. Effects of Chronic Ethanol and Allopurinol on Free Radical Formation. POBN reacts with free radicals such as ␣-hydroxyethyl radical from ethanol to produce nitroxides detectable by ESR. Radical adducts were barely detectable in rats fed an ethanol-free, high-fat control diet in both groups
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.
(data not shown). In contrast, treatment with enteral ethanol for 4 weeks caused significantly free radical formation (Fig. 7). However, this increase was blunted significantly by allopurinol. ESR hyperfine coupling constants were aN ⫽ 15.70 G, and aH ⫽ 2.72 G, characteristic of the ␣-hydroxyethyl radical adduct (Knecht et al., 1995). 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 spectrum (Fig. 8). The intensity of these signals was increased significantly by enteral ethanol but was blunted by 60% by allopurinol. Effect of Chronic Ethanol and Allopurinol on Hepatic NF-B Activity. NF-B activity was minimal after 4
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quence for NF-B binding, or with antibodies specific for p50 or p65 subunit (Fig. 9B). In the absence of nuclear proteins, no protein-DNA complex was detected (lane 1). NF-B DNA binding activity was assessed from electrophoretic mobility shift assays with nuclear extracts prepared from liver of a rat fed high-fat ethanol-containing diet (lane 2). Furthermore, addition of anti-p50 or -p65 antiserum reduced the intensity of the complex and produced supershifted complexes with a higher molecular mass, respectively (lanes 3 and 4). Moreover, unlabeled oligonucleotide that contained the NF-Bbinding site could effectively compete for DNA binding with 32 P-labeled probe (lane 5).
Discussion
Fig. 3. Effect of chronic enteral ethanol and allopurinol on serum aspartate and aminotransferase levels. Blood samples were collected via the tail vein at 4 weeks and AST and ALT were measured as described in Materials and Methods. Data represent means ⫾ S.E. (n ⫽ 6). *P ⬍ .05 compared with rats fed high-fat control diet; #P ⬍ .05 compared with high-fat ethanol-containing diet by ANOVA and Bonferroni’s post hoc test.
weeks of high-fat control diet without ethanol (Fig. 9). After 4 weeks of enteral ethanol, however, NF-B activity was increased significantly nearly 2.5-fold over control values. This increase was blunted to similar values as control by allopurinol. To confirm that protein binding to labeled oligonucleotide probe was specific for the active form NF-B, gel shift assays were carried out either in the presence of an excess of unlabeled double-stranded oligonucleotide with a consensus se-
Possible Mechanism of Effect of Allopurinol on Early Alcohol-Induced Liver Injury. Xanthine oxidase generates reactive oxygen species such as superoxide anion and hydrogen peroxide (Roy and McCord, 1983). In the liver, Kupffer cells and endothelial cells, as well as hepatocytes, contain xanthine dehydrogenase that is readily converted into xanthine oxidase (Brass et al., 1991). Importantly, hypoxia occurs in the liver during enteral ethanol feeding (Arteel et al., 1997). Enteral ethanol has been shown to cause a steepening of the oxygen gradient across the liver lobule and to cause hypoxia in downstream pericentral regions (Arteel et al., 1997). During hypoxia, xanthine dehydrogenase is converted into xanthine oxidase significantly faster in Kupffer cells than that in other cell types. Upon reoxygenation, xanthine oxidase reacts with molecular oxygen to produce a burst of superoxide radicals that mediate subsequent tissue injury (Zhong et al., 1989). Thus, xanthine oxidase in Kupffer cells could be a potent source of oxidants in the enteral alcohol model. Allopurinol, a xanthine oxidase inhibitor and a free radical scavenger (Wiezorek et al., 1994), prevents liver injury due to ischemia-reperfusion by inhibition of free radical formation (Marotto et al., 1988). Moreover, it was reported recently from this laboratory that peroxisome proliferators (e.g., WY14,643 and monoethylhexylphthalate) activate Kupffer cells directly and that oxidants from Kupffer cells play a central role in NF-B activation and cell proliferation caused by peroxisome proliferators (Rose et al., 1999). Importantly, allopurinol blocks NF-B activation caused by WY-14 643 in the liver (Rusyn et al., 1998). Thus, allopurinol prevents oxidant-dependent activation of NF-B. Oxidants produced during enteral ethanol are most likely involved in the pathogenesis of early alcohol-induced liver injury (Thurman, 1998). Indeed, free radical formation was increased in bile from rats fed enteral ethanol (Knecht et al., 1995). Furthermore, the antioxidant diphenyleneiodonium (DPI) prevented free radical formation and liver injury nearly completely in the enteral alcohol model (Kono et al., 1999). DPI is an inhibitor of NADPH oxidase that is present in high levels in Kupffer cells and is a major source of superoxide in this cell type. In this study, allopurinol also blunted free radical formation in the liver (Figs. 7 and 8). Importantly, the effect of allopurinol on changes in pathology and free radical formation showed a significant correlation (r2 ⫽ 0.998). Thus, it is concluded that allopurinol prevents liver injury, most likely by inhibition of xanthine oxidase and/or scavenging of free radicals (Fig. 10).
