SAM levels were lower in hepatocytes isolated from pyrazole-treated rats than control hepatocytes. Mato et al found that defective formation of SAM is associated with an impaired synthesis of GSH and that reduced synthesis of SAM and GSH can both act together in a self-perpetuating cycle, where a reduction in hepatic GSH leads to an inhibition of MAT activity via a mechanism involving ROS [35
]. A critical cysteine residue required for MAT1A activity is oxidized by ROS, with a subsequent decline in MAT1A activity and SAM [36
]. Pyrazole elevates expression of CYP2E1, which produces ROS during its catalytic cycle [38
]. We speculate that CYP2E1-derived ROS inhibit the MAT1A which results in lower SAM levels in the hepatocytes isolated from pyrazole-treated rats. Further experiments evaluating this are in progress. Hepatocytes from CMZ plus pyrazole treated rats show higher SAM levels than that of pyrazole treated rats, validating a role for CYP2E1 in the pyrazole-induced decline in hepatic SAM levels. Hepatocytes from CMZ plus pyrazole treated rats with lower CYP2E1 activity have lower production of intracellular ROS and O2•−
Cycloleucine is an effective MAT inhibitor and can strongly lower the SAM levels in control hepatocytes and deplete the SAM level in pyrazole hepatocytes to undetectable concentrations (). Pyrazole hepatocytes that express elevated CYP2E1 were more sensitive to cycloleucine treatment, and exhibited significant cell death compared with that of control hepatocytes (). Cell nuclear morphology, DNA ladder and caspase 3 cleavage revealed that cycloleucine treatment induced apoptosis in pyrazole hepatocytes to a much greater extent than in control hepatocytes. LDH leakage assay indicated that necrosis may also be occurring and contributing to the pyrazole hepatocyte death. The CYP2E1 inhibitor, CMZ protected pyrazole hepatocytes from cycloleucine cytotoxicity by decreasing the elevated CYP2E1 activity, maintaining SAM levels and decreasing ROS levels. It is important to emphasize the very effective hepatoprotective effects of SAM since toxicity is observed only when SAM levels are lowered to undetectable levels. Decreases of “only” 70 to 80% as found with non-treated pyrazole hepatocytes or cycloleucine-treated control hepatocytes did not result in hepatotoxicity even after 3 days in culture.
A likely explanation as to why cycloleucine toxicity is increased in the CYP2E1-expressing cells is an elevated oxidant stress, as ROS is generated via the induction of CYP2E1 in the endoplasmic reticulum, and ROS detoxification is lowered by the decline in SAM. The antioxidants Trolox, NAC and MnTMPyP could rescue pyrazole hepatocytes from cycloleucine-induced cell death. Trolox also decreased the elevated ROS and intracellular O2•− produced by cycloleucine in the pyrazole hepatocytes, indicating that enhanced ROS production appears to be central to the mechanism leading to the death of pyrazole hepatocytes. In addition to an increased production of ROS, a decrease of the intracellular GSH level, likely due to the decline in SAM, is probably an important mechanism for the cycloleucine-induced apoptosis. The lower intracellular GSH levels likely sensitize the pyrazole hepatocytes to the increase in ROS caused by the combination of CYP2E1 plus cycloleucine treatment.
TFP had been used to inhibit the mitochondrial membrane permeability transition and subsequently the loss of mitochondrial membrane potential [32
]. TFP effectively protected pyrazole hepatocytes from toxicity induced by cycloleucine treatment, which suggests that a decrease in mitochondrial membrane potential contributes to the potentiation of cell death caused by cycloleucine treatment in the pyrazole hepatocytes. TFP did not restore GSH levels or prevent the increase in ROS suggesting that mitochondrial injury is a downstream target of the elevated oxidative stress.
shows a scheme of the proposed mechanism of cycloleucine plus pyrazole induced cell death. Pyrazole induces CYP2E1, which increases intracellular ROS production. Cycloleucine and the increased CYP2E1-derived ROS inhibit MAT which results in a decrease in SAM synthesis. A decline in SAM may produce a decline of GSH. The fall in SAM and GSH sustain accumulation of ROS which further causes a loss in MAT activity. The increase in oxidant stress results in a decline in the mitochonrial membrane potential, leading to apoptosis and necrosis. The scheme depicts sites where various protective agents used in this study would operate to protect against the cycloleucine plus CYP2E1 potentiated injury.
Fig. 8 Summary of a proposed mechanism for the enhancement of cell death induced by cycloleucine treatment of pyrazole hepatocytes. Pyrazole elevates CYP2E1 expression and increases intracellular production of ROS. Increases in ROS inhibit methionine adenosyltransferase (more ...)
SAM levels were lowered in animal models of alcohol liver injury and human alcoholic liver disease [39
]. In these situations the CYP2E1 levels are usually elevated. Depleted SAM, reduced GSH levels, and elevated CYP2E1 expression combine to lead to an increased oxidative stress in hepatocytes, which plays a crucial role in execution of the downstream events of apoptosis and necrosis. That could make an important contribution to mechanisms of alcoholic liver injury.