CYTOCHROMES P450 WITH BISALLYLIC HYDROXYLATION ACTIVITY ON ARACHIDONIC AND LINOLEIC ACIDS STUDIED WITH HUMAN RECOMBINANT ENZYMES AND WITH HUMAN AND RAT-LIVER MICROSOMES

Bisallylic carbons of polyunsaturated fatty acids can be hy droxylated in NADPH-dependent reactions in liver microsomes. Human recombinant c ytochromes P450 and human and rat liver microsomes were assayed for bi sallylic hydroxylation activity. CYP1A2, CYP2C8, CYP2C9, CYP2C19 and C YP3A4 converted [C-14]linoleic acid to C-14-labeled 11-hydroxyoctadeca dienoic acid (11-HODE), whereas [C-14]arachidonic acid was oxygenated by CYP1A2 and CYP3A4 to C-14-labeled 13-hydroxyeicosatrienoic acid (13 -HETE), 10-HETE and 7-HETE as determined by HPLC. Both substrates were also converted to many other metabolites. CYP2C9 appeared to form 12R -HETE and 13-HETE, whereas CYP2C8 formed 13-HETE, 11-HETE and 15-HETE as main monohydroxy metabolites. Fetal human liver microsomes metaboli zed linoleic acid to 11-HODE as a major hydroxy metabolite, whereas ar achidonic acid appeared to be hydroxylated at C13, C20 and, to some ex tent, at C10, C19 and C7. Fetal liver microsomes mainly formed 13R-HET E, whereas adult human liver microsomes and CYP1A2 mainly formed 13S-H ETE. 7,8-Benzoflavone (5 mu M) and furafylline (20 mu M), two inhibito rs of CYP1A2, reduced the bisallylic hydroxylation activity of adult h uman liver microsomes. Treatment of rats with erythromycin or dexameth asone induced bisallylic hydroxylation of linoleic acid to 11-HODE in liver microsomes by 2- and 10-fold, respectively. The biosynthesis of 11-HODE by microsomes of dexamethasone-treated rats was inhibited by t roleandomycin (ED50 = 1 mu M) and by polyclonal antibodies against CYP 3A1, suggesting that CYP3A1 could catalyze bisallylic hydroxylations i n the dexamethasone-treated rat. We conclude from steric analysis of 1 3-HETE and the effects of CYP inhibitors on adult human liver microsom es that CYP1A2 might contribute to its bisallylic hydroxylation activi ty.