Lipeng Wang

A Critical Involvement of Oxidative Stress in Acute Alcohol-Induced Hepatic TNF-α Production

Tumor necrosis factor-α (TNF-α) 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-α 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-α 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-α 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-α 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-α 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-α production following acute alcohol administration, and the results demonstrate that oxidative stress mediates endotoxin-induced hepatic TNF-α production in acute alcohol intoxication.

Zinc supplementation prevents alcoholic liver injury in mice through attenuation of oxidative stress

Alcoholic liver disease is associated with zinc decrease in the liver. Therefore, we examined whether dietary zinc supplementation could provide protection from alcoholic liver injury. Metallothionein-knockout and wild-type 129/Sv mice were pair-fed an ethanol-containing liquid diet for 12 weeks, and the effects of zinc supplementation on ethanol-induced liver injury were analyzed. Zinc supplementation attenuated ethanol-induced hepatic zinc depletion and liver injury as measured by histopathological and ultrastructural changes, serum alanine transferase activity, and hepatic tumor necrosis factor-alpha in both metallothionein-knockout and wild-type mice, indicating a metallothionein-independent zinc protection. Zinc supplementation inhibited accumulation of reactive oxygen species, as indicated by dihydroethidium fluorescence, and the consequent oxidative damage, as assessed by immunohistochemical detection of 4-hydroxynonenal and nitrotyrosine and quantitative analysis of malondialdehyde and protein carbonyl in the liver. Zinc supplementation suppressed ethanol-elevated cytochrome P450 2E1 activity but increased the activity of alcohol dehydrogenase in the liver, without affecting the rate of blood ethanol elimination. Zinc supplementation also prevented ethanol-induced decreases in glutathione concentration and glutathione peroxidase activity and increased glutathione reductase activity in the liver. In conclusion, zinc supplementation prevents alcoholic liver injury in an metallothionein-independent manner by inhibiting the generation of reactive oxygen species (P450 2E1) and enhancing the activity of antioxidant pathways.

S-adenosylhomocysteine sensitizes to TNF-α hepatotoxicity in mice and liver cells: A possible etiological factor in alcoholic liver disease

In alcoholic liver disease, tumor necrosis factor‐α (TNFα) is a critical effector molecule, and abnormal methionine metabolism is a fundamental acquired metabolic abnormality. Although hepatocytes are resistant to TNFα‐induced killing under normal circumstances, previous studies have shown that primary hepatocytes from rats chronically fed alcohol have increased TNFα cytotoxicity. Therefore, there must be mechanisms by which chronic alcohol exposure “sensitizes” to TNFα hepatotoxicity. S‐adenosylhomocysteine (SAH) is product of methionine in transsulfuration pathway and a potent competitive inhibitor of most methyltransferases. In this study, we investigated the effects of increased SAH levels on TNFα hepatotoxicity. Our results demonstrated that chronic alcohol consumption in mice not only decreased hepatic S‐adenosylmethionine levels but also increased hepatic SAH levels, which resulted in a significantly decreased S‐adenosylmethionine‐to‐SAH ratio. This was associated with significant increases in hepatic TNFα levels, caspase‐8 activity, and cell death. In vitro studies demonstrated that SAH‐enhancing agents sensitized hepatocytes to TNFα killing, and the death was associated with increased caspase‐8 activity, which was blocked by a caspase‐8 inhibitor. In addition, increased intracellular SAH levels had no effect on nuclear factor κB activity induced by TNFα. In conclusion, these results provide a new link between abnormal methionine metabolism and abnormal TNFα metabolism in alcoholic liver disease. Increased SAH is a potent and clinically relevant sensitizer to TNFα hepatotoxicity. These data further support improving the S‐adenosylmethionine‐to‐SAH ratio and removal of intracellular SAH as potential therapeutic options in alcoholic liver disease. Supplementary material for this article can be found on the HEPATOLOGYwebsite (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;40:989–997.)

