Either LPS/TNF-α or CPY2E1 are considered independent risk factors involved in liver toxicity and alcoholic liver disease but mutual relationships or interactions between them have not been clearly defined. Increased oxidative stress from induction of CYP2E1 in vivo by pyrazole administration sensitizes hepatocytes to LPS and TNF-α toxicity and activation of JNK MAP kinase is a downstream mediator of this potentiated hepatotoxicity (12
). Activation of the MAP kinase kinase kinase (ASK-1) was found in livers from mice treated with pyrazole plus TNF-α but not in livers from mice treated with pyrazole alone or TNF-α alone or in CYP2E1 knockout mice treated with pyrazole plus TNF-α (24
). A model in which pyrazole plus TNF-α-elevated oxidative stress first activates ASK-1 via dissociation of the complex of thioredoxin, followed by ultimate activation of downstream MAP kinase kinase and then JNK was proposed as part of the injury mechanism. In these time course experiments, it was noted that JNK1 became activated prior to JNK2 (24
). Whether this earlier and more pronounced activation of JNK1, relative to JNK2, plays a role in the elevation of hepatotoxicity and oxidative stress was the goal of this study.
JNK2 KO mice displayed hepatotoxicity similar to WT mice when treated with pyrazole plus TNF-α, however, JNK1 KO mice did not display this hepatotoxicity. No mice showed significant liver injury when treated with pyrazole alone or TNF-α alone. TUNEL positive staining was observed for the WT and JNK2 KO mice treated with pyrazole plus TNF-α but not the JNK1 KO mice. The elevated TUNEL staining may reflect necrotic but not caspase-dependent apoptotic DNA damage since activities of caspases 3 and 8 were similar for the three genotypes. The mitochondrial dysfunction in the pyrazole plus TNF-α-treated WT and JNK2 KO mice may have caused a necrotic rather than an apoptotic mode of cell injury. Levels of ATP were lowest in all the pyrazole plus TNF-α treated mice which likely limits full blown apoptosis. Levels of CYP2E1 protein and activity were similar in the three genotypes treated with TNF-α alone. Induction of CYP2E1 protein and activity by pyrazole was found with all three genotypes. However, although CYP2E1 protein was increased, CYP2E1 catalytic activity was not elevated in the WT mice or JNK2 KO mice treated with pyrazole plus TNF-α. We speculate that this may be due to the hepatotoxicity and oxidative stress found with these two genotypes. Heme proteins such as cytochrome P450 are denatured by oxidants such as lipid hydroperoxides with a subsequent loss of catalytic activity (25
). Thus, CYP2E1 catalytic activity but not CYP2E1 protein may not be elevated 24 hrs after addition of TNF-α to pyrazole-treated WT mice or JNK2 KO mice because of the hepatotoxicity and oxidative stress found with these mice but not in JNK1 KO mice at this time point. CYP2E1 catalytic activity is comparably elevated in pyrazole alone treated mice with all three genotypes, likely because there is no hepatotoxicity in these mice. In any event, the lack of hepatotoxicity (and elevated oxidative stress) in the pyrazole plus TNF-α-treated JNK1 KO mice is not due to failure to elevate CYP2E1 in these mice.
While ROS play an important role in activating JNK, increases in JNK can further elevate ROS (26
). Several parameters associated with oxidative/nitrosative stress were elevated in the WT and the JNK2 KO mice, but not the JNK1 KO mice treated with pyrazole plus TNF-α. The increases in 4-HNE and 3-NT protein adducts and iNOS were largely in the pericentral zone of the liver acinus, the area where the liver injury was most pronounced. It is likely that the elevated oxidative/nitrosative stress is playing a central role in the hepatotoxicity found with the JNK2 KO mice, while the lack of an increase in oxidative/nitrosative stress in the JNK1 KO mice is preventive against hepatotoxicity. Oxidative stress may be enhanced because of decreases in survival factors such as pAkt or decreases in antioxidant protection e.g. GSH, Trx-2, GPx-4. Catalase activity was lowered in the WT and JNK2 KO mice compared to the JNK1 KO mice. The decline in catalase activity and levels of GSH, GPx-4 and Trx-2 would impact on effective removal of H2
in the WT and JNK2 KO mice. Note that catalase activity but not catalase protein was lowered in the WT and JNK2 KO mice (but not the JNK1 KO mice), results similar to the decline in CYP2E1 activity but not protein previously discussed. Catalase, like CYP2E1, is a heme enzyme; activity likely declines because of denaturation of the heme by the elevated oxidative stress as discussed for CYP2E1 catalytic activity.
