HepG2 cells overexpressing cytochrome P450 2E1 (CYP2E1) were generated by transfection with a pCI expression vector containing the human CYP2E1 cDNA, and selected with the use of geneticin. Overexpression of CYP2E1 was confirmed at the RNA level by reverse-transcriptase PCR, at the protein level by western blot, and at the enzymatic activity level by 7-methoxy-4-trifluorocoumarin O-demethylation. Control HepG2 cells were transfected with the empty pCI plasmid. The content of mitochondrial DNA in CYP2E1-overexpressing HepG2 cells (determined by real-time PCR and expressed relative to nuclear DNA content), was two-fold higher than that of control HepG2 cells. In parallel, the level of reactive oxygen species expressed as dichlorodihydrofluorescein oxidation rate and determined by flow cytometry increased two-fold with respect to control HepG2 cells. These results support a working hypothesis where CYP2E1 overexpression induces mitochondrial DNA synthesis, probably by increasing oxidative stress levels.

CYP2E1 overexpression induces mitochondrial biogenesis in HepG2 cells

245th National Meeting (2013)

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