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Fig. 4. Photomicrographs of livers after ethanol treatment. Livers from rats given high-fat control or high-fat ethanol-containing diets are shown. Original magnification, 100⫻. Representative photomicrographs of high-fat control diet ⫹ vehicle (A), high-fat control diet ⫹ allopurinol (B), high-fat ethanol-containing diet ⫹ vehicle (C), and high-fat ethanol-containing diet ⫹ allopurinol (D). With higher magnification (200⫻), E and F show inflammation (open arrow) and necrosis (filled arrow) in rats fed high-fat ethanol-containing diet ⫹ vehicle, and G and H depict histology without inflammation and necrosis in rats fed high-fat ethanol-containing diet ⫹ allopurinol.
Role of NF-B in Early Alcohol-Induced Liver Injury. NF-B is rapidly activated in response to immunological stimuli such as lipopolysaccharide, cytokines, and oxidants (Baldwin, 1996). Binding sites for NF-B have been identi-
fied within the regulatory elements of genes for several inflammatory cytokines such as TNF-␣. Thus, NF-B plays an important role in regulation of inflammatory responses. Increases in production of inflammatory cytokines and adhe-
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Fig. 5. Effect of chronic enteral ethanol and allopurinol on hepatic pathology scores. Pathology was scored as described in Materials and Methods. Steatosis, inflammation, and necrosis are shown individually. Data represent means ⫾ S.E. (n ⫽ 5). E, high-fat control diet; 䡺, high-fat control diet with allopurinol; F, high-fat ethanol-containing diet; f, and high-fat ethanol-containing diet with allopurinol. *P ⬍ .05 compared with rats fed high-fat control diet; #P ⬍ .05 compared with rats fed high-fat ethanol-containing diet by the Mann-Whitney rank sum test. Fig. 7. Effect of chronic enteral ethanol and allopurinol on ESR spectra. Rats were fed enteral liquid diets for 4 weeks intragastrically. After the spin trap reagent (POBN injection, 1 g/kg i.v.), bile was collected into Desferal (deferoxamine mesylate, 0.5mM) and analysis of ESR spectra was performed as described in Materials and Methods. Representative ESR spectra.
Fig. 6. Effect of chronic enteral ethanol and allopurinol on the number of neutrophils in the liver. The number of neutrophils observed in H&E sections of liver in control and ethanol-fed rats are shown. Values were determined by counting neutrophils in three high power fields (400⫻) per slide. The number of hepatocytes also was counted in each field and the number of cells was expressed per 100 hepatocytes. Data represent means ⫾ S.E. (n ⫽ 5). *P ⬍ .05 compared with rats fed high-fat control diet; #P ⬍ .05 compared with rats fed high-fat ethanol-containing diet with vehicle by ANOVA with Bonferroni’s post hoc test.
sion molecules by NF-B activated by oxidants could be one explanation for the pathogenesis of early alcohol-induced liver injury. Indeed, NF-B activation was increased significantly by enteral ethanol (Kono et al., 1999). Furthermore, DPI prevented free radical formation, NF-B activation, and
Fig. 8. Effect of chronic enteral ethanol and allopurinol on average radical adduct signal intensity. Conditions were the same as for Fig. 7. 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. Data represent means ⫾ S.E. (n ⫽ 4). VEH, vehicle; ALLO, allopurinol. *P ⬍ .05 compared with rats fed high-fat ethanol-containing diet with vehicle by ANOVA and Bonferroni’s post hoc test.
