Ketoconazole as a possible universal inhibitor of cytochrome P-450 dependent enzymes: its mode of inhibition

Modes of inhibition and binding of ketoconazole, an orally antimycotic agent, to NADPH-cytochrome P-450 dependent enzymes were investigated using subcellular fractions of human and rat testes, human adrenocortical adenoma tissue and rat adrenals and livers. Ketoconazole competitively inhibited the activities of steroid 17 alpha-hydroxylase and C17-20 lyase in rat and human testes, 16 alpha-hydroxylase in human testes and 21-hydroxylase in rat adrenal glands. Ki values were in the order of 10(-8)M for human testicular enzymes, while the order was 10(-7)-10(-6) M for rat adrenal and testicular enzymes. Kinetic studies indicated that ketoconazole bound to cytochrome P-450 and not to other components of monooxygenase systems. Spectrophotometric studies also revealed direct binding of ketoconazole to cytochrome P-450 component by inducing type II difference spectra in all tissue preparations examined, indicating that ketoconazole is possibly a universal inhibitor of NADPH-cytochrome P-450 dependent monooxygenases which are involved in metabolism of many substances including steroids, toxins, carcinogens and others.     

Inhibition mechanism of reconstituted cytochrome P-450scc-linked monooxygenase system by antimycotic reagents and other inhibitors.

The effects of various antimycotic reagents and some other reagents on a cytochrome P-450-linked monooxygenase system were investigated with respect to the activities of NADPH-ferricyanide reductase. NADPH-cytochrome c reductase of NADPH-adreno-ferredoxin reductase from NADPH to cytochrome c via adreno-ferredoxin, NADPH-cytochrome P-450-phenylisocyanide complex reductase, and the cholesterol side chain cleavage of the cytochrome P-450scc-linked monooxygenase system. No reagents inhibited the NADPH-ferricyanide reductase activity. Only cloconazole inhibited about 50% of NADPH-cytochrome c reductase activity. Cloconazole, econazole, clotrimazole, etomidate and ketoconazole inhibited both NADPH-cytochrome P-450-phenylisocyanide complex reductase and the side chain cleavage activity of cholesterol of the cytochrome P-450scc-linked monooxygenase system. Cloconazole, econazole, etomidate and ketoconazole behaved like non-competitive inhibitors for NADPH-cytochrome P-450-phenylisocyanide reductase activities and their Ki values were 10(-4)-10(-6) M. Cloconazole was a non-competitive inhibitor of NADPH-cytochrome c reductase and its Ki value was 8.3 x 10(-4) M. Cloconazole, clotrimazole, econazole, etomidate, ketoconazole and mitotane completely inhibited the side chain cleavage activity of cholesterol.     

Inhibition of hCG- and cAMP-stimulated progesterone production in MA-10 mouse Leydig tumor cells by ketoconazole

In this study we attempted to examine the effects of ketoconazole on steroid biosynthesis and to determine which steps in the steroidogenic pathway were blocked using MA-10 Mouse Leydig tumor cells. This cloned cell line produces progesterone as the major steroid following stimulation by hCG or dbcAMP. At a concentration of 1 microM ketoconazole completely inhibited the hCG- and dbcAMP-stimulated progesterone synthesis in MA-10 Leydig cells. The conversion of 25-hydroxycholesterol and 22R-hydroxycholesterol into progesterone was also suppressed by this drug. The presence of ketoconazole inhibited mitochondrial steroid synthesis but required high concentrations of the drug as compared to inhibition in intact cells. No accumulation of pregnenolone was observed in the presence of ketoconazole indicating that the activity of 3 beta-hydroxysteroid dehydrogenase was not affected. We conclude that ketoconazole directly inhibits the activity of cholesterol side-chain cleavage enzyme (CSCC), a rate-determining enzymatic step in steroidogenesis, by interacting with cytochrome P-450scc.     

