Transgenic expression of human cytochrome P450 2E1 in C. elegans and rat PC-12 cells sensitizes to ethanol-induced locomotor and mitochondrial effects.

Chronic alcohol (ethanol) use is increasing in the United States and has been linked to numerous health issues in multiple organ systems including neurological dysfunction and diseases. Ethanol toxicity is mainly driven by the metabolite acetaldehyde, which is generated through three pathways: alcohol dehydrogenase (ADH2), catalase (CAT), and cytochrome P450 2E1 (CYP2E1). ADH2, while the main ethanol clearance pathway in the liver, is not expressed in the mammalian brain, resulting in CAT and CYP2E1 driving local metabolism of ethanol in the central nervous system. CYP2E1 is known to generate reactive metabolites and reactive oxygen species and localizes to the mitochondria (mtCYP2E1) and endoplasmic reticulum (erCYP2E1). We sought to understand the consequences of mtCYP2E1 and erCYP2E1 in the nervous system during acute ethanol exposure. To answer this question, we generated transgenic Caenorhabditis elegans roundworms expressing human CYP2E1 in the mitochondria, endoplasmic reticulum, or both and exposed them to ethanol. We found that at lower concentrations, wild-type and mtCYP2E1-expressing worms had a small but significant inhibition of locomotion, whereas the erCYP2E1-expressing worms showed protection from this inhibition. At higher doses, all strains had reduced locomotion, but the erCYP2E1-expressing worms recovered faster than wild-type controls. CYP2E1 expression, regardless of organellar targeting, reduced mitochondrial respiration in response to ethanol. Similarly, transgenic expression of CYP2E1 in either organelle in PC-12 rat neuronal cell lines sensitized them to ethanol-induced cell death. Together, these findings suggest that subcellular localization of CYP2E1 impacts behavioral effects of ethanol and should be further studied in the mammalian central nervous system.

Subcellular expression of CYP2E1 in HepG2 cells impacts response to free oleic and palmitic acid.

Aims: Cytochrome P450 2E1 (CYP2E1) is a mammalian monooxygenase expressed at high levels in the liver that metabolizes low molecular weight pollutants and drugs, as well as endogenous fatty acids and ketones. Although CYP2E1 has been mainly studied in the endoplasmic reticulum (ER, microsomal fraction), it also localizes in significant amounts to the mitochondria, where it has been far less studied. We investigated the effects of CYP2E1 expression in mitochondria, endoplasmic reticulum, or both organelles in transgenic HepG2 cells exposed to free oleic and palmitic acid, including effects on cytotoxicity, lipid storage, respiration, and gene expression. Results: We found that HepG2 cells expressing CYP2E1 in both the ER and mitochondria have exacerbated levels of palmitic acid cytotoxicity and inhibited respiration. CYP2E1 expression did not impact lipid accumulation from fatty acid exposures, but mitochondrial CYP2E1 expression promoted lipid droplet depletion during serum starvation. In contrast to HepG2 cells, differentiated HepaRG cells express abundant CYP2E1, but they are not sensitive to palmitic acid cytotoxicity. Oleic acid exposure prompted less cytotoxicity, and CYP2E1 expression in the ER prevented an oleic-acid-induced increase in respiration. HepG2 cells exposed to mixtures of palmitic and oleic acid are protected from palmitic acid cytotoxicity. Additionally, we identified that CYP2E1 was decreased at the gene and protein level in hepatocellular carcinoma. Moreover, patients with tumors that had higher CYP2E1 expression had a better prognosis compared to patients with lower CYP2E1 expression. Innovation: This study has demonstrated that transgenic CYP2E1 subcellular localization plays an important role in sensitivity to cytotoxicity, lipid storage, and respiration in the hepatoma cell line HepG2 exposed to palmitic and oleic acid. HepaRG cells, in contrast, were insensitive to palmitic acid. This work demonstrates the clear importance of CYP2E1 in dictating lipotoxicity and differential roles for the mitochondrial and ER forms of the enzyme. Additionally, our data supports a potentially unique role for CYP2E1 in cancer cells. Conclusion: There lies a role for CYP2E1 in altering lipotoxicity, and since CYP2E1 is known to be upregulated in both liver disease and hepatocellular carcinoma, it is important to better define how the role of CYP2E1 changes during disease progression.