Chronic alcoholism induces hepatic mitochondrial biogenesis in vivo. However, the mechanisms by which ethanol induces mitochondrial biogenesis are unknown. Chronic ethanol consumption also enhances oxidative stress in the liver. A central pathway by which ethanol generates oxidative stress is the induction of the cytochrome P450 isoform 2E1 (CYP2E1). Recently, our laboratory provided evidence that hepatic oxidative stress as a result of chronic ethanol exposure triggers mitochondrial biogenesis. However, the source of oxidants due to ethanol that triggers these events is unknown. The central hypothesis of this work is that oxidants derived from CYP2E1 induce mitochondrial biogenesis in hepatocytes. To test our hypothesis we use two cell lines: HepG2 cells that were transfected with a CYP2E1-containing plasmid to constitutively express CYP2E1 (E47 cells), or an empty plasmid as a control cell line (C34 cells). Cells were plated in in MEM supplemented with docosahexaenoic acid (DHA, as a pro-oxidant) up to 10 uM for up to 3 days. Viability assessed by MTT reduction was not affected by exposure to DHA, confirming that the growing conditions were non-cytotoxic. Lipid oxidation assessed with C11-BODIPY 581/591 by flow cytometry increased only in E47 cells exposed to DHA, confirming the generation of CYP2E1-dependent oxidative stress. Mitochondrial transcription factor-A (TFAM) mRNA levels (marker of mitochondrial biogenesis) increased in E47 cells exposed to DHA, suggesting the role of CYP2E1 and DHA in inducing mitochondrial biogenesis. These results suggest that CYP2E1-derived DHA oxidation products can induce mitochondrial biogenesis in hepatocytes.

Docosahexaenoic acid induces mitochondrial biogenesis in HepG2 cells that overexpress cytochrome P450 2E1

253rd National Meeting (2017)

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