Characterization of Arachidonic Acid Metabolism by Rat Cytochrome P450 Enzymes: The Involvement of CYP1As

Abstract

Cytochrome P450 (P450) enzymes mediate arachidonic acid (AA) oxidation to several biologically active metabolites. Our aims in this study were to characterize AA metabolism by different recombinant rat P450 enzymes and to identify new targets for modulating P450-AA metabolism in vivo. A liquid chromatography-mass spectrometry method was developed and validated for the simultaneous measurements of AA and 15 of its P450 metabolites. CYP1A1, CYP1A2, CYP2B1, CYP2C6, and CYP2C11 were found to metabolize AA with high catalytic activity, and CYP2A1, CYP2C13, CYP2D1, CYP2E1, and CYP3A1 had lower activity. CYP1A1 and CYP1A2 produced v-1→4 hydroxyeicosatetraenoic acids (HETEs) as 88.7 and 62.7%, respectively, of the total metabolites formed. CYP2C11 produced epoxyeicosatrienoic acids (EETs) as 61.3%, and CYP2C6 produced midchain HETEs and EETs as 48.3 and 29.4%, respectively, of the total metabolites formed. The formation of CYP1A1, CYP1A2, CYP2C6, and CYP2C11 major metabolites followed an atypical kinetic profile of substrate inhibition. CYP1As inhibition by a-naphthoflavone or anti-CYP1As antibodies significantly reduced v-1→4 HETE formation in the lungs and liver, whereas CYP1As induction by 3-methylcholanthrene resulted in a significant increase in v-1→4 HETEs formation in the heart, lungs, kidney, and livers by 370, 646, 532, and 848%, respectively. In conclusion, our results suggest that CYP1As and CYP2Cs are major players in the metabolism of AA. The significant contribution of CYP1As to AA metabolism and their strong inducibility suggest their possible use as targets for the prevention and treatment of several diseases.

Omeprazole increases the efficacy of a soluble epoxide hydrolase inhibitor in a PGE2 induced pain model

“…Compared to the PEG400 vehicle control, OME significantly increased (p < 0.05) levels of 14, 15-EET and 12,13-EpOME in plasma. It has been reported that P450 1A and 2B generate more hydroxy metabolites than epoxy metabolites in vitro (El-Sherbeni and El-Kadi, 2014). Similarly, TPPU alone resulted in a significant increase (p < 0.05) in the level of 12,13-EpOME, a linoleic acid epoxy metabolite (Table 1).…”

“…EROD, MROD, PROD and BFCOD activities, consistent with P450 1A1, P450 1A2, P450 2B1 and P450 3A1 + P450 1A2 (Yang et al, 2003; Masubuchi et al, 1997; Petrulis et al, 2001; Baer-Dubowska et al, 1998; Donato et al, 2004) were induced with OME; however, this induction was short lived (2 days). These isoforms of P450s also generate EETs (Diani-Moore et al, 2006; El-Sherbeni and El-Kadi, 2014; Mitra et al, 2011) in addition to P450 2D (Thompson et al, 2000). In contrast, AMMCOD activity, indicative of P450 2D2 activity, was significantly suppressed by OME because the proton pump inhibitor at different concentrations (10 nM-1 µM) did not inhibit formation of the product AHMC when AMMC (substrate) was incubated with liver microsomes from untreated rats in vitro .…”

“…Rate of epoxidation/hydroxylation of ARA by rat recombinant P450 1A1, P450 1A2, P450 2B1, P450 2C6 and P450 2C11 is approximately 1/11, 1.5/6.5, 0.5/1.5, 3/7, and 12/8 pmole/pmole P450/min respectively (El-Sherbeni and El-Kadi et al, 2014). The rate of epoxidation/hydroxylation by P450 3A4 is reported as ~ 2/0.1 pmol/pmol CYP3A4/min (Mitra et al, 2011).…”

Dietary Omega-3 Polyunsaturated Fatty Acids Prevent Vascular Dysfunction and Attenuate Cytochrome P4501A1 Expression by 2,3,7,8-Tetrachlorodibenzo-P-Dioxin

“…Although TCDD induces CYP1A1, 1A2, and 1B1 and studies show they can produce 19-HETE, there is no evidence these P450s can produce 20-HETE (Choudhary et al, 2004;El-Sherbeni et al, 2014;Falck et al, 1990;Schwarz et al, 2004). CYP4A12 is the major P450 isoform that generates 20-HETE in male mice (Muller et al, 2007); however, it was not induced by TCDD in either the liver or kidney.…”

Cardiotonic Pill Reduces Myocardial Ischemia-Reperfusion Injury via Increasing EET Concentrations in Rats

“…CYP2C11 and CYP2J3 are two main AA epoxygenases in the rat heart (El-Sherbeni et al, 2013). Cardiac CYP1A1, CYP2B1, and CYP2E1 have also been reported to display AA epoxygenase activity (El-Sherbeni and El-Kadi, 2014). CYP1B1 has been found able to metabolize AA into both EETs and 20-HETE simultaneously (Choudhary et al, 2004).…”