Abrogation of Nuclear Factor-κB Activation Is Involved in Zinc Inhibition of Lipopolysaccharide-Induced Tumor Necrosis Factor-α Production and Liver Injury

Endotoxin (lipopolysaccharide, LPS)-induced tumor necrosis factor-α (TNF-α) release from Kupffer cells is critically involved in the pathogenesis of alcohol-induced liver injury. We recently reported that inhibition of alcohol-induced plasma endotoxin elevation contributes to the protective action of zinc against alcoholic hepatotoxicity. The present study was undertaken to determine whether zinc interferes with the endotoxin-TNF-α signaling pathway, and possible mechanism(s) by which zinc modulates the endotoxin-TNF-α signaling. Administration of LPS to metallothionein (MT)-knockout (MT-KO) mice and 129/Sv wild-type (WT) controls at 4 mg/kg induced hepatic TNF-α elevation at 1.5 hours, followed by liver injury at 3 hours. Zinc pretreatment (two doses at 5 mg/kg) attenuated TNF-α production and liver injury in both MT-KO and WT mice, indicating a MT-independent action of zinc. Immunohistochemical detection of the phosphorylation of I-κB and nuclear factor (NF)-κB in the liver of MT-KO mice demonstrated that zinc pretreatment abrogated LPS-induced NF-κB activation in the Kupffer cells. Fluorescent microscopy of superoxide by dihydroethidine and of zinc ions by Zinquin in the liver of MT-KO mice showed that zinc pretreatment increased the intracellular labile zinc ions and inhibited LPS-induced superoxide generation. These results demonstrate that zinc inhibits LPS-induced hepatic TNF-α production through abrogation of oxidative stress-sensitive NF-κB pathway, and the action of zinc is independent of MT. Thus, zinc may be beneficial in the treatment of LPS-induced liver injuries, such as sepsis and alcoholism.

Myocardial toxicity of arsenic trioxide in a mouse model

Arsenic trioxide is highly effective in the treatment of acute promyelocytic leukemia (APL). In September 2000, the Trisenox brand of arsenic trioxide for the treatment of relapsed and refractory APL was approved in the United States. Arecent clinical report has shown a serious ventricular tachycardia at the therapeutic doses of arsenic trioxide in APL patients. The present study was undertaken to investigate the cardiotoxic effect of arsenic trioxide using a mouse model. Animals were injected intraperitoneally with arsenic trioxide 5 mg/kg/d for 30 d, a dose regiment that has been shown to produce plasma concentrations of arsenic within the range of those present in arsenic-treated APL patients. Analysis of myocardial function revealed that arsenic caused a significant decrease in the maximum rate of rise in intraventricular pressure during ventricular contraction (MAX dP/dt), and significant increases in the end diastolic pressure and ventricle minimum diastolic pressure. In response to β-adrenergic stimulation by isoproterenol, the arsenic-treated heart did not show increase in MAX dP/dt, which was observed as a stress response in the saline-treated controls. The functional alterations were accompanied by cardiomyopathy, as revealed by histopathological and ultrastructural examination. Furthermore, arsenic caused myocardial apoptosis, as determined by a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, which was confirmed by caspase-3 activation detected by enzymatic assay. Our study thus demonstrates that arsenic trioxide, in a dose that could produce clinically comparable serum concentrations to those observed in humans, causes cardiotoxicity.