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Fig. 10. Working hypothesis. Chronic ethanol administration causes free radical formation in the liver. These radicals could be involved in triggering liver injury by increasing transcription factors such as NF-B, which induce inflammatory cytokine productions. Kupffer cells play an important role in this process because xanthine oxidase in Kupffer cells responds to stress much faster than in hepatocytes. In this study, allopurinol, a xanthine oxidase inhibitor and a free radical scavenger, prevented NF-B activity and early alcohol-induced liver injury, most likely by inhibition of free radical formation or scavenging of radical species.
Fig. 9. Effect of chronic enteral ethanol and allopurinol on hepatic NF-B activity. A, nuclear extracts (40 g 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 NF-B/DNA complex image in livers of male rats fed high-fat control diet ⫹ vehicle was set to 100%. Data represent means ⫾ S.E. (n ⫽ 4). VEH, vehicle; ALLO, allopurinol. *P ⬍ .05 compared with rats given high-fat control diet; #P ⬍ .05 compared with male rats given ethanol-containing diet by ANOVA with Bonferroni’s post hoc test. B, lane 1 shows labeled probe with no nuclear extract NF-B added. NF-B DNA-binding activity was assessed from electrophoretic mobility shift assays with nuclear extracts prepared from livers from of rats fed high-fat control diet (lane 2). Nuclear extracts from rats fed high-fat ethanol-containing diet were used for supershift experiments with antibodies specific for p50 or p65 subunit as described in Materials and Methods (lanes 3 and 4). In competition assays, 200-fold excess of the unlabeled oligonucleotide was used (lane 5).
liver injury in the enteral alcohol model. Moreover, allopurinol blunted these increases significantly in this study (Figs. 5, 7, and 9). Furthermore, the effect of allopurinol on the activation of NF-B by ethanol correlated with both changes in pathology (r2 ⫽ 0.659) and free radical formation (r2 ⫽
0.622). These results indicate that oxidant-dependent activation of NF-B plays an important role in early alcohol-induced liver injury (Fig. 10). NF-B could increase TNF-␣ production (Watanabe et al., 1996), which plays an important role in the inflammatory cytokine cascade (Decker et al., 1989). Recent evidence has accumulated supporting the hypothesis that TNF-␣ plays an essential role in early alcohol-induced liver injury. Indeed, ethanol increases TNF-␣ mRNA expression in the liver in the Tsukamoto-French model (Iimuro et al., 1997a,b). Furthermore, anti-TNF-␣ antibody reduces inflammatory cell infiltration and necrosis in the enteral alcohol model (Iimuro et al., 1997a). Moreover, alcohol-induced liver injury in wildtype mice fed ethanol is prevented in TNF receptor-1 knockout mice (Yin et al., 1999). TNF-␣ stimulates endothelial cells to synthesize adhesion molecules [e.g., intercellular adhesion molecule (ICAM-1)], leading to liver injury (Bevilacqua et al., 1987; Dustin and Springer, 1988; Yu et al., 1995). Indeed, expression of TNF-␣ mRNA and ICAM-1 as well as the number of neutrophils in the liver are increased in the Tsukamoto-French enteral model (Fig. 6) (Iimuro et al., 1997a,b). Furthermore, alcohol-induced liver injury in wild-type mice fed enteral ethanol was prevented in ICAM-1 knockout mice (data not shown). Taken together, TNF-␣ plays an essential role in the mechanism of early alcohol-induced liver injury. Allopurinol most likely inhibits TNF-␣ production by oxidant-dependent NF-B activation in the liver (See Fig. 10).
Conclusion We propose that allopurinol prevents early alcohol-induced liver injury by preventing NF-B activation, most likely by inhibiting xanthine oxidase and/or scavenging oxidants. Kupffer cells most likely play a pivotal role in this process, resulting in subsequent increase in inflammatory cytokines such as TNF-␣ (Fig. 10).
