Cytochrome P4502E1 is responsible for miscoding etheno-Dna adducts induced by ethanol in the liver

Abstract

Chronic alcohol consumption leads to hepatic cirrhosis and hepatocellular cancer (HCC) possibly via oxidative stress. Ethanol induces cytochrome P4502E1 (CYP2E1) resulting in the generation of reactive oxygen species (ROS) which cause lipidperoxidation. Lipid peroxidation products such as 4-hydroxynonenal (4HNE) can finally react with DNA forming exocyclic etheno-DNA adducts with high mutagenic and carcinogenic properties (1). Such adducts have been detected in hepatic biopsies from patients with alcoholic liver disease (ALD) (2). To study whether alcohol metabolism via CYP2E1 results in the generation of 1,N6-ethenodeoxyadenosine (εdA) and 1,N6-ethenodeoxycitosine (εdC), we used HEP G–2 cells, overexpressing CYP2E1 (E47) and mok transfected control cells (C34). The cells were incubated with ethanol concentrations between 0 and 25 mM for 0, 24, 48 and 72 hours. Chlormethiazole (CMZ) was used to inhibit CYP2E1. εdA, εdC and CYP2E1 were determined by immunohistochemistry (3). In addition, glutathione levels were also determined by staining cells with monobromobimane. Ethanol results in an increase in εdA and εdC in a dose and time dependent manner in E47 cells, but not in C34 cells. CMZ in a concentration of 20µM resulted in a significant inhibition of εdA and εdC at each ethanol concentration. This inhibition was 100% at 2.5 and 5.0 mM ethanol and 66–75% at 10 mM and 77–90% at 25 mM ethanol. Although some E47 cells did only reveal minor amounts of CYP2E1, they still showed adduct formation. This was probably due to a decrease in glutathione levels as part of the antioxidative defense system observed in E47 cells in the presence of ethanol. A significant co-localization between exocylic etheno-DNA adducts and CYP2E1 as well as between the two adducts εdA and εdC was noted. The results support the hypothesis that CYP2E1 is responsible for the generation of highly mutagenic and carcinogenic exocyclic etheno-DNA adducts possibly via the generation of ROS with consecutive lipidperoxidation. It is concluded that excessive hepatic DNA damage as assessed by miscoding etheno DNA adducts contributes to the genomic instability and acts as a driving force towards malignancy in ALD. Induction of CYP2E1 by chronic alcohol consumption seems to be a major mechanism for this finding.