Hoopes Samantha L, Gruzdev Artiom, Edin Matthew L, Graves Joan P, Bradbury J Alyce, Flake Gordon P, Lih Fred B, DeGraff Laura M, Zeldin Darryl C
Division of Intramural Research, National Institute of Environmental Health Sciences, Research Triangle Park, Durham, NC, United States of America.
PLoS One. 2017 Apr 6;12(4):e0175348. doi: 10.1371/journal.pone.0175348. eCollection 2017.
Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play an important role in blood pressure regulation, protection against ischemia-reperfusion injury, angiogenesis, and inflammation. Epoxide hydrolases metabolize EETs to their corresponding diols (dihydroxyeicosatrienoic acids; DHETs) which are biologically less active. Microsomal epoxide hydrolase (EPHX1, mEH) and soluble epoxide hydrolase (EPHX2, sEH) were identified >30 years ago and are capable of hydrolyzing EETs to DHETs. A novel epoxide hydrolase, EPHX3, was recently identified by sequence homology and also exhibits epoxide hydrolase activity in vitro with a substrate preference for 9,10-epoxyoctadecamonoenoic acid (EpOME) and 11,12-EET. EPHX3 is highly expressed in the skin, lung, stomach, esophagus, and tongue; however, its endogenous function is unknown. Therefore, we investigated the impact of genetic disruption of Ephx3 on fatty acid epoxide hydrolysis and EET-related physiology in mice. Ephx3-/- mice were generated by excising the promoter and first four exons of the Ephx3 gene using Cre-LoxP methodology. LC-MS/MS analysis of Ephx3-/- heart, lung, and skin lysates revealed no differences in endogenous epoxide:diol ratios compared to wild type (WT). Ephx3-/- mice also exhibited no change in plasma levels of fatty acid epoxides and diols relative to WT. Incubations of cytosolic and microsomal fractions prepared from Ephx3-/- and WT stomach, lung, and skin with synthetic 8,9-EET, 11,12-EET, and 9,10-EpOME revealed no significant differences in rates of fatty acid diol formation between the genotypes. Ephx3-/- hearts had similar functional recovery compared to WT hearts following ischemia/reperfusion injury. Following intranasal lipopolysaccharide (LPS) exposure, Ephx3-/- mice were not different from WT in terms of lung histology, bronchoalveolar lavage fluid cell counts, or fatty acid epoxide and diol levels. We conclude that genetic disruption of Ephx3 does not result in an overt phenotype and has no significant effects on the metabolism of EETs or EpOMEs in vivo.
细胞色素P450（CYP）环氧合酶将花生四烯酸代谢为环氧二十碳三烯酸（EETs），其在血压调节、抗缺血再灌注损伤、血管生成和炎症中发挥重要作用。环氧水解酶将EETs代谢为相应的二醇（二羟基二十碳三烯酸；DHETs），后者的生物活性较低。微粒体环氧水解酶（EPHX1，mEH）和可溶性环氧水解酶（EPHX2，sEH）在30多年前就已被鉴定出来，能够将EETs水解为DHETs。最近通过序列同源性鉴定出一种新型环氧水解酶EPHX3，其在体外也表现出环氧水解酶活性，对9,10-环氧十八碳单烯酸（EpOME）和11,12-EET具有底物偏好性。EPHX3在皮肤、肺、胃、食管和舌中高度表达；然而，其内源性功能尚不清楚。因此，我们研究了Ephx3基因敲除对小鼠脂肪酸环氧化水解和EET相关生理学的影响。使用Cre-LoxP方法切除Ephx3基因的启动子和前四个外显子，从而产生Ephx3-/-小鼠。对Ephx3-/-心脏、肺和皮肤裂解物进行液相色谱-串联质谱（LC-MS/MS）分析，结果显示与野生型（WT）相比，内源性环氧化物：二醇比率没有差异。相对于WT，Ephx3-/-小鼠血浆中脂肪酸环氧化物和二醇水平也没有变化。用合成的8,9-EET、11,12-EET和9,10-EpOME孵育从Ephx3-/-和WT胃、肺和皮肤制备的胞质和微粒体部分，结果显示不同基因型之间脂肪酸二醇形成速率没有显著差异。在缺血/再灌注损伤后，Ephx3-/-心脏与WT心脏的功能恢复相似。经鼻内给予脂多糖（LPS）后，Ephx3-/-小鼠在肺组织学、支气管肺泡灌洗液细胞计数或脂肪酸环氧化物和二醇水平方面与WT没有差异。我们得出结论，Ephx3基因敲除不会导致明显的表型，并且对体内EETs或EpOMEs的代谢没有显著影响。
