ABSTRACT
The fungistatic drug clotrimazole (1-[(o-chlorophenyl)diphenylmethyl]imidazole) in concentrations of 5 or 50 microM completely prevented the formation of benzo[a]pyrene metabolite-DNA adducts in vitro catalyzed by liver microsomes from phenobarbital-or 3-methylcholanthrene-treated rats, respectively. Microsomal 7-ethoxycoumarin de-ethylase and aryl hydrocarbon hydroxylase were effectively inhibited by clotrimazole and three clotrimazole derivatives in all induction states tested, with I50 values down to 7 x 10(-8) M. The mechanism of inhibition was noncompetitive in phenobarbital-stimulated microsomes. Microsomal epoxide hydratase in vitro was enhanced up to 450% by clotrimazole and one of the analogues in concentrations between 5 and 500 microM. Clotrimazole spectrally interacted with reduced cytochrome P-450, exhibiting a double-banded Soret region with peaks at 427 and 446 nm, and partially prevented cytochrome P-450-CO complex formation. When administered in vivo, clotrimazole effectively induced cytochrome P-450 content, mono-oxygenase activity and epoxide hydratase activity in rat liver microsomes. The induction pattern was similar to that obtained with phenobarbital. The analogues were less potent inducers.
Subject(s)
Benzopyrenes/metabolism , Clotrimazole/pharmacology , DNA/metabolism , Imidazoles/pharmacology , Microsomes, Liver/metabolism , Mixed Function Oxygenases/antagonists & inhibitors , Oxidoreductases/antagonists & inhibitors , Animals , Clotrimazole/analogs & derivatives , Cytochrome P-450 Enzyme System/metabolism , In Vitro Techniques , Kinetics , Male , Microsomes, Liver/drug effects , Phenobarbital/pharmacology , RatsABSTRACT
The murine Ah complex represents a 'cluster' of genes controlling the induction of numerous cytochrome P-450-mediated monooxygenase 'activities' by polycyclic aromatic compounds. These forms of cytochrome represent structural gene products. A major regulatory gene product is a cytosolic receptor to which radiolabeled 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) binds with very high affinity. Developmental differences in mouse, rat and rabbit liver have been previously reported, indicating that some form of temporal control exists during the induction of enzymes associated with the ah locus. The cytosolic receptor levels were determined by sucrose density gradient centrifugation after dextran-charcoal treatment. Hepatic receptor levels, beta-naphthoflavone-inducible and control aryl hydrocarbon hydroxylase (EC 1.14.14.2) and acetanilide 4-hydroxylase activities and total P-450 content were studied in the Sprague-Dawley rat, C57BL/6N mouse, New Zealand White rabbit and Sigmoden hispedis (cotton rat) as a function of age. 3-Methylcholanthrene-inducible and control aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase activities in nonhepatic tissues of the neonatal and adult rabbit were examined. beta-Naphthoflavone-inducible hepatic microsomal proteins detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were also studied in the four species. Taken altogether, the developmental data indicate that: the form of P-450 responsible for induced aryl hydrocarbon hydroxylase activity is not the same as that for induced acetanilide 4-hydroxylase, and neither one is the same as induced cytochrome P-448; the presence of the TCDD-specific cytosolic receptor per se (detected as a distinct high-specificity saturable peak on sucrose density gradients) does not guarantee the expression of inducible aryl hydrocarbon hydroxylase or acetanilide 4-hydroxylase activity; although interesting developmental differences exist among all four species examined, the TCDD-specific receptor is maximal between the neonatal and weaning period, is considerably decreased in the adult, and is suppressed even more during the latter half of pregnancy; in general, the times at which the cytosolic receptor is highest or lowest parallels quite closely the well-known increases in inducible drug-metabolizing enzymes that have been commonly observed in the rat, mouse and other laboratory animals, and more studies are necessary before we understand what the 'TCDD-specific binding peak' (as observed on the sucrose density gradients) actually represents.(ABSTRACT TRUNCATED AT 400 WORDS)
