Acute ethanol can potentiate the hepatic apoptosis and necrosis by Jo2 Fas antibody treatment as shown by three fold increases of ALT or AST and more extensive degeneration, apoptotic necrosis of hepatocytes, and focal hemorrhages of the hepatic lobule in the Fas Jo2 plus acute ethanol treatment group compared to the Jo2 alone group or ethanol alone group. Only small increases of transaminases and mild changes of morphology were observed in either the Jo2 alone or ethanol alone group. Thus, the enhanced toxicity found in the Eth/Jo2 group is not merely the addition of toxicity produced by Jo2 plus the toxicity produced by ethanol. A low concentration of Jo2 was used in order to minimize toxicity and thus allow evaluation of potentiation of injury by acute ethanol pretreatment. These findings suggest that ethanol pretreatment sensitizes the liver to toxic challenge by Jo2 so as to potentiate liver damage.
The Fas/Fas-L system plays a central role in ethanol-induced hepatic apoptosis [21
]. Binding of Fas to its ligand (FasL) or Fas antibody results in receptor cross-linking and apoptosis of Fas positive cells via cellular pathways including receptor oligomerization and recruitment of the Fas-associated protein with death domain, which eventually leads to the activation of caspase-8 and downstream caspases such as caspase-3 [33
]. Liver is particularly sensitive to Fas-induced apoptosis and hepatotoxicity and the injection of Fas antibody in mice results in extensive hepatocyte necrosis and even fulminant liver failure [39
]. In the current study, Fas Jo2 induced-liver injury was potentiated by acute ethanol pretreatment as development of apoptotic acidophilic necrosis in hepatocytes including focal hemorrhages and small amounts of inflammatory cell infiltration in damaged areas were observed. Injury appeared to be largely necrotic. Results from caspase activities and DNA fragmentation indicated that there were increases of caspase-8, and caspase-3 activities, and TUNEL positive cells, in the Eth/Jo2 group compared to the Jo2 alone group, suggesting apoptosis is also occurring in the Jo2 plus ethanol group. It is possible that an initial apoptotic mode of cell death may switch to a necrotic mode (or oncotic cell death) under conditions of massive toxicity or oxidative stress, as ATP which is necessary for apoptosis to proceed becomes depleted. The results from CYP2E1 knockout mice indicated that the enhanced hepatotoxicity was significantly decreased compared to the WT mice but the caspase activities were not different compared to the WT mice. This suggests that the decreased hepatotoxicity of Jo2 plus ethanol in the CYP2E1 KO mice did not involve the down-regulation of apoptotic pathways in the liver. The results also suggest that CYP2E1, unlike the liver necrosis toxicity, does not play a major role in the apoptotic toxicity induced by the Jo2 plus ethanol treatment.
Ethanol-induced oxidative stress appears to play an important role in mechanisms by which ethanol causes liver injury [40
]. Induction of CYP2E1 contributes to the ethanol-induced oxidant stress. CYP2E1 levels were elevated after acute ethanol treatment as there was an increase of enzyme catalytic activity and an increase of CYP2E1 protein expression. The increase and location of the liver histopathology i.e., centrilobular zone of the liver acinus, is associated with the elevation and location of CYP2E1 in this zone. In CYP2E1 knockout mice, the enhanced hepatotoxicity by Jo2 plus ethanol was partially (but not completely) decreased compared to the CYP2E1 wild-type mice. These results suggest that one mechanism by which acute ethanol administration increases the susceptibility of the liver to hepatotoxicity by Fas Jo2 antibody is due to increases in the expression of CYP2E1 by the ethanol pretreatment. CYP2E1 may promote Jo2 toxicity even in the absence of acute ethanol pretreatment. When higher concentrations of Jo2 alone were administered, hepatic toxicity was produced in the WT mice, however, injury was considerably reduced in the Jo2-treated CYP2E1 KO mice. Thus, even basal non-induced levels of CYP2E1 appear to play a role in potentiating Jo2 toxicity. Formation of peroxynitrite anion and nitrotyrosine adducts would result from interaction between superoxide anion and NO. Increases in superoxide production would occur as a result of induction of CYP2E1 while increases in NO occur when iNOS is induced.
