Regulation of Cytochrome P-450-supported 11P-Hydroxylation

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 11b-hydroxylase (cytochrome P-450118) was investigated. Treatment of cultures with 30 PM cortisol reduced the half-life of llb-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 p ~ . Deoxycortisol, deoxycorticosterone, androstenedione, and testosterone or their 11b-hydroxylated derivatives also caused losses of lib-hydroxylase activity after 24-h treatment, while the lla-hydroxylated derivatives and the 11-ketones were inactive. The antioxidants dimethyl sulfoxide and butylated hydroxyanisole prevented the cortisol-induced loss of lip-hydroxylase activity, partially at 19% O2 and almost completely at l% 02. Adrenocorticotropin did not induce lip-hydroxylase in bovine adrenocortical cell cultures under standard conditions. Dimethyl sulfoxide, butylated hydroxyanisole, and ascorbic acid allowed some induction by adrenocorticotropin at 19% 02. At 1% O2 there was much higher induction by adrenocorticotropin and also by cholera toxin, monobutyryl CAMP, prostaglandin El, and angiotensin 11. Experiments on fetal human adrenocortical cells in culture also demonstrated the protective effects of antioxidants during cortisol-induced loss of 11b-hydroxylase activity and during adrenocorticotropin induction of 11S-hydroxylase. It is concluded that cytochrome P45OIl8 is subject to destruction or inactivation through interaction with 11p-hydroxylated steroids, ie. 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.