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Selective inactivation of four rat liver microsomal androstenedione hydroxylases by chloramphenicol analogs.
Mol Pharmacol. 1988 Jan; 33(1):103-10.MP

Abstract

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.

Authors+Show Affiliations

Stevens JC
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721.
Halpert J
No affiliation info available

MeSH

AndrostenedioneAnimalsAryl Hydrocarbon HydroxylasesChloramphenicolCytochrome P-450 Enzyme InhibitorsIsoenzymesKineticsMicrosomes, LiverRatsSteroid 16-alpha-HydroxylaseSteroid HydroxylasesStructure-Activity Relationship

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

3336347

Citation

Stevens, J C., and J Halpert. "Selective Inactivation of Four Rat Liver Microsomal Androstenedione Hydroxylases By Chloramphenicol Analogs." Molecular Pharmacology, vol. 33, no. 1, 1988, pp. 103-10.
Stevens JC, Halpert J. Selective inactivation of four rat liver microsomal androstenedione hydroxylases by chloramphenicol analogs. Mol Pharmacol. 1988;33(1):103-10.
Stevens, J. C., & Halpert, J. (1988). Selective inactivation of four rat liver microsomal androstenedione hydroxylases by chloramphenicol analogs. Molecular Pharmacology, 33(1), 103-10.
Stevens JC, Halpert J. Selective Inactivation of Four Rat Liver Microsomal Androstenedione Hydroxylases By Chloramphenicol Analogs. Mol Pharmacol. 1988;33(1):103-10. PubMed PMID: 3336347.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Selective inactivation of four rat liver microsomal androstenedione hydroxylases by chloramphenicol analogs. AU - Stevens,J C, AU - Halpert,J, PY - 1988/1/1/pubmed PY - 1988/1/1/medline PY - 1988/1/1/entrez SP - 103 EP - 10 JF - Molecular pharmacology JO - Mol Pharmacol VL - 33 IS - 1 N2 - 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. SN - 0026-895X UR - https://www.unboundmedicine.com/medline/citation/3336347/Selective_inactivation_of_four_rat_liver_microsomal_androstenedione_hydroxylases_by_chloramphenicol_analogs_ DB - PRIME DP - Unbound Medicine ER -