Effects of two oral antimycotics, ketoconazole and fluconazole, upon steroidogenesis in rat adrenal cells in vitro

Rat adrenal cells were incubated with various concentrations of two orally active azole antimycotics in order to evaluate the effects on steroidogenesis. The first compound was ketoconazole, a well-known inhibitor not only of fungal cytochrome P-450 but at higher concentrations also of mammalian cytochrome P-450 dependent enzymes. The second was fluconazole, a newly developed oral antimycotic with a triazole structure, which likewise inhibits fungal cytochrome P-450. The influence of both drugs on mammalian cytochrome P-450 dependent enzymes was investigated in this study. Ketoconazole inhibited ACTH-stimulated corticosterone (IC50 = 0.9 microM) and aldosterone secretion (IC50 = 1.4 microM) and enhanced 11-deoxycorticosterone output at low concentrations but reduced it at higher concentrations. Radiotracer experiments with [3H]pregnenolone or [3H]11-deoxycorticosterone as exogenous substrates revealed a 50% inhibition of the oxidative substrate metabolism at about 1 microM ketoconazole. These effects could also be observed with fluconazole but occurred at concentrations approximately two orders of magnitude higher as compared to ketoconazole. We conclude that fluconazole has a much higher selectivity for fungal cytochrome P-450 than ketoconazole. The order of sensitivity of the cytochrome P-450 dependent enzymes of rat adrenal steroidogenesis to ketoconazole was the 11 beta/18-hydroxylase, the cholesterol side chain cleavage enzyme and the 21-hydroxylase with decreasing sensitivities.     

Topological studies of cytochromes P-450scc and P-45011 beta in bovine adrenocortical inner mitochondrial membranes. Effects of controlled tryptic digestion.

The topology of the steroid hydroxylase complexes in bovine adrenocortical mitochondria were studied by using controlled digestion with trypsin of purified inner mitochondrial membranes. Inhibition of steroid hydroxylase activity by trypsin was only observed in inner mitochondrial membranes which had been disrupted by various techniques. The steroid hydroxylase activity of intact inner membranes was not inhibited by trypsin. The effect of tryptic digestion was monitored by measuring 11 beta-hydroxylase and cholesterol side chain cleavage activities, as well as cytochrome P-450 reduction. The effect of trypsin on the steroid-induced difference spectra using pregnenolone, 20 alpha-hydroxycholesterol, and deoxycorticosterone was also measured. The results were similar regardless of which procedure was utilized and strongly suggest that both cytochrome P-45011 beta and cytochrome P-450scc are located on the matrix side of the mitochondrial inner membrane.     

Selective chemical modification of a functionally linked lysine in cytochrome P-450 LM2

Fluorescein isothiocyanate (FITC) has been selectively bound to the epsilon-amino group of lysine-382 in cytochrome P-450 LM2 (RH, reduced-flavoprotein: oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) at pH 8.15. Benzphetamine N-demethylase activity of the reconstituted FITC-modified cytochrome P-450 LM2 was inhibited by 25%. This inhibition has been shown to be due to an impaired electron transfer from the NADPH-cytochrome P-450 reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) to the haemoprotein. The data indicate that cytochrome P-450 interacts with the flavoprotein via electrostatic interactions.     

Occurrence of hydroxysteroid oxidoreductases in liver of turtles

1. Hydroxysteroid oxidoreductases have been partially purified from the cytosol fraction (105,000 g supernatant) of liver from a fresh-water turtle (Podocnemis expansa) and a sea-water turtle (Chelonia mydas mydas) by precipitation with ammonium sulphate (AS, 10-80% saturation). 2. The following enzymes were detected (substrates in brackets): 3 alpha-hydroxysteroid oxidoreductase (androsterone), 3 beta-hydroxysteroid oxidoreductase (DHEA) and 17 beta-hydroxysteroid oxidoreductase (testosterone, oestradiol-17 beta). NAD as well as NADP were effective as cofactors. 3. In fresh-water turtle, highest activities of the 3 alpha-enzyme were measured in the 20% AS fraction (cofactor NAD), of the 3 beta-enzyme in the 60% AS fraction (cofactor NAD) and of the 17 beta-enzyme in the 40% AS fraction (cofactor NADP). 4. In sea-water turtle, highest activities were observed for all three enzymes in the 60% AS fraction. 5. Generally, enzyme activities were higher in sea-water turtles than in fresh-water turtles. The most active enzyme in both turtles was found to be the 3 alpha-hydroxysteroid oxidoreductase, followed by the 17 beta- and the 3 beta-hydroxysteroid oxidoreductases.     

