Abstract
Cytochrome P-450-mediated arachidonic acid metabolism in chick embryo liver microsomes was increased by both Ah receptor-dependent (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-naphthoflavone) and independent (phenobarbital) P-450 inducers. Arachidonic acid epoxides and monohydroxyeicosatetraenoic acids were increased 9-12-fold. omega-1-OH arachidonic acid was also significantly increased by TCDD and beta-naphthoflavone while omega-OH arachidonic acid, the main metabolite in uninduced livers, was decreased by all three agents. The P-450s catalyzing the enhanced arachidonate metabolism in beta-naphthoflavone- and phenobarbital-treated liver were investigated in reconstituted systems containing wholly or partially purified P-450s. beta-Naphthoflavone induced formation of a 55-kDa P-450 selective for arachidonate metabolism and for epoxygenation in particular. This P-450 was purified (beta NFAA). It was found to be distinct from a 54.5-kDa beta-naphthoflavone-induced P-450 catalyzing aryl hydrocarbon hydroxylase and 7-ethoxyresorufin deethylase (designated NF1). Mean turnover numbers for arachidonate epoxygenase, aryl hydrocarbon hydroxylase, and 7-ethoxyresorufin deethylase were 11.2, 0.56, and 0.04, respectively, for reconstituted beta NFAA and 0.33, 11.8, and 2.4 for NF1. beta NFAA and NF1 also differed in chromatography elution characteristics and N-terminal amino acid sequences. Both were low spin, with carbon monoxide binding peaks at 448 nm. The phenobarbital-induced arachidonate epoxygenation was catalyzed by P-450 fractions containing the main 48- and 49-kDa phenobarbital-induced P-450s; fractions in which the 49-kDa P-450 predominated were the most active. Turnover numbers for arachidonic acid epoxygenation were not correlated with those for aminopyrine demethylation or 7-ethoxycoumarin deethylation for P-450s from phenobarbital-treated livers or with aryl hydrocarbon hydroxylase, 7-ethoxyresorufin deethylase, or 7-ethoxycoumarin deethylase for P-450s from beta-naphthoflavone-treated livers. Also, different P-450s catalyzed the epoxygenation and the omega-hydroxylation of arachidonic acid in both beta-naphthoflavone- and phenobarbital-treated livers. The findings support a physiologic role for P-450-induced arachidonate metabolism and provide a basis for a possible link between TCDD's induction of P-450 and alterations of cellular homeostasis.
Highlights
PROCEDURESMaterials-Sources of materials were: fertilized eggs, White Leghorn strain, H n’ R Poultrv Farms
1-OH arachidonic acid was significantly increased by TCDD and B-naphthoflavone while w-OH arachidonic acid, the main metabolite in uninduced livers, was decreased by all three agents
We recently reported that treatment of chick embryos close to hatching with Ah receptor ligands, TCDD, or /?-NF, or with phenobarbital, which induces different P-450s independently of the Ah receptor (2), markedly increased hepatic P-450-mediated arachidonic acid metabolism and EET formation in particular (7)
Summary
Materials-Sources of materials were: fertilized eggs, White Leghorn strain, H n’ R Poultrv Farms. ”. day-old chick embryos were treated with phenobarbital sodium at 20 mg/egg in 0.4 ml of H20, P-NF at 6.7 mg/egg in 0.1 ml of Me&O or TCDD at 1 nmol (322 ng) per egg in 0.005 ml dioxane. G-lv_ceraldehvde-3--phosphat-e - dehvdrogenase (36 kDa), carbonic anhydrase (29 kDa), and trypsinogen (24-kDa) as molecular mass markers; NADPH cytochrome c (P-450) reductase (13) and protein (14) using BSA as a standard with appropriate additions to correct for interfering glycerol, salts, and detergents (15). Sodium cholate in 0.; M kPi with 1 mM EDTA and 1 mM dithiothreitol (DTT) (EDTA and DTT, each at 1 mM were present in all P-450 purification buffers, unless otherwise indicated). The column was washed with 1 volume of 0.1 M KPi with 0.4% sodium cholate.
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