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Generation and characterization of epoxide hydrolase 3 ( EPHX3 )-deficient mice

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


LC-MS/MS analysis shows no differences in endogenous epoxide:diol ratios in the Ephx3-/- mice.Endogenous epoxide:diol ratios for (A) 8,9-EET/8,9-DHET, (B) 11,12-EET/11,12-DHET, (C) 14,15-EET/14,15-DHET, (D) 9,10-EpOME/9,10-DiHOME, (E) 12,13-EpOME/12,13-DiHOME, (F) 17,18-EpETE/17,18-DHET, and (G) 19,20-EpDPE/19,20-DiHDPA were determined in the heart, lung, skin, and plasma by LC-MS/MS in Ephx3-/- and WT mice (n = 3–10, p = NS).
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pone.0175348.g005: LC-MS/MS analysis shows no differences in endogenous epoxide:diol ratios in the Ephx3-/- mice.Endogenous epoxide:diol ratios for (A) 8,9-EET/8,9-DHET, (B) 11,12-EET/11,12-DHET, (C) 14,15-EET/14,15-DHET, (D) 9,10-EpOME/9,10-DiHOME, (E) 12,13-EpOME/12,13-DiHOME, (F) 17,18-EpETE/17,18-DHET, and (G) 19,20-EpDPE/19,20-DiHDPA were determined in the heart, lung, skin, and plasma by LC-MS/MS in Ephx3-/- and WT mice (n = 3–10, p = NS).

Mentions: Previous in vitro studies showed that EPHX3 has epoxide hydrolase activity with high catalytic efficiency for 8,9-EET, 11,12-EET, and 9,10-EpOME. To determine if EPHX3 performs significant hydrolysis of fatty acid epoxides in vivo, LC-MS/MS analysis was performed on tissue lysates prepared from lung, skin, stomach, and heart samples. We quantified levels of arachidonic acid (Fig 5A–5C), linoleic acid (Fig 5D and 5E), EPA (Fig 5F), and DHA (Fig 5G)-derived epoxides and diols. There were no differences in epoxide:diol ratios between Ephx3-/- and WT mice in any of these tissues.


Generation and characterization of epoxide hydrolase 3 ( EPHX3 )-deficient mice
LC-MS/MS analysis shows no differences in endogenous epoxide:diol ratios in the Ephx3-/- mice.Endogenous epoxide:diol ratios for (A) 8,9-EET/8,9-DHET, (B) 11,12-EET/11,12-DHET, (C) 14,15-EET/14,15-DHET, (D) 9,10-EpOME/9,10-DiHOME, (E) 12,13-EpOME/12,13-DiHOME, (F) 17,18-EpETE/17,18-DHET, and (G) 19,20-EpDPE/19,20-DiHDPA were determined in the heart, lung, skin, and plasma by LC-MS/MS in Ephx3-/- and WT mice (n = 3–10, p = NS).
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Related In: Results  -  Collection

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pone.0175348.g005: LC-MS/MS analysis shows no differences in endogenous epoxide:diol ratios in the Ephx3-/- mice.Endogenous epoxide:diol ratios for (A) 8,9-EET/8,9-DHET, (B) 11,12-EET/11,12-DHET, (C) 14,15-EET/14,15-DHET, (D) 9,10-EpOME/9,10-DiHOME, (E) 12,13-EpOME/12,13-DiHOME, (F) 17,18-EpETE/17,18-DHET, and (G) 19,20-EpDPE/19,20-DiHDPA were determined in the heart, lung, skin, and plasma by LC-MS/MS in Ephx3-/- and WT mice (n = 3–10, p = NS).
Mentions: Previous in vitro studies showed that EPHX3 has epoxide hydrolase activity with high catalytic efficiency for 8,9-EET, 11,12-EET, and 9,10-EpOME. To determine if EPHX3 performs significant hydrolysis of fatty acid epoxides in vivo, LC-MS/MS analysis was performed on tissue lysates prepared from lung, skin, stomach, and heart samples. We quantified levels of arachidonic acid (Fig 5A–5C), linoleic acid (Fig 5D and 5E), EPA (Fig 5F), and DHA (Fig 5G)-derived epoxides and diols. There were no differences in epoxide:diol ratios between Ephx3-/- and WT mice in any of these tissues.

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.