Modulation of Caspase‐3 Activity and Fas Ligand mRNA Expression in Rat Liver Cells In Vivo by Alcohol and Lipopolysaccharide

Abstract

The purpose of this study was to determine if exacerbation of apoptosis precedes liver injury during chronic exposure of rats to alcohol. After 7 weeks of feeding an alcohol- or dextrin-containing liquid diet, the animals were treated with gram-negative bacterial lipopolysaccharide (1 mg x kg(-1) body weight, intravenously) or sterile saline and sacrificed 3 hr after the treatment. Alanine:2-oxoglutarate aminotransferase (ALT) and lactate:NAD oxidoreductase [lactate dehydrogenase (LDH)] were measured in plasma. The caudate lobe of the liver was resected for histology, while the rest of the organ was perfused with collagenase to isolate hepatocytes, Kupffer cells (KCs), and sinusoidal endothelial cells (SECs) by centrifugal elutriation. Hepatocyte mitochondria were isolated by differential centrifugation of the cell homogenate. Reduced and oxidized glutathione (GSH and GSSG) in isolated hepatocytes and hepatocyte mitochondria, and malondialdehyde in hepatocytes were assayed. Caspase-3 activity and Fas ligand mRNA expression were determined in hepatocytes, KCs, and SECs. Plasma ALT and LDH activity, liver histology, GSH, GSSG and their ratio, and malondialdehyde content were not affected by alcohol treatment Caspase-3 activity was significantly increased in alcohol-treated rats in all three cell types, with the lowest response observed in hepatocytes and the highest in KCs. Fas ligand mRNA expression, which had the highest level in SECs, followed by KCs and hepatocytes, was not affected by alcohol administration. Lipopolysaccharide had the following effects: an increase in ALT in both pair- and alcohol-fed rats, and LDH only in alcohol-fed rats, a decrease in GSH + GSSG levels in both mitochondria and hepatocytes, an elevation of malondialdehyde content in hepatocytes, a raise in caspase-3 activity in all groups and cell types, and an augmentation of Fas ligand expression in hepatocytes and KCs, but not in SECs. These data suggest that, during chronic alcohol consumption, an exacerbated apoptosis precedes alcohol-induced liver injury.

Mitochondrial dysfunction and oxidative stress in the pathogenesis of alcohol- and obesity-induced fatty liver diseases

“…Importantly, it is proposed that loss of mitochondrial GSH sensitizes hepatocytes from alcohol-fed animals to TNFα induced cell death [78,79], specifically when mitochondria are "loaded" with free cholesterol [80]. In contrast, several studies have shown that mitochondrial GSH depletion may not be a consistent outcome following chronic alcohol consumption as studies have shown increased mitochondrial GSH in response to alcohol consumption [81][82][83][84]. Similarly, Cederbaum and colleagues have shown increased GSH in HepG2 cells over-expressing CYP450 2E1 due to upregulation of glutamate cysteine ligase [85].…”

The protective efficacy of vitamins (C and E), selenium and silymarin supplements against alcohol toxicity

“…Potential mechanisms of acute ethanolinduced liver apoptosis include increased cytokine activity, Fas ligand (FasL) expression, and/or oxidative stress (Kurose et al, 1997, Neuman et al, 2001. Ethanol-induced liver apoptosis involves the activation of cysteine proteases or caspases (Deaciuc et al, 1999, Zhou et al, 2001. Endonucleases and DNA fragmentation factors are activated during apoptosis, resulting in degradation of chromatin DNA into internucleosomal units (Cohen andDuke, 1984, Liu et al, 1997).…”

Distinct methylation patterns in histone H3 at Lys-4 and Lys-9 correlate with up- & down-regulation of genes by ethanol in hepatocytes

“…Thus changes in the expression of genes have been implicated in the development of alcoholic liver diseases (ALD). Genes that are involved in ethanol metabolism (Morimoto et al, 1993;Ronis et al, 1993;Deaciuc et al, 2004a,b) cell signaling (Mochly-Rosen et al, 1988;Gordon et al, 1986;Hong et al, 2002a,b) and apoptosis (Yacoub et al, 1995;Deaciuc et al, 1999;Deaciuc et al, 2002a,b;Koteish et al, 2002) are highly sensitive to alcohol. Micro-array studies have also shown profile of hepatic gene expressions induced by alcohol consumption (Tadic et al, 2002;Deaciuc et al, 2004a,b;French et al, 2005).…”