Kanetoshi A, Ward A M, May B K, Rifkind A B
Department of Pharmacology, Cornell University Medical College, New York, New York 10021.
Mol Pharmacol. 1992 Dec;42(6):1020-6.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (beta NF), and phenobarbital (PB) cause marked induction of cytochrome P-450 (P-450)-mediated arachidonic acid metabolism in chick embryo liver. We show here that the P-450 arachidonic acid epoxygenases induced by TCDD and beta NF are immunochemically indistinguishable from each other and unrelated to the arachidonic acid epoxygenase induced by PB. On Western blots, IgG from an antiserum against beta NFAA, a 55-kDa P-450 arachidonic acid epoxygenase purified from beta NF-treated chick embryo liver, immunoreacted selectively and to the same extent with a 55-kDa band in liver microsomes from chick embryos treated with TCDD or beta NF. It failed to react with proteins from untreated, solvent-treated, or PB-treated embryos on immunoblots or to immunoinhibit PB-induced arachidonic acid metabolism. Anti-beta NFAA IgG immunoinhibited all arachidonic acid metabolism by reconstituted beta NFAA and formation of arachidonic epoxides (EETs) and monohydroxylated derivatives (HETEs) by microsomes from TCDD- and beta NF-treated livers; it did not inhibit omega-hydroxylation. In contrast, IgG from an antiserum against the major PB-induced chicken P-450s, 2H1 and 2H2, immunoreacted with two major PB-induced P-450s, of 48 and 49 kDa, on Western blots. It also immunoinhibited formation of EETs and HETEs by PB-treated microsomes entirely and omega-hydroxylation by 50%. It failed to react with TCDD- or beta NF-induced P-450s on Western blots or to immunoinhibit TCDD- or beta NF-induced arachidonic acid metabolism. Because other P-450s with which anti-beta NFAA and anti-PB IgG cross-reacted were inactive in arachidonic acid epoxygenation, the findings are consistent with beta NFAA being principally responsible for the epoxygenation induced by TCDD and beta NF and 2H1 and/or 2H2 being responsible for epoxygenation induced by PB. Further, the P-450 arachidonate omega-hydroxylase and the epoxygenase in livers of TCDD- or beta NF-treated embryos are immunochemically unrelated, whereas those in livers of PB-treated embryos may be partly related.
2,3,7,8-四氯二苯并-对-二恶英（TCDD）、β-萘黄酮（βNF）和苯巴比妥（PB）可显著诱导鸡胚肝脏中细胞色素P-450（P-450）介导的花生四烯酸代谢。我们在此表明，TCDD和βNF诱导的P-450花生四烯酸环氧化酶在免疫化学上彼此无法区分，且与PB诱导的花生四烯酸环氧化酶无关。在蛋白质免疫印迹分析中，从用βNF处理的鸡胚肝脏中纯化得到的一种55 kDa的P-450花生四烯酸环氧化酶βNFAA抗血清中的IgG，与用TCDD或βNF处理的鸡胚肝脏微粒体中的一条55 kDa条带发生选择性且程度相同的免疫反应。它在免疫印迹中不与未处理、溶剂处理或PB处理的胚胎中的蛋白质发生反应，也不能免疫抑制PB诱导的花生四烯酸代谢。抗βNFAA IgG可免疫抑制重组βNFAA的所有花生四烯酸代谢，以及TCDD和βNF处理的肝脏微粒体中花生四烯酸环氧化物（EETs）和单羟基化衍生物（HETEs）的形成；它不抑制ω-羟基化。相比之下，针对主要由PB诱导的鸡P-450s（2H1和2H2）的抗血清中的IgG，在蛋白质免疫印迹分析中与两条主要的由PB诱导的48 kDa和49 kDa的P-450s发生免疫反应。它还完全免疫抑制PB处理的微粒体中EETs和HETEs的形成，并使ω-羟基化抑制50%。它在蛋白质免疫印迹中不与TCDD或βNF诱导的P-450s发生反应，也不能免疫抑制TCDD或βNF诱导的花生四烯酸代谢。由于抗βNFAA和抗PB IgG发生交叉反应的其他P-450s在花生四烯酸环氧化中无活性，这些发现表明βNFAA主要负责TCDD和βNF诱导的环氧化，而2H1和/或2H2负责PB诱导的环氧化。此外，TCDD或βNF处理的胚胎肝脏中的P-450花生四烯酸ω-羟化酶和环氧化酶在免疫化学上无关，而PB处理的胚胎肝脏中的那些酶可能部分相关。