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References
attenuates hepatic necrosis and inflammation caused by chronic intragastric ethanol exposure in rats. Clin Nutr 17:127. Kono H, Rusyn I and Thurman RG (1999) Diphenyleneiodonium, an NADPH oxidase inhibitor, prevents early alcoholic liver injury by chronic intragastric ethanol exposure in rats. Toxicologist 48:257. Marotto M, Thurman RG and Lemasters JJ (1988) Early midzonal cell death during low-flow hypoxia in the isolated, perfused rat liver: Protection by allopurinol. Hepatology 8:585–590. Morimoto M, Zern MA, Hagbjork AL, Ingelman-Sundberg M and French SW (1994) Fish oil, alcohol, and liver pathology: Role of cytochrome P4502E1. Proc Soc Exp Biol Med 207:197–205. Nanji AA, Khettry U and Sadrzadeh SMH (1994) Lactobacillus feeding reduces endotoxemia and severity of experimental alcoholic liver disease. Proc Soc Exp Biol Med 205:243–247. Nanji AA, Mendenhall CL and French SW (1989) Beef fat prevents alcoholic liver disease in the rat. Alcohol Clin Exp Res 13:15–19. Nanji AA, Rahemtulla A, Maio L, Khwaja S, Zhao S, Tahan SR and Thomas P (1997) Alterations in thromboxane synthase and thromboxane A2 receptors in experimental alcoholic liver disease. J Pharmacol Exp Ther 282:1037–1043. Rivera CA, Bradford BU, Seabra V and Thurman RG (1998) Role of endotoxin in the hypermetabolic state following acute ethanol exposure. Am J Physiol 275:G1252– G1258. Rose ML, Rivera CA, Bradford BU, Graves LM, Cattley RC, Schoonhoven R, Swenberg JA and Thurman RG (1999) Kupffer cell oxidant production is central to the mechanism of peroxisome proliferators. Carcinogenesis 20:27–33. Roy RS and McCord JM (1983) Superoxide and ischemia: Conversion of xanthine dehydrogenase to xanthine oxidase, in Oxy Radicals and Their Scavenger Systems. Vol. 2: Cellular and Medical Aspects (Greenwald RA and Cohen G eds) pp 145–153, Elsevier Publishing Co., Alabama. Rusyn I, Tsukamoto H and Thurman RG (1998) WY-14,643 rapidly activates nuclear factor B in Kupffer cells before hepatocytes. Carcinogenesis 19:1217–1222. Thompson JA and Reitz RC (1978) Effects of ethanol ingestion and dietary fat levels on mitochondrial lipids in male and female rats. Lipid 13:540 –550. Thurman RG (1998) Mechanisms of hepatic toxicity. II. Alcoholic liver injury involves activation of Kupffer cells by endotoxin. Am J Physiol 275:G605–G611. Tsukamoto H, French SW, Reidelberger RD and Largman C (1985) Cyclical pattern of blood alcohol levels during continuous intragastric ethanol infusion in rats. Alcohol Clin Exp Res 9:31–37. Tsukamoto H, Gaal K and French SW (1990) Insights into the pathogenesis of alcoholic liver necrosis and fibrosis: Status report. Hepatology 12:599 – 608. Tsukamoto H, Reiderberger RD, French SW and Largman C (1984) Long-term cannulation model for blood sampling and intragastric infusion in the rat. Am J Physiol 247:R595–R599. Watanabe N, Suzuki J and Kobayashi Y (1996) Role of calcium in tumor necrosis factor-␣ produced by activated macrophages. J Biochem 120:1190 –1195. Wiezorek JS, Brown DH, Kupperman DE and Brass CA (1994) Rapid conversion to high xanthine oxidase activity in viable Kupffer cells during hypoxia. J Clin Invest 94:2224 –2230. Yin M, Wheeler MD, Kono H, Bradford BU, Gallucci RM, Luster MI and Thurman RG (1999) Essential role of tumor necrosis factor ␣ in alcohol-induced liver injury. Gastroenterology 117:942–952. Yu K, Bayona W, Kallen CB, Harding HP, Ravera CP, McMahon G, Brown M and Lazar MA (1995) Differential activation of peroxisome proliferator-activated receptor by eicosanoids. J Biol Chem 270:23975–23983. Zabel U, Schreck R and Baeuerle PA (1991) DNA binding of purified transcription factor NF-B. Affinity, specificity, Zn2⫹ dependence, and differential half-site recognition. J Biol Chem 266:252–260. Zhong Z, Lemasters JJ and Thurman RG (1989) Role of purines and xanthine oxidase in reperfusion injury in perfused rat liver. J Pharmacol Exp Ther 250: 470 – 475.