The involvement of cytochrome P-450 in hepatic microsomal steroid hydroxylation reactions supported by sodium periodate, sodium chlorite, and organic hydroperoxides.

The mechanism of steroid hydroxylation in rat liver microsomes has been investigated by employing NaIO4, NaClO2, and various organic hydroperoxides as hydroxylating agents and comparing the reaction rates and steroid products formed with those of the NADPH-dependent reaction. Androstenedione, testosterone, progesterone, and 17beta-estradiol were found to act as good substrates. NaIO4 was by far the most effective hydroxylating agent followed by cumene hydroperoxide, NADPH, NaClO2, pregnenolone 17alpha-hydroperoxide, tert-butyl hydroperoxide, and linoleic acid hydroperoxide. Androstenedione was chosen as the model substrate for inducer and inhibitor studies. The steroid was converted to its respective 6beta-, 7alpha, 15-, and 16alpha-hydroxy derivatives when incubated with microsomal fractions fortified with hydroxylating agent. Evidence for cytochrome P-450 involvement in androstenedione hydroxylation included a marked inhibition by substrates and modifiers of cytochrome P-450 and by reagents which convert cytochrome P-450 to cytochrome P-420. The ratios of the steroid products varied according to the type of hydroxylating agent used and were also modified by in vivo phenobarbital pretreatment. It was suggested that multiple forms of cytochrome P-450 exhibiting different affinities for hydroxylating agent are responsible for these different ratios. Horse-radish peroxidase, catalase, and metmyoglobin could not catalyze androstenedione hydroxylation. Addition of NaIO4, NaClO2, cumene hydroperoxide and other organic hydroperoxides to microsomal suspensions resulted in the appearance of a transient spectral change in the difference spectrum characterized by a peak at about 440 nm and a trough at 420 nm. The efficiency of these oxidizing agents in promoting steroid hydroxylation in microsomes appeared to be related to their effectiveness in eliciting the spectral complex. Electron donors, substrates, and modifiers of cytochrome P-450 greatly diminished the magnitude of the spectral change. It is proposed that NaIO4, NaClO2, and organic hydroperoxides promote steroid hydroxylation by forming a transient ferryl ion (compound I) of cytochrome P-450 which may be the common intermediate hydroxylating species involved in hydroxylations catalyzed by cytochrome P-450.     

Reasons for reduced activities of 17 alpha-hydroxylase and C17-C20 lyase in spite of increased contents of cytochrome P-450 in mature rat testis fetally irradiated with 60Co

Pregnant rats received whole body irradiation with 2.6 Gy gamma-ray from a 60Co source at Day 20 of gestation. When pups were 4 months old, activities of electron transport system and steroid monooxygenase in tests were assayed. The content of total cytochrome P-450 in the irradiated testes had increased to 170% of that in non-irradiated rats, but NADPH-cytochrome P-450 reductase activity had reduced to 36% of the control. Also, amounts of cytochrome b5 in testicular microsomal fraction were decreased markedly after irradiation, but no significant change of NADH-cytochrome b5 reductase activity was observed in the treated pups. Because both 17 alpha-hydroxylase and C17-C20 lyase activities tended to be decreased by fetal irradiation, testosterone production from progesterone and 17 alpha-hydroxyprogesterone was reduced to about 30% of the control. From these results, it has been suggested that the testicular cytochrome P-450 is radioresistant but steroid monooxygenase activities are reduced after the fetal irradiation. We propose that the discrepancy arises from the marked decrement of NADPH-cytochrome P-450 reductase activity.     

