RT Journal Article SR Electronic T1 Selective inactivation of four rat liver microsomal androstenedione hydroxylases by chloramphenicol analogs. JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 103 OP 110 VO 33 IS 1 A1 J C Stevens A1 J Halpert YR 1988 UL http://molpharm.aspetjournals.org/content/33/1/103.abstract AB The steroid androstenedione has been shown to be a valuable tool for the study of the selective inactivation of cytochrome P-450 isozymes in intact rat liver microsomes. The validity of this approach was investigated using microsomes, purified cytochrome P-450 isozymes, antibodies to particular cytochromes P-450, and the known mechanism-based inactivator chloramphenicol. Enzyme inactivation and antibody inhibition studies show that microsomes from both phenobarbital- and non-phenobarbital-treated rats are needed to accurately monitor the inactivation of the major phenobarbital-inducible cytochrome P-450 isozyme (PB-B) and of the major constitutive androstenedione 16 alpha-hydroxylase (UT-A). Similar experiments indicate that, although isozyme P-450g does catalyze the 6 beta-hydroxylation of androstenedione in a reconstituted system, this cytochrome appears to make only a minimal contribution to microsomal 6 beta-hydroxylase activity, which reflects instead the activity of pregnenolone-16 alpha-carbonitrile-induced isozymes. With these parameters investigated, initial enzyme inactivation studies showed that the antibiotic chloramphenicol caused different rates of NADPH-dependent enzyme inactivation among the four androstenedione hydroxylases monitored (16 beta greater than 6 beta greater than 16 alpha greater than 7 alpha). Based on these data, 12 chloramphenicol analogs were examined, and the results with these compounds show that their selectivity as cytochrome P-450 inactivators is a function of at least three structural features: 1) the number of halogen atoms, 2) the presence of a para-nitro group on the phenyl ring, and 3) substitutions on the ethyl side chain. For example, the compound N-(2-phenethyl)dichloroacetamide was shown to reversibly inhibit but not inactivate the cytochrome(s) P-450 responsible for androstenedione 6 beta-hydroxylase activity, whereas N-(2-p-nitrophenethyl) and N-(1,2-diphenethyl)dichloroacetamide rapidly inactivated the 6 beta-hydroxylase. The ability to monitor the activity of multiple isozymes with a single substrate should allow the development of a systematic approach to the design of selective inactivators of rat liver cytochromes P-450.