2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Induces Hepatic Cytochrome P450-Dependent Arachidonic Acid Epoxygenation in Diverse Avian Orders: Regioisomer Selectivity and Immunochemical Comparison of the TCDD-Induced P450s to CYP1A4 and 1A5

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) markedly induced cytochrome P450 (CYP)-dependent arachidonic acid metabolism in liver microsomes from hatchlings of four avian species belonging to four different orders: chick, pigeon, cormorant, and great blue heron, increasing formation of arachidonic acid epoxides (EETs), monohydroxyeicosatetraenoic acids (HETEs), ω−1, and ω−2 OH arachidonic acid products by fivefold or more. Microsomes from TCDD-induced hatchling chicks had the highest activity and the least restricted EET regioselectivity. ω-OH arachidonic acid, the principal constitutive metabolite in chick and pigeon liver microsomes and a major product for cormorant and great blue heron was not induced by TCDD. Constitutive EET formation in avian liver microsomes was very low except in cormorant microsomes where 8,9-EET was generated almost exclusively. Western blots of liver microsomes using polyclonal antisera to chick embryo-derived CYP1A4 and 1A5 recognized two TCDD-induced bands in each of the species. The chick bands had the same molecular weights as CYP1A4 and 1A5 (55 and 55.5 kDa, respectively) but those of the other species differed. Immunopurified antiserum monospecific for CYP1A5 recognized a band in microsomes from all of the avian species, and monospecific antiserum for CYP1A4 recognized a band in microsomes from chick, pigeon, and great blue heron. AntiCYP1A4 and 1A5 IgG immunoinhibited TCDD-induced mixed function oxidase activity completely in chick and chick embryo microsomes and only partially in the other avian microsomes. The results demonstrate that (1) TCDD causes much greater induction of CYP-dependent arachidonic acid metabolism, and of arachidonic acid epoxygenation in particular, in avian than in mammalian species; (2) TCDD induces two CYP1A-related enzymes in birds as in mammals; (3) CYP1A enzymes in the birds other than chicks are not identical to CYP1A4 and 1A5 but share some enzymatic and immunochemical characteristics with them; (4) constitutive ω-OH arachidonic acid in all of the avian species and 8,9-EET in cormorant are formed by CYP enzymes unrelated to CYP1A; and (5) two distinct characteristics of avian CYP1A enzymes are the acquisition by avian CYP1A4-related P450 of unique epitope(s) and by CYP1A5-related P450 of unusual catalytic effectiveness for arachidonic acid epoxygenation.