Rifkind A B, Lee C, Chang T K, Waxman D J
Department of Pharmacology, Cornell University Medical College, New York, New York 10021, USA.
Arch Biochem Biophys. 1995 Jul 10;320(2):380-9. doi: 10.1016/0003-9861(95)90023-3.
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)
膜结合的内源性脂肪酸花生四烯酸可通过磷脂酶从膜中释放出来，然后由环氧化酶、脂氧合酶和细胞色素P450（CYP）酶代谢为生物活性化合物。在肝脏中，CYP途径最为重要。肝脏CYP花生四烯酸产物包括环氧二十碳三烯酸（EETs）和单羟基化产物（HETEs）。我们检测了在HepG2细胞中表达的一组10种人类CYP酶对[1-14C]花生四烯酸的代谢情况。在未表达CYP酶的情况下，对照HepG2细胞微粒体仅产生少量的ω-和ω-1-羟基花生四烯酸（比例为2:1）。表达CYP2C8、2C9、1A2和2E1的HepG2细胞微粒体的活性比HepG2对照微粒体高7至21倍。CYP2C8、2C9和1A2主要产生环氧化酶产物；36%至48%为EET-二醇形式，反映了宿主HepG2微粒体环氧化物水解酶的活性。CYP2C8和2C9形成的14,15-和11,12-EET比CYP1A2多，而CYP1A2形成的8,9-EET更多。CYP2C9还产生了一个保留时间与12-HETE相同的峰。CYP2E1产生ω-1-羟基花生四烯酸，在较小程度上产生ω-羟基花生四烯酸（比例为2:1）。还检测到CYP2B6有少量环氧化酶活性；然而，其总体活性仅约为对照水平的两倍。CYP2A6、3A3、3A4和3A5的活性较低，仅限于ω-/ω-1-羟基花生四烯酸峰；CYP2D6无活性。从三个个体的肝脏制备的微粒体在花生四烯酸总代谢方面相差三倍。对于所有三个肝脏，ω-羟基花生四烯酸是主要产物（占总代谢物的74%）。环氧化酶产物占总产物的14%至28%；其中60%至83%为EET-二醇，表明人肝微粒体具有大量的EET-环氧化物水解酶活性。11,12-EET是两个肝脏的主要EET，14,15-EET是第三个肝脏的主要EET。CYP2C抑制剂磺胺苯唑在50 microM时可使人类肝微粒体环氧化酶活性降低50%，而α-萘黄酮在2 microM时可使花生四烯酸环氧化酶活性降低27%，在10 microM时降低32%。总体而言，这些发现表明人类肝微粒体花生四烯酸代谢主要由CYP2C酶催化。CYP1A2、CYP2E1以及可能的CYP2B6可能起较小作用，尽管接触这些酶的诱导剂可能会增强它们的作用。CYP2A6、CYP2D6和CYP3A酶不太可能有任何显著贡献。（摘要截短至400字）
