The role of Cytochrome P450 2E1 in hepatic ischemia reperfusion injury and reactive oxygen species formation

Abstract

The mechanisms of hepatic ischemia/reperfusion (I/R) injury, which occurs during liver transplantation or surgery, are poorly understood. The central pathophysiological phenomenon of this injury is the formation of reactive oxygen species (ROS). Recent studies suggest that P450 enzymes may contribute to ROS generation, which may occur through their uncoupling and/or release of heme and iron after their degradation. Among P450 enzymes, CYP2E1 is unique because it is considered a leaky enzyme, producing significant ROS through uncoupling in the absence of substrates, accelerating its own degradation and reducing its half-life. In this dissertation, we generated and characterized a HepG2 cell line with stable overexpression of CYP2E1 to investigate the role of the enzyme in I/R injury in an ex vivo setting. As a result, GFP-tagged CYP2E1 and Control clones were developed, and their gene expression and protein levels of GFP and CYP2E1 were determined using RT-PCR and ELISA/Western blot analysis, respectively. Additionally, the CYP2E1 catalytic activity was determined by UPLC-MS/MS analysis of 6-hydroxychlorzoxazone formed from the chlorzoxazone substrate. The CYP2E1 and Control clones were subjected to hypoxia (10 h) and reoxygenation (0.5 h), and cell death and ROS generation were quantitated using LDH and flow cytometry, respectively. Compared with Control clone, the selected CYP2E1 clone showed a 720-fold increase in CYP2E1 expression and a prominent band in the Western blot analysis, which was associated with a 150-fold increase in the CYP2E1 catalytic activity. CYP2E1 clone produced 2.3-fold more ROS and 1.9-fold more cell death in the H/R model. To investigate the potential role of CYP2E1 degradation in the H/R injury, we also attempted to mutate the wild-type enzyme to prevent its degradation through ubiquitindependent 26S proteasomal system (UPS). Ten lysine residues, which reportedly participate in ubiquitination and degradation of CYP2E1, were successfully mutated to arginine by site-directed mutagenesis PCR. However, multiple attempts at CYP2E1 protein expression were challenging, which hindered further investigations. In conclusion, our HepG2 cellular studies suggest that the constitutive CYP2E1 in the liver may play a detrimental role in hepatic I/R injury.