A critical involvement of oxidative stress in acute alcohol-induced hepatic TNF-alpha production

Abstract

Tumor necrosis factor-alpha (TNF-alpha) production is a critical factor in the pathogenesis of alcoholic liver injury. Both oxidative stress and endotoxin have been implicated in the process of alcohol-induced TNF-alpha production. However, a cause-and-effect relationship between these factors has not been fully defined. The present study was undertaken to determine the mediators of acute alcohol-induced TNF-alpha production using a mouse model of acute alcohol hepatotoxicity. Alcohol administration via gavage at a dose of 6 g/kg to 129/Sv mice induced hepatic TNF-alpha production in Kupffer cells as demonstrated by measuring protein levels, immunohistochemical localization, and mRNA expression. Alcohol intoxication caused liver injury in association with increases in plasma endotoxin and hepatic lipid peroxidation. Treatment with an endotoxin neutralizing protein significantly suppressed alcohol-induced elevation of plasma endotoxin, hepatic lipid peroxidation, and inhibited TNF-alpha production. Treatment with antioxidants, N-ACETYL-L-CYSTEINE, or dimethylsulfoxide, failed to attenuate plasma endotoxin elevation, but significantly inhibited alcohol-induced hepatic lipid peroxidation, TNF-alpha production and steatosis. All treatments prevented alcohol-induced necrotic cell death in the liver. This study thus systemically dissected the relationship among plasma endotoxin elevation, hepatic oxidative stress, and TNF-alpha production following acute alcohol administration, and the results demonstrate that oxidative stress mediates endotoxin-induced hepatic TNF-alpha production in acute alcohol intoxication.

Figure 1.

Time-course changes in hepatic TNF-α levels after acute alcohol administration. One gastric alcohol was given at 6 g/kg, and hepatic TNF-α levels at different time-points were measured by ELISA. The data were analyzed by analysis of variance and Newman-Keuls’ multiple-comparison test. Significant difference (P < 0.05) is identified by various superscript letters. Cont, control.

Figure 2.

Alcohol-induced TNF-α mRNA expression in the liver. Livers were removed at 3 or 6 hours after alcohol administration (6 g/kg), and TNF-α and housekeeping gene mRNA were determined by RT-PCR analysis. Alcohol administration increased TNF-α mRNA expression in the liver at both 3 and 6 hours after treatment. Cont, control.

Figure 3.

Immunohistochemical staining of TNF-α-positive cells in the liver. Livers were removed 6 hours after alcohol administration (6 g/kg) and 7-μm cryostat section were made. Sections were incubated with a rabbit polyclonal anti-mouse TNF-α antibody, followed by incubation with HRP-conjugated goat anti-rabbit IgG antibody. A: Control liver. B: Alcohol-treated liver. Arrowheads: TNF-α-positive cells. Arrows: Liver sinusoid. Magnification, ×260.

Figure 4.

Time-course changes in plasma ALT activities after acute alcohol administration. One gastric alcohol was given at 6 g/kg, and plasma ALT activity was measured by using a Sigma diagnostic kit. The data were analyzed by analysis of variance and Newman-Keuls’ multiple-comparison test. Significant difference (P < 0.05) is identified by different letter superscripts. Cont, control.

Figure 5.

Alcohol-induced histopathological changes in the liver. One gastric alcohol was given at 6 g/kg. A: Control. B: Alcohol for 3 hours. C: Alcohol for 6 hours. D: Alcohol for 12 hours. Alcohol treatment induced prominent microvesicular steatosis (arrows) along with necrosis (arrowheads) in the liver. The necrotic hepatocytes are characterized by cell enlargement and nuclear dissolution. Hematoxylin and eosin staining; magnification, ×260.

Figure 6.

Time-course changes in plasma endotoxin levels after acute alcohol administration. One gastric alcohol was given at 6 g/kg, and plasma endotoxin levels at different time-points were measured with a chromogenic detection kit based on limulus amebocyte lysate assay. The data were analyzed by analysis of variance and Newman-Keuls’ multiple-comparison test. Significant difference (P < 0.05) is identified by various superscript letters. Cont, control.

Figure 7.

Time-course changes in hepatic lipid peroxidation after acute alcohol administration. One gastric alcohol was given at 6 g/kg, and hepatic lipid peroxidation at different time-points was evaluated by measuring TBARS concentrations. The data were analyzed by analysis of variance and Newman-Keuls’ multiple-comparison test.

Figure 8.

Effects of endotoxin neutralization and antioxidants on alcohol-induced plasma endotoxin elevation. One gastric alcohol was given at 6 g/kg for 1.5 hours. ENP (10 mg/kg) was injected intravenously after alcohol administration. NAC (300 mg/kg) or DMSO (2 g/kg) was injected intravenously before alcohol treatment. Plasma endotoxin levels were measured with a chromogenic detection kit based on limulus amebocyte lysate assay. The data were analyzed by analysis of variance and Newman-Keuls’ multiple-comparison test. Significant difference (P < 0.05) is identified by various superscript letters.

Figure 9.

Effects of endotoxin neutralization and antioxidants on alcohol-induced hepatic TNF-α production. One gastric alcohol was given at 6 g/kg for 1.5 hours. ENP (10 mg/kg) was injected intravenously after alcohol administration. NAC (300 mg/kg) or DMSO (2 g/kg) was injected intravenously before alcohol treatment. Hepatic TNF-α levels were measured by ELISA. The data were analyzed by analysis of variance and Newman-Keuls’ multiple-comparison test. Significant difference (P < 0.05) is identified by various superscript letters.

Figure 10.

Effects of endotoxin neutralization and antioxidants on alcohol-induced hepatic lipid peroxidation. One gastric alcohol was given at 6 g/kg for 1.5 hours. ENP (10 mg/kg) was injected intravenously after alcohol administration. NAC (300 mg/kg) or DMSO (2 g/kg) was injected intravenously before alcohol treatment. Hepatic lipid peroxidation was evaluated by measuring TBARS concentrations. The data were analyzed by analysis of variance and Newman-Keuls’ multiple-comparison test. Significant difference (P < 0.05) is identified by various superscript letters.

Figure 11.

Effects of endotoxin neutralization and antioxidants on alcohol-induced plasma ALT elevation. One gastric alcohol was given at 6 g/kg for 1.5 hours. ENP (10 mg/kg) was injected intravenously after alcohol administration. NAC (300 mg/kg) or DMSO (2 g/kg) was injected intravenously before alcohol treatment. Plasma ALT activity was measured using a Sigma diagnostic kit. The data were analyzed by analysis of variance and Newman-Keuls’ multiple-comparison test. Significant difference (P < 0.05) is identified by various superscript letters.

Figure 12.

Effects of ENP and NAC on alcohol-induced histopathological changes in the liver. One gastric ethanol was given at 6 g/kg. ENP (10 mg/kg) was injected intravenously after alcohol administration. NAC (300 mg/kg) was injected intravenously before alcohol treatment. Livers were removed at 6 hours after alcohol administration. A: Control. B: Alcohol. C: ENP/alcohol. D: NAC/alcohol. Endotoxin neutralization by ENP resulted in abrogation of necrotic cell death and inhibition of lipid accumulation. NAC treatment attenuated alcohol-induced liver injury, with only minor microvesicular steatosis being found. Hematoxylin and eosin staining; magnification, ×260.