CYP2E1 is induced under a variety of pathophysiological conditions such as fasting, diabetes, obesity and high-fat diet [26
]. CYP2E1 protein expression and activity are elevated after ethanol exposure [29
]. CYP2E1 is responsible for metabolizing and activating a large number of chemical solvents, industrial monomers and precarcinogens [31
] and is active in catalyzing the production of ROS and lipid peroxidation in microsomal membranes [32
]. In vitro, ethanol-mediated production of ROS, cell death and GSH depletion were observed in both the cytosolic and mitochondrial fraction of CYP2E1 transfected cell lines [8
]. The in vivo role of CYP2E1 in alcohol-induced liver injury is controversial, especially in the early stage of experimental ALD. In this study, we used a chronic ethanol feeding plus Jo2 treatment model in wild type mice and in CYP2E1 knockout mice to evaluate whether chronic ethanol treatment increases Jo2 hepatotoxicity, whether CYP2E1 or CYP2E1-mediated ROS contributes to an increased liver injury formed after chronic ethanol consumption, and what key factors participate in this potentiated hepatotoxicity. Hepatotoxicity occurs in the wild type mice fed the alcohol diet chronically and treated with Jo2 compared to the wild type mice fed dextrose and treated with Jo2 or CYP2E1 knockout mice fed the dextrose or alcohol diet followed by treatment with Jo2. Steatosis was more severe in the CYP2E1 wild-type mice than that in the CYP2E1 knockout mice after alcohol feeding and treatment with either saline or Jo2. We have previously shown that CYP2E1 plays a role in ethanol-induced fatty liver [36
]. Interestingly, no significant toxicity was found in the saline treated ethanol-fed groups, suggesting that steatotitic histopathological changes alone did not promote further liver injury. However, the fatty liver may provide a basis for a second toxic challenge e.g. with Jo2. CYP2E1 protein and catalytic activity were highly induced in wild type mice compared to the CYP2E1 knockout mice fed alcohol and treated with either saline or Jo2 treatment. These results show that a) chronic ethanol feeding produced more steatosis in wild type mice than in CYP2E1 knockout mice; b) the ethanol feeding elevated CYP2E1 in wild type mice; c) the increase in fatty liver and in CYP2E1 are not sufficient by themselves to result in significant liver injury i.e. necrosis; d) however, the increases in fatty liver and CYP2E1 may set the stage for the liver to become sensitive to a second challenge as provided by Jo2 administration at concentrations which do not cause liver injury to dextrose-fed mice without fatty liver or elevated CYP2E1.
Experiments were subsequently carried out to evaluate possible mechanisms which play a role in the elevated Jo2 toxicity in the ethanol-fed WT mice. Since CYP2E1 contributes to this enhanced toxicity, our initial hypothesis was that oxidative stress was elevated in the ethanol plus Jo2-treated wild type mice. Indeed, protein carbonyl, MDA or lipid peroxidation and GSH depletion were highly elevated in the wild type mice fed ethanol and treated with Jo2, as compared to the CYP2E1 knockout mice fed ethanol and treated with Jo2. However, similar increases in these markers of oxidative stress also occur in the saline-treated ethanol-fed mice (without Jo2 treatment) as compared to saline-treated ethanol-fed CYP2E1 knockout mice. These results show that CYP2E1 plays a major role in the elevation of oxidative stress produced by chronic ethanol feeding. However, since this elevation was not different between the saline-ethanol-fed mice which do not show liver injury, and the Jo2-ethanol-fed mice which do show liver injury, differences in the extent of oxidative stress alone can not explain the liver injury formed in the ethanol/Jo2 mice compared to ethanol/saline mice (although differences in oxidative stress can contribute to the liver injury in the ethanol/Jo2 mice compared to the dextrose/Jo2 mice).
