Cytochrome P450 (P450)-derived arachidonic acid (AA) metabolites serve pivotal physiological roles. Therefore, it is important to determine the dominant P450 AA monooxygenases in different organs. We investigated the P450 AA monooxygenases protein expression as well as regioselectivity, immunoinhibition, and kinetic profile of AA epoxygenation and hydroxylation in rat heart, lung, kidney, and liver. Thereafter, the predominant P450 epoxygenases and P450 hydroxylases in these organs were characterized. Microsomes from heart, lung, kidney, and liver were incubated with AA. The protein expression of CYP2B1/2, CYP2C11, CYP2C23, CYP2J3, CYP4A1/2/3, and CYP4Fs in the heart, lung, kidney, and liver were determined by Western blot analysis. The levels of AA metabolites were determined by liquid chromatography- electrospray ionization mass spectroscopy. This was followed by determination of regioselectivity, immunoinhibition effect, and the kinetic profile of AA metabolism. AA was metabolized to epoxyeicosatrienoic acids and 19- and 20-hydroxyeicosatetraenoic acid in the heart, lung, kidney, and liver but with varying metabolic activities and regioselectivity. Anti-P450 antibodies were found to differentially inhibit AA epoxygenation and hydroxylation in these organs. Our data suggest that the predominant epoxygenases are CYP2C11, CYP2B1, CYP2C23, and CYP2C11/CYP2C23 for the heart, lung, kidney, and liver, respectively. On the other hand, CYP4A1 is the major ω-hydroxylase in the heart and kidney; whereas CYP4A2 and/or CYP4F1/4 are probably the major hydroxlases in the lung and liver. These results provide important insights into the activities of P450 epoxygenases and P450 hydroxylases-mediated AA metabolism in different organs and their associated P450 protein levels.
Bibliographical noteFunding Information:
The authors wish to thank Dr. Dion Brocks for his valuable comments on this manuscript. This work was supported by a grant from the Canadian Institutes of Health Research (CIHR) MOP 106665 to AOSE. AAE is the recipients of Egyptian Government Scholarship. BNMZ is the recipient of Alberta Innovates-Health Solutions Studentship. AA-M is the recipient of Alberta Ingenuity Graduate Scholarship and Izaak Walton Killam Memorial Graduate Scholarship.
Louis, MO). AA metabolite standards: 5,6-epoxyeicosatrienoic acid (5,6-EET), 8,9-epoxyeicosatrienoic acid (8,9-EET), 11,12-epoxyeicosatrienoic acid (11,12-EET), 14,15-epoxyeicosatrienoic acid (14,15-EET), 5,6-dihydroxyeicosa-trienoic acid (5,6-DHET), 8,9-dihydroxyeicosatrienoic acid (8,9-DHET), 11,12-dihydroxyeicosatrienoic acid (11,12-DHET), 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), 19-HETE, and 20-HETE, were purchased from Cayman Chemical (Ann Arbor, MI). High-performance liquid chromatography grade reagents were used for liquid chromatography–electrospray ionization mass spectrometry (LC-ESI-MS). Acetonitrile, methanol, and ethyl acetate were purchased from EM Scientific (Gibbstawn, NJ). Potassium chloride, calcium chloride dihydrate, potassium dihydrogen orthophosphate, dipotassium hydrogen orthophosphate, and magnesium chloride hexahydrate were obtained from BDH (Toronto, ON, Canada). Acrylamide, N′N′-bis-methylene-acrylamide, ammonium persulphate, β-mercaptoethanol, glycine, nitrocellulose membrane (0.45 μm), and TEMED were purchased from Bio-Rad Laboratories (Hercules, CA). Chemiluminescence Western blotting detection reagents were purchased from GE Healthcare Life Sciences, Piscataway, NJ. CYP2C11, CYP2C23, and CYP4F2 primary antibodies were purchased from Abcam (Cambridge, UK). CYP2Js primary antibody was a generous gift from Dr. Darryl Zeldin (National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC). CYP2B1/2, CYP4A1/2/3, secondary antibodies, and control IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Other chemicals were purchased from Fisher Scientific Co. (Toronto, ON, Canada).
- P450 epoxygenase activity
- P450 hydroxylase activity
- arachidonic acid metabolism
- cytochrome P450