Adachi Y, Bradford BU, Gao W, Bojes HK and Thurman RG (1994) Inactivation of Kupffer cells prevents early alcohol-induced liver injury. Hepatology 20:453– 460. Adachi Y, Moore LE, Bradford BU, Gao W and Thurman RG (1995) Antibiotics prevent liver injury in rats following long-term exposure to ethanol. Gastroenterology 108:218 –224. Arteel GE, Iimuro Y, Yin M, Raleigh JA and Thurman RG (1997) Chronic enteral ethanol treatment causes hypoxia in rat liver tissue in vivo. Hepatology 25:920 – 926. Badger TM, Crouch J, Irby D and Shahare M (1993) Episodic excretion of ethanol during chronic intragastric ethanol infusion in the male rat: Continuous vs. cyclic ethanol and nutrient infusions. J Pharmacol Exp Ther 264:938 –943. Baeuerle PA and Baltimore D (1989) A 65 KD subunit of active NF-B is required for inhibition of NF-B by IB. Genes Dev 3:1689 –1698. Baldwin AS (1996) The NF-B and IB proteins: New discoveries and insights. Annu Rev Immunol 14:649 – 681. Bergmeyer HU (1988) Methods of Enzymatic Analysis. Academic Press, New York. Bevilacqua MP, Pober JS, Mendrick DL, Cotran RS and Gimbrone MA (1987) Identification of an inducible endothelial-leukocyte adhesion molecule. Proc Natl Acad Sci USA 84:9238 –9242. Bode JC, Bode C, Heidelbach R, Durr H-K and Martini GA (1984) Jejunal microflora in patients with chronic alcohol abuse. Hepatogastroenterology 31:30 –34. Bradford BU, Enomoto N, Ikejima K, Rose ML, Bojes HK, Forman DT and Thurman RG (1999) Peroxisomes are involved in the swift increase in alcohol metabolism. J Pharmacol Exp Ther 288:254 –259. Bradford BU, Misra UK and Thurman RG (1993) Kupffer cells are required for the swift increase in alcohol metabolism. Res Commun Subst Abuse 14:1– 6. Brass CA, Narciso J and Gollan JL (1991) Enhanced activity of the free radical producing enzyme xanthine oxidase in hypoxic rat liver. J Clin Invest 87:424 – 431. Cohen PJ (1992) Allopurinol administered prior to hepatic ischemia in the rat prevents chemiluminescence following restoration of circulation. Can J Anaesth 39:1090 –1093. Decker T, Lohmann-Matthes ML, Karck U, Peters T and Decker K (1989) Comparative study of cytotoxicity, tumor necrosis factor, and prostaglandin release after stimulation of rat Kupffer cells, murine Kupffer cells, and murine inflammatory liver macrophages. J Leukoc Biol 45:139 –146. Duling DR (1994) Simulation on multiple isotropic spin-trap EPR spectra. J Magn Reson 104:105–110. Dustin ML and Springer TA (1988) Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecule-1 (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells. J Cell Biol 107:321–331. French SW, Miyamoto K and Tsukamoto H (1986) Ethanol-induced hepatic fibrosis in the rat: role of the amount of dietary fat. Alcohol Clin Exp Res 10:S13–S19. Fukui H, Brauner B, Bode JC and Bode C (1991) Plasma endotoxin concentrations in patients with alcoholic and non-alcoholic liver disease: Reevaluation with an improved chromogenic assay. J Hepatol 12:162–169. Glassman EB, McLaughlin GA, Forman DT, Felder MR and Thurman RG (1989) Role of alcohol dehydrogenase in the swift increase in alcohol metabolism (SIAM): Studies with deermice deficient in alcohol dehydrogenase. Biochem Pharmacol 34:3523–3526. Iimuro Y, Frankenberg Mv, Arteel GE, Bradford BU, Wall CA and Thurman RG (1997b) Female rats exhibit greater susceptibility to early alcohol-induced injury than males. Am J Physiol 272:G1186 –G1194. Iimuro Y, Gallucci RM, Luster MI, Kono H and Thurman RG (1997a) Antibodies to tumor necrosis factor-␣ attenuate hepatic necrosis and inflammation due to chronic exposure to ethanol in the rat. Hepatology 26:1530 –1537. Karwinski W, Farstad M, Ulvik R and Soreide O (1991) Sixty-minute normothermic liver ischemia in rats. Evidence that allopurinol improves liver cell energy metabolism during reperfusion but that timing of drug administration is important. Transplantation 52:231–234. Knecht KT, Adachi Y, Bradford BU, Iimuro Y, Kadiiska M, Qun-hui X and Thurman RG (1995) Free radical adducts in the bile of rats treated chronically with intragastric alcohol: Inhibition by destruction of Kupffer cells. Mol Pharmacol 47:1028 – 1034. Kono H, Rusyn I and Thurman RG (1998) Allopurinol, a xanthine oxidase inhibitor,
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