JNK kinase activity was elevated in the JNK1 KO mice after treatment with pyrazole plus TNF-α, but decreased in the WT and the JNK2 KO mice. This may suggest that JNK2 signalling events after pyrazole plus TNF-α treatment are more pronounced upon depletion of JNK1. Loss of JNK2 caused increased activation of JNK1 in a mouse steatohepatitis model (17
), and JNK2 interferred with JNK1 activation by TNF-α in fibroblasts (28
). In general, JNK1, not JNK2, is believed to be most active in phosphorylation of c-Jun (17
). Further studies are required to determine why JNK kinase activity is activated by pyrazole plus TNF-α in JNK1 KO mice and whether this plays a role in blunting the pyrazole plus TNF-α hepatotoxicity and oxidative stress in these mice. Recent studies have evaluated whether JNK1 or JNK2 play the more predominant role in potentiation of liver injury. In fibroblasts, JNK1 but not JNK2 appears to be essential for TNF-α-induced apoptosis (28
). However, liver injury produced by either LPS/D-galactosamine or TNF-α/D-galacosamine was the same in WT and JNK1 KO mice but lower in JNK2 KO mice (29
). JNK1 but not JNK2 promoted the development of steatohepatitis in mice fed a methionine choline-deficient diet (17
). Singh et al (30
) reported that JNK1 KO mice fed a high fat diet did not gain weight or develop steatohepatitis as did the WT and JNK2 KO mice. JNK2 was found to be predominant in acetaminophen toxicity (18
). 6-hydroxydopamine-induced apoptosis in PC12 cells was JNK2 but not JNK1 dependent (31
). While it has been suggested that JNK1 may promote cell death and JNK2 may promote proliferation and survival (21
), it appears that depending on the toxin and cell type, either JNK1 or JNK2 or both play the major role in cell injury. Our results show that JNK1 plays the major role in the pyrazole plus TNF-α-induced liver injury, elevated oxidative stress and mitochondrial dysfunction.
It is not clear what activity JNK1 may catalyze that is not being catalyzed by JNK2, or vice versa, to explain the predominant role of JNK1 or JNK2 in promoting hepatotoxicity of a particular toxin or condition. Failure to activate caspase-8 or -3 or -7 was suggested as a possible reason why LPS/D-galactosamine was not toxic in the JNK2 KO mice (29
), however, JNK1 appeared to be important for this activation in a steatohepatitis model (30
). Activities of caspase-3 and -8 were comparable in the WT, JNK1 KO and JNK2 KO mice treated with pyrazole plus TNF-α. Toxicity in the WT and JNK2 KO mice was largely necrotic, not apoptotic as TUNEL staining did not correlate with caspase activities. We did not find significant differences in the levels of Bcl-2, Bcl-XL, Bax and Bak in livers of the three genotypes treated with pyrazole plus TNF-α (data not shown). Elevation of CYP2E1 protein levels was the same by pyrazole or pyrazole plus TNF-α in the three genotypes. There were no differences in TNF-α levels or levels of TNFR1 between the three genotypes to explain why JNK1 KO mice were protected against the pyrazole plus TNF-α toxicity whereas JNK2 KO mice were not. Levels of endoplasmic reticulum stress markers were not significantly elevated by pyrazole plus TNF-α treatment in all three genotypes (data not shown). In prostate cancer cells, it was concluded that TNF-α-induced ROS formation was mediated by JNK1 but not JNK2 and a mechanism involving ferritin degradation and increase in cellular non-heme iron pools was suggested (33
). Hepatic iron levels were not determined in our study, nor whether iron chelators mitigate the pyrazole plus TNF-α toxicity and oxidative stress Proteomic and molecular modeling experiments may be helpful in identifying JNK1 target(s). Use of a liver specific JNK1 KO mouse model may also be helpful in determining whether differences between JNK1 and JNK2 in promoting the pyrazole plus TNF-α hepatotoxicity is specific to JNK actions in the liver, and not extrahepatic JNK actions. While elevated oxidative/nitrosative stress, decline in antioxidant protection, and mitochondrial dysfunction occur in the JNK2 but not JNK1 KO mice treated with pyrazole plus TNF-α, suggesting a role for JNK1 in interacting with and potentiating CYP2E1 toxicity, additional studies are necessary to identify specific target(s) for JNK1, but not JNK2, which play critical roles in the CYP2E1 plus TNF-α toxicity. Identifying JNK1 as the JNK isoform responsible for the liver injury, the elevated oxidative stress and the mitochondrial dysfunction, may have relevance in attempts to prevent or lower the hepatotoxicity by specifically inhibiting JNK1 without affecting JNK2.