Abnormal cytokine metabolism is a major feature of ALD, especially LPS mediated-hepatitis [8
], and increased serum TNF-α levels are associated with the disease severity and mortality of alcoholic hepatitis [39
]. Rats after chronic alcohol feeding are more sensitive to the hepatotoxic effects of administration of LPS and have higher plasma levels of TNF-α than control rats [23
]. Gadolinium chloride was found to protect against alcoholic liver injury via down-regulation of TNF-α production in Kupffer cells [11
]. Anti-TNF-α antibody prevented alcohol liver injury in rats [42
], and mice lacking the TNF-R1 receptor did not develop alcohol liver injury [43
]. These studies clearly indicate TNF-α as a major risk factor for the development of alcohol liver injury. Could differences in TNF-α levels be important for the liver injury found in the ethanol/Jo2 group compared to the other groups ? There was a small increase in serum TNF-α levels in wild type mice and CYP2E1 knockout mice fed the dextrose diet followed by Jo2 treatment as compared to the wild type or knockout mice fed the dextrose diet and treated with saline. Thus, Jo2 elevates TNF-α comparably in the absence or presence of CYP2E1 in dextrose fed mice. Chronic ethanol feeding alone did not elevate serum TNF-α levels in wild type or knockout mice compared to the dextrose controls, showing that fatty liver or elevated CYP2E1 are not sufficient to increase TNF-α in this model. However, Jo2 elevated serum TNF-α levels to high levels in wild type mice fed ethanol; this increase was not observed in the ethanol/Jo2-treated knockout mice indicating a role for CYP2E1 in the ethanol/Jo2-mediated elevation of TNF-α. CYP2E1 is present in hepatic Kupffer cells and is inducible by ethanol [44
]. How CYP2E1 and Jo2 may interact to elevate production of TNF-α will require further study. Interestingly, the extent of TNF-α elevation is associated with the severity of hepatotoxicity being highest in CYP2E1 wild-type mice fed alcohol which display liver injury, and low in the 3 other groups which do not show liver injury. These results suggest that the elevation of TNF-α may contribute to the increased hepatotoxicity in the wild type ethanol/Jo2 mice.
The Fas/Fas-L system may play a key role in ethanol-induced hepatic apoptosis [13
]. Fas recognizes Fas ligand (FasL) or Fas antibody to initiate the receptor cross-linking and cell apoptosis via 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 [45
]. Liver is particularly sensitive to Fas-mediated toxicity and the injection of Fas antibody in mice results in extensive hepatocyte necrosis and even fulminant liver failure [48
]. The current study shows that Jo2 Fas antibody-mediated toxicity was potentiated by chronic alcohol feeding with development of extensive acidophilic necrosis, hemorrhage and infiltration of inflammatory cells in damaged areas. Levels of Fas were similar in all groups. While there were increases of caspase-8, and caspase-3 activities in the alcohol/Jo2 mice, there were similar increases in the dextrose/Jo2 mice, thus elevated caspase activities cannot explain the differences in liver injury between the alcohol/Jo2 versus the dextrose/Jo2. There was no significant difference in the Jo2 elevation of caspase 3 or 8 activities between wild type mice and CYP2E1 knockout mice, thus the Jo2 elevation of caspase 3 or 8 activities is independent of CYP2E1 status. c-FLIP has been identified as a regulator of death ligand-induced apoptosis downstream of death receptors and FADD [49
]. c-FLIP was decreased after alcohol feeding plus Jo2 treatment compared to the dextrose feeding plus Jo2 treatment. However, there was no significant difference in downregulation of c-FLIP between wild type alcohol/Jo2 mice with liver injury and CYP2E1 knockout alcohol/Jo2 mice without liver injury. These results suggest that the enhanced hepatotoxicity found in the CYP2E1 wild type mice fed alcohol followed by Jo2 treatment is not due to the altered levels of Fas, the upregulation of caspase-8, −3 and the decreased levels of cFLIP. These results do not rule out an important role for any of the above in the overall injury, but appear to rule out an important role for any of the above in explaining the differential liver injury produced by Jo2 in the ethanol-fed mice.
