ARACHIDONIC-ACID METABOLISM BY HUMAN CYTOCHROME P450S-2C8, CYTOCHROMEP450S-2C9, CYTOCHROME P450S-2E1, AND CYTOCHROME P450S-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 relea sed from membranes by phospholipases and then metabolized to biologica lly active compounds by cyclooxygenases, lipoxygenases, and cytochrome P450 (CYP) enzymes. In the liver the CYP pathway is the most signific ant. Liver CYP arachidonate products include epoxyeicosatrienoic acids (EETs) and monohydroxylated products (HETEs). We examined metabolism of [1-C-14]arachidonic acid by a panel of 10 human CYP enzymes express ed in HepG2 cells. In the absence of expressed CYP enzymes, control He pG2 cell microsomes generated only small amounts of omega- and omega - 1-OH arachidonic acid (ratio 2:1). Microsomes from HepG2 cells expres sing CYP2C8, 2C9, 1A2, and 2E1 were 7-21 times more active than micros omes from the HepG2 controls. CYP2C8, 2C9, and 1A2 principally generat ed epoxygenase products; 36 to 48% were in the form of EET-diols, refl ecting host HepG2 microsomal epoxide hydrolase activity. CYP2C8 and 2C 9 formed more 14,15- and 11,12-EET than did CYP1A2, while CYP1A2 forme d more 8,9-EET. CYP2C9 also generated a peak with the retention time o f 12-HETE. CYP2E1 generated omega - 1-OH arachidonic acid and, to a le sser extent, omega-OH arachidonic acid (ratio 2:1). A small amount of epoxygenase activity was also detected for CYP2B6; its overall activit y, however, was only about twice control levels. Activities of CYP2A6, 3A3, 3A4, and 3A5 were low and limited to the omega-/omega - 1-OH ara chidonic acid peak; CYP2D6 was inactive. Microsomes prepared from thre e individual human livers varied threefold in total arachidonic acid m etabolism. For all three livers omega-OH arachidonic acid was the majo r product (up to 74% of total metabolites). Epoxygenase products const ituted 14 to 28% of the total products; 60 to 83% of those were EET-di ols, indicating that the human liver microsomes have substantial EET-e poxide hydrolase activity. 11,12-EET was the major EET for two livers and 14,15-EET for the third. The CYP2C inhibitor sulfaphenazole depres sed human liver microsomal epoxygenase activity by 50% at 50 mu M, whi le alpha-naphthoflavone inhibited arachidonic acid epoxygenase activit y by 27% at 2 mu M and by 32% at 10 mu M. Collectively, these findings suggest that human liver microsomal arachidonic acid metabolism is ca talyzed principally by CYP2C enzymes. CYP1A2, CYP2E1, and possibly CYP 2B6 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. The studies suggest further that the CYP composition of the liver may affect the arachidonic acid metabolite profile and in turn the cellula r effects resulting from arachidonic acid metabolism. (C) 1995 Academi c Press, Inc.