Alcohol-Induced Myocardial Fibrosis in Metallothionein-Null Mice: Prevention by Zinc Supplementation

Alcohol-induced cardiomyopathy including fibrosis has been recognized clinically for a long time, but its pathogenesis is incompletely understood. Studies using experimental animals have not fully duplicated the pathological changes in humans, and animal models of alcoholic cardiac fibrosis are not available. In the present study, we have developed a mouse model in which cardiac hypertrophy and fibrosis were produced in metallothionein-knockout (MT-KO) mice fed an alcohol-containing liquid diet for 2 months. The same alcohol feeding did not produce cardiac fibrosis in the wild-type (WT) control mice, although there was no difference in the alcohol-induced heart hypertrophy between the WT controls and the MT-KO mice. Zinc supplementation prevented cardiac fibrosis but did not affect heart hypertrophy in the alcohol-fed MT-KO mice, suggesting a specific link between zinc homeostasis and cardiac fibrosis. Serum creatine phosphokinase activity was significantly higher in the alcohol-administered MT-KO mice than in the WT mice, and zinc supplementation decreased serum creatine phosphokinase activities and eliminated the difference between the groups. Thus, disturbance in zinc homeostasis due to the lack of MT associates with alcohol-induced cardiac fibrosis and more severe cardiac injury, making the MT-KO mouse model of alcohol-induced cardiac fibrosis a useful tool to investigate specific factors involved in the alcoholic cardiomyopathy.

Preservation of Intestinal Structural Integrity by Zinc Is Independent of Metallothionein in Alcohol-Intoxicated Mice

Intestinal-derived endotoxins are importantly involved in alcohol-induced liver injury. Disruption of intestinal barrier function and endotoxemia are common features associated with liver inflammation and injury due to acute ethanol exposure. Zinc has been shown to inhibit acute alcohol-induced liver injury. This study was designed to determine the inhibitory effect of zinc on alcohol-induced endotoxemia and whether the inhibition is mediated by metallothionein (MT) or is independent of MT. MT knockout (MT-KO) mice were administered three oral doses of zinc sulfate (2.5 mg zinc ion/kg body weight) every 12 hours before being administered a single dose of ethanol (6 g/kg body weight) by gavage. Ethanol administration caused liver injury as determined by increased serum transaminases, parenchymal fat accumulation, necrotic foci, and an elevation of tumor necrosis factor (TNF-alpha). Increased plasma endotoxin levels were detected in ethanol-treated animals whose small intestinal structural integrity was compromised as determined by microscopic examination. Zinc supplementation significantly inhibited acute ethanol-induced liver injury and suppressed hepatic TNF-alpha production in association with decreased circulating endotoxin levels and a significant protection of small intestine structure. As expected, MT levels remained undetectable in the MT-KO mice under the zinc treatment. These results thus demonstrate that zinc preservation of intestinal structural integrity is associated with suppression of endotoxemia and liver injury induced by acute exposure to ethanol and the zinc protection is independent of MT.

Elevation of serum endothelins and cardiotoxicity induced by particulate matter (PM2.5) in rats with acute myocardial infarction

Epidemiological studies have defined a significant positive association of acute exposure to ambient concentrations of particulate matter (PM) with increased daily mortality and hospital admission for cardiovascular diseases. Experimental studies have shown that animals with pre-existing cardiovascular diseases are more susceptible to the cardiac effect of PM exposure. The present study was undertaken to investigate possible involvement of upregulation of the endothelin system in PM exposure-induced cardiotoxicity in rats with acute myocardial infarction (MI). Adult male Sprague-Dawley rats were subjected to occlusion of the left coronary artery and displayed myocardial infarction 12 h after the surgery. The heart rate significantly decreased and premature ventricular complexes of the electrocardiogram occurred in the myocardial infarct animals. Exposure to PM2.5 via intratracheal instillation with 2.0 mg in 0.3 mL normal saline significantly worsened the ventricular arrhythmia along with a further decrease in heart rate. The same PM exposure only caused slight cardiac changes in the sham-operated animals. Serum total endothelin concentrations were significantly elevated in both myocardial infarct rats and sham-operated controls in response to PM exposure. However, increased numbers of the endothelin receptor type A on the cardiomyocytes were observed only in the infarct myocardium. This study thus suggests that upregulation of the endothelin system in rats with MI is likely involved in the PM exposure-induced cardiotoxicity.

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

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.