Effects of n3/n6‐Derived Epoxy‐Fatty Acids on Hepatocyte Cell Death

Abstract

Background Epoxy-fatty acids (EpFAs) are bioactive lipid mediators that are derived from n3- and n6-polyunsaturated fatty acids (PUFAs) via CYP450 epoxygenation. EpFAs are thought to be beneficial lipids by virtue of their systemic anti-inflammatory, anti-fibrotic, and tissue-regenerative effects. EpFAs are endogenously deactivated by soluble epoxide hydrolase (sEH) to their less biologically active, or in some cases deleterious, dihydroxy-fatty acid (dihydroxy-FAs). Recent work from our group discovered an imbalance in the EpFA/dihydroxy-FA pool in patients with alcohol-associated liver disease (ALD). Importantly, little is known regarding the mechanisms associated with this imbalance or the role of individual EpFAs or dihydroxy-FAs in ALD. Therefore, the objective of this study was to interrogate the effects of EpFAs and dihydroxy-FAs on hepatocyte cell death, a key pathological contributor to liver injury in ALD. Hypothesis EpoxyFAs and dihydroxyFAs will alter hepatocyte survival in vitro following toxic insult in primary or immortalized hepatocytes. Methods Primary mouse hepatocytes, human HepG2, or rat H4IIE cells were treated with 8,9-EET, 8,9-DiHETE, 17,18-EpETE, 19,20-EpDPA, or a vehicle control. Effects on baseline or palmitic acid (PA, 150 µM)-induced cell death were measured by MTT assay and apoptotic cell death was identified by Annexin-V staining (quantified by cellomics analysis). Additionally, primary hepatocytes were isolated from WT and Ephx2-/- mice (global sEH knockout), then exposed to 400 µM PA to determine differences in cell death between genotypes. Cell death was determined by supernatant alanine aminotransferase (ALT) activity and confirmed by MTT assay (n = 3 mice per genotype). Results In both HepG2 cells and primary mouse hepatocytes, n-6-PUFA-derived 8,9-EET had no effect on PA-induced cell death by MTT assay, but its sEH degradation product 8,9-DiHETE significantly decreased cell viability (by 29% in primary cells and 38% in HepG2 cells). By contrast, in H4IIE or HepG2 cells, cellomics analysis showed that n-3-PUFA-derived 19,20-EpDPA reduced PA-induced apoptosis by 21%. Primary mouse hepatocytes derived from Ephx2-/- mice had only a 31% increase in cell death following PA treatment, whereas WT-derived hepatocytes had a 110% increase. These results suggest that with respect to hepatocellular death, n3-derived EpFAs are more beneficial than n6-derived EpFAs. Conclusions PUFA-derived EpFAs and their sEH degradation products have diverse effects in vitro with respect to hepatocyte cell death. In multiple cell lines and in primary mouse hepatocytes, dihydroxy-FAs exacerbated cell death, whereas EpFAs ameliorated it, with n3-derived EpFAs having a more potent effect than those derived from n6-PUFAs. Future studies will further characterize these lipid mediators in liver cell health and determine their mechanisms of action.