Immunochemical identity of the 2,3,7,8-tetrachlorodibenzo-p-dioxin- and β-naphthoflavone-induced cytochrome P-450 arachidonic acid epoxygenases in chick embryo liver: Distinction from the ω-hydroxylase and the phenobarbital-induced epoxygenase
Cytochrome P-450 Enzyme System
Mixed Function Oxygenases
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.