title: Mode of action of butylated hydroxyanisole (BHA) and other phenols in preventing loss of 11 beta-hydroxylase activity in cultured bovine adrenocortical cells.
abstract: When cultured bovine adrenocortical cells are incubated with cortisol, or other steroids that are pseudosubstrates for 11 beta-hydroxylase (cytochrome P-45011 beta), the activity of the enzyme decreases. In previous experiments, three substances were shown to protect 11 beta-hydroxylase against loss of enzymatic activity in the presence of pseudosubstrates:BHA (butylated hydroxyanisole,2(3)-tert-butyl-4-methoxyphenol), dimethyl sulfoxide (DMSO), and metyrapone. The present experiments examine the protective effects of several phenolic analogs of BHA in this system, and compare their activities to that of DMSO and metyrapone. When a variety of analogs of BHA were tested for their abilities to prevent loss of 11 beta-hydroxylase activity in cultured adrenocortical cells incubated with 50 microM cortisol for 24 hr, phenol itself was found to be about equipotent with BHA. Addition of methyl, methoxy and benzyl groups to phenol did not diminish protective activity of the compound, but addition of one and particularly two tert-butyl groups greatly diminished activity. Thus, BHT(2,6-di-t-butyl-4-methylphenol) was inactive, in contrast to BHA. The hydroxy group of phenol was essential since benzene and fluorobenzene were inactive. Compounds with multiple hydroxyl groups were not as active as phenol itself, with the exception of catechol. No products of phenol formed during incubations of cells with cortisol were detected by high performance liquid chromatography. Estimated EC50 values for protection of 11 beta-hydroxylase by phenols were about 100 microM, whereas the EC50 values for dimethyl sulfoxide and metyrapone were 10 mM and 300 nM respectively. On a semilogarithmic plot, the dose-response curves for all these compounds were approximately parallel. To aid in determining the mechanism of protection of 11 beta-hydroxylase, phenols and DMSO were tested for prevention of loss of 11 beta-hydroxylase activity at three different oxygen concentrations (2, 5, and 19% O2). Lowering the oxygen concentration itself resulted in a small diminution of the loss of 11 beta-hydroxylase. Phenols and dimethyl sulfoxide were more effective at low oxygen and less effective in air. Because the cytochrome P-450 inhibitor metyrapone was found previously to be very effective in protecting 11 beta-hydroxylase against loss of activity, we examined whether phenols and dimethyl sulfoxide may act by directly inhibiting 11 beta-hydroxylase activity. In a 1-hr incubation with cells, BHA, phenol, and dimethyl sulfoxide all inhibited 11 beta-hydroxylase, but at concentrations that ranged from 4- to greater than 100-fold higher than those required for protection.(ABSTRACT TRUNCATED AT 400 WORDS)
mesh_terms: D000302:Adrenal Cortex / Q000201:enzymology*; D000818:Animals; D000840:Anisoles / Q000494:pharmacology*; D002083:Butylated Hydroxyanisole / Q000494:pharmacology*; D002250:Carbon Radioisotopes; D002417:Cattle; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006854:Hydrocortisone / Q000494:pharmacology; D010100:Oxygen / Q000494:pharmacology; D019800:Phenol; D010636:Phenols / Q000378:metabolism / Q000494:pharmacology*; D013252:Steroid 11-beta-Hydroxylase / Q000032:analysis* / Q000037:antagonists & inhibitors; D013250:Steroid Hydroxylases / Q000032:analysis*
chemical_list: D000840:Anisoles; D002250:Carbon Radioisotopes; D010636:Phenols; D002083:Butylated Hydroxyanisole; D019800:Phenol; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D013252:Steroid 11-beta-Hydroxylase; D010100:Oxygen; D006854:Hydrocortisone; D004121:Dimethyl Sulfoxide