Arachidonic acid metabolism by human cytochrome P450s 2C8, 2C9, 2E1, and 1A2: regioselective oxygenation and evidence for a role for CYP2C enzymes in arachidonic acid epoxygenation in human liver microsomes.
The membrane-bound endogenous fatty acid arachidonic acid can be released from membranes by phospholipases and then metabolized to biologically active compounds by cyclooxygenases, lipoxygenases, and cytochrome P450 (CYP) enzymes. In the liver the CYP pathway is the most significant. Liver CYP arachidonate products include epoxyeicosatrienoic acids (EETs) and monohydroxylated products (HETEs). We examined metabolism of [1-14C]arachidonic acid by a panel of 10 human CYP enzymes expressed in HepG2 cells. In the absence of expressed CYP enzymes, control HepG2 cell microsomes generated only small amounts of omega- and omega--1-OH arachidonic acid (ratio 2:1). Microsomes from HepG2 cells expressing CYP2C8, 2C9, 1A2, and 2E1 were 7-21 times more active than microsomes from the HepG2 controls. CYP2C8, 2C9, and 1A2 principally generated epoxygenase products; 36 to 48% were in the form of EET-diols, reflecting host HepG2 microsomal epoxide hydrolase activity. CYP2C8 and 2C9 formed more 14,15- and 11,12-EET than did CYP1A2, while CYP1A2 formed more 8,9-EET. CYP2C9 also generated a peak with the retention time of 12-HETE. CYP2E1 generated omega--1-OH arachidonic acid and, to a lesser extent, omega-OH arachidonic acid (ratio 2:1). A small amount of epoxygenase activity was also detected for CYP2B6; its overall activity, however, was only about twice control levels. Activities of CYP2A6, 3A3, 3A4, and 3A5 were low and limited to the omega-/omega--1-OH arachidonic acid peak; CYP2D6 was inactive. Microsomes prepared from three individual human livers varied threefold in total arachidonic acid metabolism. For all three livers omega-OH arachidonic acid was the major product (up to 74% of total metabolites). Epoxygenase products constituted 14 to 28% of the total products; 60 to 83% of those were EET-diols, indicating that the human liver microsomes have substantial EET-epoxide hydrolase activity. 11,12-EET was the major EET for two livers and 14,15-EET for the third. The CYP2C inhibitor sulfaphenazole depressed human liver microsomal epoxygenase activity by 50% at 50 microM, while alpha-naphthoflavone inhibited arachidonic acid epoxygenase activity by 27% at 2 microM and by 32% at 10 microM. Collectively, these findings suggest that human liver microsomal arachidonic acid metabolism is catalyzed principally by CYP2C enzymes. CYP1A2, CYP2E1, and possibly CYP2B6 are likely to play more minor roles, though their contribution may be enhanced by exposure to inducers of those enzymes. CYP2A6, CYP2D6, and CYP3A enzymes are unlikely to make any significant contribution.(ABSTRACT TRUNCATED AT 400 WORDS)
Rifkind
A B
AB
Department of Pharmacology, Cornell University Medical College, New York, New York 10021, USA.
Lee
C
C
Chang
T K
TK
Waxman
D J
DJ
D016718:Arachidonic Acid / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases; D019388:Cytochrome P-450 CYP1A2; D065727:Cytochrome P-450 CYP2C8; D065729:Cytochrome P-450 CYP2C9; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000378:metabolism*
D016718:Arachidonic Acid; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; C504573:CYP1A2 protein, human; C450259:CYP2C8 protein, human; D019388:Cytochrome P-450 CYP1A2; D065727:Cytochrome P-450 CYP2C8; D039181:Steroid 16-alpha-Hydroxylase; D010089:Oxidoreductases, N-Demethylating