Fig 1.
A) Wild type Ephx3 locus on the reverse strand of chromosome 17:b1. B) Targeting vector to introduce LoxP sites flanking the promoter region and first four exons of Ephx3. Targeting vector constructed with FRT-site flanked neomycin resistance positive selection and MC1-DTA negative selection. C) Targeted Ephx3 locus following homologous recombination. D) Ephx3 “flox” locus following excision of neomycin resistance cassette via in vivo FLP recombinase exposure. E) Ephx3 null allele generated by breeding to germline expressing Cre recombinase mouse line (Prm-Cre). F) Representative Southern blots using both the 5’ and 3’ probes to confirm proper recombination event. Only relevant restriction enzymes sites shown with expected fragment sizes.
Fig 2.
Expression of epoxide hydrolase isoforms in Ephx3-/- mice.
Quantitative RT-PCR of (A) Ephx3, (B) Ephx1, (C) Ephx2, and (D) Ephx4 in Ephx3-/- and WT mice. (*p<0.05; n = 3–4).
Fig 3.
Disruption of Ephx3 does not alter mRNA levels of CYP epoxygenases.
Quantitative RT-PCR of Cyp2c and Cyp2j isoforms in (A) lung, (B) skin, (C) stomach, (D) heart, (E) esophagus, (F) tongue, and (G) liver of Ephx3-/- and WT mice. (*p<0.05; n = 3–5, p = NS).
Fig 4.
Body weight and organ weight:body weight ratios are not altered in Ephx3-/- mice.
(A) Body weight of both male and female WT and Ephx3-/- mice. (B) Heart weight:body weight ratio and (C) kidney weight:body weight ratio in Ephx3-/- and WT mice (n = 5–10, p = NS).
Fig 5.
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).
Fig 6.
Incubation with synthetic EETs/EpOMEs showes no changes in diol formation rate in Ephx3-/- mice.
LC-MS/MS analysis of diol formation after a 5 minute incubation with synthetic EETs or EpOMEs in (A) lung, (B) skin, and (C) stomach lysates from Ephx3-/- and WT mice. (n = 5–10, p = NS).
Fig 7.
11,12-DHET formation rates are largely unchanged in microsomal and cytosolic fractions from various tissues of Ephx3-/- mice.
LC-MS/MS analysis of 11,12-DHET formation in microsomal and cytosolic fractions after a 5 minute incubation with synthetic 11,12-EET with/without the EPHX2 inhibitor t-AUCB in (A) lung, (B) skin, and (C) stomach samples (n = 3–6, *p<0.05).
Fig 8.
Bronchoalveolar lavage fluid cells, lung histology, and plasma epoxide:diol ratios are unchanged in Ephx3-/- mice after LPS treatment.
(A) Bronchoalveolar lavage fluid cell counts and differentials, (B) lung histology, and (C) LC-MS/MS epoxide/diol ratios in plasma from Ephx3-/- and WT mice four hours after intranasal LPS exposure (n = 4–6; p = NS, original magnification = 10x).
Table 1.
Inflammation scores in LPS-induced lung injury model.
Table 2.
Eicosanoids and Prostaglandins (pg/ml) in Plasma from Ephx3-/- and WT mice treated with or without LPS.
Fig 9.
Cardiac recovery is unchanged in Ephx3-/- hearts after global ischemia/reperfusion.
(A) Hearts were allowed to equilibrate for 40 min, subjected to 20 min of global, no-flow ischemia, and then reperfused for 40 min using the Langendorff system. (B) Time course for recovery of left ventricular developed pressure. (C) Time course for recovery of rate pressure product (n = 3–4, p = NS).