Stable expression of human cytochrome P4502E1 in HepG2 cells: Characterization of catalytic activities and production of reactive oxygen intermediates

Abstract

Experiments were carried out to stably and constitutively express the coding sequence of the human cytochrome P4502E1 in HepG2, a human-hepatoma-derived cell line, by recombinant retroviral expression. Southern blot analysis showed a successful integration of a single copy of unaltered viral DNA into the genome of each transduced clone tested. Northern blot analysis showed that the transduced clones produced an RNA species which hybridized to the CYP2E1 cDNA probe. Western blot analysis using anti-human P4502E1 IgG indicated that the transduced clones produced a protein band with molecular weight of 54 000. Microsomes from transduced clones were catalytically active with p-nitrophenol, dimethylnitrosamine, aniline, and ethanol as substrates; little or no activity was found with control clones. Oxidation of p-nitrophenol was inhibited by anti-human P4502E1 IgG, diethyl dithiocarbamate, 4-methylpyrazole, and ethanol. ESR spectroscopy showed that microsomes from clone MV2E1-9 produced superoxide radical. Rates were an order of magnitude higher than that for control microsomes, most likely reflecting the loose coupling associated with P4502E1. The rate of H2O2 production by microsomes from MV2E1-9 was 2-fold greater than that of control clones. The elevated rate of H2O2 production in clone MV2E1-9 is about half the rate of superoxide radical production, suggesting that this H2O2 is largely derived from superoxide radical dismutation. Microsomal lipid peroxidation was determined using ferric-ATP as the iron catalyst. When the concentration of iron was "high" (0.025 mM), rates of production of thiobarbituric acid reactive components were identical for microsomes from MV2E1-9 and control clones. However, when the concentration of iron was lowered to 0.005 mM, control clones did not display lipid peroxidation, whereas microsomes from MV2E1-9 were reactive. This peroxidation was sensitive to antioxidants such as trolox, propyl gallate, and glutathione but not to catalase or superoxide dismutase. Rates of superoxide and H2O2 production and of lipid peroxidation were 7-20-fold higher on a per nanomole of P450 basis with clone MV2E1-9 compared to human liver microsomes, indicating that the human P4502E1 is especially reactive in production of reactive oxygen intermediates and in catalysis of lipid peroxidation.