Chronic alcohol induces hepatic oxidative stress, characterized by increased concentration of reactive oxygen species. Cytochrome P450 2E1 (CYP2E1, both microsomal and mitochondrial) contribute to this increased oxidative stress. The mechanisms by which chronic alcohol induces mitochondrial oxidative stress are unclear. In this work, we evaluated the hypothesis that mitochondrial reactive oxygen species derived from mitochondrial CYP2E1 trigger the induction of mitochondrial oxidative stress in hepatocytes. HepG2 cells overexpressing mitochondrial CYP2E1 (mE10 cells, transfected with an expression vector containing the human CYP2E1 cDNA lacking the coding sequence for amino acids 2-34) and HepG2 cells not expressing any cytochrome P450 (C34 cells, transfected with an empty expression vector) were used in this study. Overexpression of mitochondrial CYP2E1 in mE10 cells was confirmed by western blot, RT-PCR and enzymatic analysis. Overexpression of mitochondrial CYP2E1 in HepG2 cells occurred together with increased mitochondrial reactive oxygen species evaluated by flow cytometry using MitoSox Red. Mitochondrial oxidative stress was further increased in mE10 cells by incubation with docosahexaenoic acid, a polyunsaturated fatty acid. These results show a correlation between mitochondrial CYP2E1 expression and mitochondrial oxidative stress. The mechanisms by which mitochondrial CYP2E1 induces mitochondrial oxidative stress is under further investigation in our lab.

Effect of mitochondrial CYP2E1 overexpression on mitochondrial oxidative stress in HepG2 cells

253rd National Meeting (2017)

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