Mitogen activated protein kinases (MAPKs), JNK, p38 and ERK, are important cellular signaling molecules that convert various extracellular signals into intracellular responses via serial phosphorylation [51
]. Basically, prolonged activation of either JNK or p38 MAPK promotes cell death with an associated decrease in mitochondial membrane protential, whereas ERK activation may serve as a cell survival factor [55
]. But the exact role of MAPK in the cellular response in different hepatotoxicity models remains controversial. As to the role of MAPK in CYP2E1–dependent toxicity, Hoek and colleagues found that ethanol increased TNF-α toxicity in CYP2E1 expressing liver cells via a p38 MAPK-dependent pathway [58
]. Czaja and colleagues showed that increased CYP2E1 expression sensitized hepatocytes to TNF-α toxicity through prolonged activation of JNK [10
]. CYP2E1 plus arachidonic acid toxicity was mediated either by ERK or p38 MAPK [59
]. Koteish, et al [23
] reported that chronic ethanol exposure potentiated LPS liver injury in mice despite inhibiting JNK activity whereas Chung, et al [61
] reported that six months of ethanol treatment in rats increased JNK activation. With respect to the role of MAPK on Fas-induced liver damage, Brunner, et al reported that TRAIL-induced amplification of Fas-induced liver damage required JNK activation and phosphorylation of its downstream target and proapoptotic Bcl-2 homolog Bim [62
]. We evaluated the possible role of MAPK on the ethanol/Jo2 enhanced liver injury. Jo2 alone was able to promote the activation of JNK and p38 MAPK in both the ethanol fed and dextrose fed mice; there was no further increase in activation of JNK or p38 MAPK in the ethanol/Jo2 mice which display elevated hepatotoxicity. In order to explore the exact role and dynamic changes of JNK or p38 MAPK activation on the enhanced hepatotoxicity, time course experiments and inhibitors experiments were carried out. The results showed that chronic ethanol feeding was able to significantly promote the activation of JNK at an early stage after Jo2 treatment, with activation reaching a peak value at 2 h followed by gradual decline at 4 and 8 h. A time course of liver injury in the ethanol-fed mice showed injury occurring at 8 h but not at 2 or 4 h after addition of Jo2. Thus, the liver injury occurs after the activation of JNK and it is possible that the early activation of JNK in the ethanol/Jo2 mice may play a role in the developing liver injury. Treatment with inhibitors of JNK or p38 MAPK partially but not completely prevented the liver injury in the ethanol/Jo2 mice. These results suggest that JNK or p38 MAPK activation, at least partially, contribute to the enhanced Jo2-mediated hepatotoxicity following chronic alcohol feeding, however, detail mechanisms of the enhanced hepatotoxicity remain to be further defined.
In conclusion, chronic alcohol feeding leading to induction of CYP2E1 and to liver steatosis potentiates Fas Jo2-mediated hepatotoxicity in mice. Chronic alcohol feeding produces extensive steatosis and over-expression of CYP2E1 in liver, events which promote the early stage of ALD. CYP2E1 plays an important role in the steatosis, mediating oxidative stress and lipid peroxidation, events independent of Jo2 administration. Jo2 activates Fas internalization and increases activation of caspases 8 and 3, activation of JNK and P38 MAPK, and decreases GSH, events independent of CYP2E1. The combination of alcohol induction of CYP2E1 plus Jo2 administration leads to an increase in TNF-α levels. A scheme to summarize the enhanced hepatotoxicity and events envisioned to occur in the chronic alcohol diet feeding plus Jo2 treatment model is shown in . Neither of the factors associated with the induction of CYP2E1 (fatty liver, oxidative stress) or addition of Jo2 (activation of MAPK, caspase activation, decline in GSH) are sufficient to promote significant liver injury. However, the combination of alcohol, CYP2E1 and Jo2 is sufficient to promote liver injury. Thus, the enhanced hepatotoxicity is associated with the over-expression of CYP2E1, increase of TNF-α production and upregulation of MAPK activation especially JNK.
Model for chronic ethanol diet feeding plus Jo2 induced liver toxicity and potential mechanisms.