Alcohol-induced liver injury is associated with enhanced lipid peroxidation, protein carbonyl formation, production of reactive oxygen species (ROS), and decreases in hepatic antioxidant defense especially GSH [41
]. Mitochondrial MDA, protein carbonyl formation, 3-NT adducts, iNOS and 4-HNE adducts were higher in the Jo2 plus ethanol group compared to the Jo2 alone or ethanol alone group. GSH was lower in all the treated groups and lowest levels were in the the Jo2 plus ethanol group. These results suggest that the enhanced hepatotoxicity might come from an increase of oxidative and nitrosative stress and perhaps decrease of antioxidant defense in the liver of mice treated with Jo2 plus ethanol. TNF-α levels were also elevated in the Jo2 plus ethanol treated group compared to the saline controls, which may also contribute to an increase in oxidative stress. TNF-α levels were decreased in the CYP2E1 KO mice treated with Eth/Jo2 compared to the WT, which may play a role in the lowering of the hepatic injury. Future experiments with TNF-α receptor knockout mice and neutralizing TNF-α antibodies are planned to further evaluate a role for TNF-α in the potentiation of Jo2 toxicity by acute ethanol pretreatment. In order to evaluate whether oxidative or nitrosative stress contribute to or are merely associated with the enhanced hepatotoxicity by Jo2 plus ethanol pretreatment, an antioxidant (NAC) or an inhibitor of iNOS or cytokine (TNF-α) production (GdCl3
, PTX) were administrated prior to the Jo2 treatment. There was at least a partial decrease of hepatotoxicity after using GdCl3,
1400W and NAC, respectively, consistent with the idea that the increase of Jo2 plus ethanol hepatotoxicity involves oxidative stress (protection by NAC), nitrosative stress (protection by 1400W) and TNF-α/inflammatory reactions (protection by GdCl3
). PTX was recently shown to prevent the increase in plasma TNF-α, triglycerides and caspase-3 activity when alcohol was administrated by gastric intubation to obese mice [44
]. PTX was slightly but not significantly protective against the Jo2 plus ethanol toxicity. GdCl3
pretreatment in mice to inactivate Kupffer cells resulted in a significantly decrease of plasma ALT after endotoxin administration [45
also blocked alcohol toxicity in rats treated chronically with intragastric alcohol [46
]. 1400W, a selective iNOS inhibitor, markedly reduced the increased plasma NO levels after administration in endotoxin-injected mice [47
] and decreased alcohol liver toxicity in mice fed ethanol intragastrically by inhibiting iNOS in the Kupffer cells [48
]. NAC was shown to decrease ethanol hepatotoxicity and oxidative stress in rats fed ethanol via total enteral nutrition [49
As mentioned above, the Jo2 plus ethanol toxicity was lower in CYP2E1 knockout mice as compared to WT-treated mice. However, significant toxicity by Jo2 plus ethanol persisted in the CYP2E1 null mice. This decrease of hepatotoxicity is associated with a decrease of oxidation protein adducts such as 3-NT and 4-HNE as well as TNF-α levels in the CYP2E1 knockout mice, but again significant increases in 3-NT and 4-HNE protein adducts persisted in the Jo2 plus ethanol CYP2E1 knockout mice compared to saline-treated wild type or CYP2E1 knockout mice. This suggests that the enhanced oxidative and nitrosative stress is derived, in part, from CYP2E1-derived ROS and lipid peroxidation, but also by CYP2E1-independent pathways. The latter may reflect Jo2-derived ROS and NO, or cytokine-derived ROS. Alternatively, other CYPs may be induced in the CYP2E1 knockout mice e.g. CYP4A10 and CYP4A14 were elevated in CYP2E1 knockout mice fed a methionine choline deficient diet [50
]. To evaluate a potential role for other CYPS, we treated the CYP2E1-knockouts with aminobenzotriazole (ABT), which is known to decrease total P450 levels [51
]. Total P450 levels were lowered about 62% after treatment of CYP2E1 knockout mice with 100 mg/kg ABT (once a day for 3 days). The residual liver injury in the CYP2E1 knockout mice was lowered about 32% by the ABT treatment, indeed suggesting a role for other CYPS in the Jo2 plus ethanol toxicity. Some injury still remained compared to the saline controls which suggests non-P450 dependent mechanisms. Further studies are needed to define these non-P450 dependent pathways. We believe that ethanol pretreatment potentiates Jo2-induced hepatic toxicity by elevating CYP2E1-dependent and CYP2E1-independent pathways.