Regulation of cytochrome P-450-supported 11β-hydroxylation of deoxycortisol by steroids, oxygen, and antioxidants in adrenocortical cell cultures

It has been observed that bovine adrenocortical cells in culture fail to synthesize cortisol, while synthesizing deoxycortisol and other precursor steroids. The reason for this loss of activity of 11β-hydroxylase (cytochrome P-450₁₁β) was investigated. Treatment of cultures with 30 μM cortisol reduced the half-life of 11β-hydroxylase activity from 40 h to 9 h. The concentration of cortisol causing 50% loss of activity after 24 h of treatment was found to be 7 μM. Deoxycortisol, deoxycorticosterone, androstenedione, and testosterone or their 11β-hydroxylated derivatives also caused losses of 11β-hydroxylase activity after 24-h treatment, while the 11α-hydroxylated derivatives and the 11-ketones were inactive. The antioxidants dimethyl sulfoxide and butylated hydroxyanisole prevented the cortisol-induced loss of 11β-hydroxylase activity, partially at 19% O₂ and almost completely at 1% O₂. Adrenocorticotropin did not induce 11β-hydroxylase in bovine adrenocortical cell cultures under standard conditions. Dimethyl sulfoxide, butylated hydroxyanisole, and ascorbic acid allowed some induction by adrenocorticotropin at 19% O₂. At 1% O₂ there was much higher induction by adrenocorticotropin and also by cholera toxin, monobutyryl cAMP, prostaglandin E₁, and angiotensin II. Experiments on fetal human adrenocortical cells in culture also demonstrated the protective effects of antioxidants during cortisol-induced loss of 11β-hydroxylase activity and during adrenocorticotropin induction of 11β-hydroxylase. It is concluded that cytochrome P-450₁₁β is subject to destruction or inactivation through interaction with 11β-hydroxylated steroids, i.e. the enzyme products, which act as pseudosubstrates. This appears to involve lipid peroxidation initiated by oxygen-derived free radicals released from the cytochrome P-450·pseudosubstrate·oxygen complex due to the incapacity of the pseudosubstrate to be oxygenated.