The reoxidation of cytochrome P-450 by paraquat inhibits aldosterone biosynthesis from 18-hydroxycorticosterone

Paraquat is an artificial electron carrier that captures electrons from reduced cytochrome P-450 instead of the natural acceptors, thus decreasing the concentration of reduced mitochondrial cytochrome P-450. In the present study, paraquat inhibited the biosynthesis of aldosterone from 18-hydroxycorticosterone by mitochondria from duck adult adrenal gland, under aerobic conditions. Since paraquat did not induce any change in the absorption spectrum of highly purified cytochrome P-450 11 beta, the possibility of a displacement of steroid by the drug is ruled out. Moreover, paraquat did not affect oxidative phosphorylating chain nor did it alter by itself the chemical structure of 18-hydroxycorticosterone. In our conditions, the inhibitory role of paraquat seems restricted to a capture of electrons from reduced cytochrome P-450. Under the same conditions metopirone and spironolactone, known to bind cytochrome P-450 11 beta at the steroid binding site, also inhibited the reaction. Altogether these results show that for aldosterone synthesis from 18-hydroxycorticosterone to take place, the steroid binding site on cytochrome P-450 must be accessible to 18-hydroxycorticosterone and that the cytochrome P-450 must be the direct donor of reducing equivalents. Hence, cytochrome P-450 appears as the final linking point between 18-hydroxycorticosterone and the reducing equivalents provided by NADPH.     

Induction of the phenobarbital form of cytochrome P-450 by chemically unrelated compounds

The induction of the phenobarbital form of cytochrome P-450 by xenobiotics (phenobarbital, PB, hexachlorobenzene, HCB; hexachlorocyclohexane. HCCH, and aroclor 1016, Ar) was studied. It was demonstrated that administration of these compounds to animals is accompanied by an increase in the total cytochrome P-450, NADPH-cytochrome P-450 reductase, benzphetamine-N-demethylase and aldrin-epoxidase activities. Using monospecific antibodies against the cytochrome P-450 form isolated from PB-induced microsomes (PB-cytochrome P-450), a double immunodiffusion test revealed immunological identity of cytochrome P-450 forms induced by phenobarbital and other xenobiotics. The content of this form determined by rocket immunoelectrophoresis increased markedly and made up to 20-40% of the total cytochrome P-450 content. Antibodies against PB-cytochrome P-450 inhibited by 50-70% the benzphetamine-N-demethylase and aldrin-epoxidase activities, whereas the antibodies to methylcholanthrene-induced cytochrome P-450 were fairly ineffective. It was concluded that the chemically unrelated compounds induce in liver microsomes a cytochrome P-450 form, whose immunological properties and substrate specificity are close to the PB-form of cytochrome P-450.     

Heterogeneity of cytochrome P-450 in rat testis microsomes.

Cytochrome P-450 appears to be a component of the steroid-coverting enzymes, 17alpha-hydroxylase and 17,20-lyase, which catalyze sequential steps in sex hormone synthesis. Further evidence indicates that the steroid substrates of these enzymes bind to cytochrome P-450 during catalysis. The present report deals with the problem of whether a single form of cytochrome P-450 mediates both enzyme reactions or whether two enzymes are involved. Both activities are competitively inhibited by a number of the same inhibitors. Because K1 values of competitive inhibitors are dissociated constants, and thus a property of the cytochrome, different magnitudes of K1, determined for the same inhibitor with each enzyme, are consistent with the participation of more than one form of cytochrome P-450. Differences in the K1 values were found to be statistically significant and varied from 3- to 10-fold. Two competitive inhibitors retarded velocities with one reaction but not the other. In addition, the enzyme activities were markedly different in their sensitivity to carbon monoxide inhibition. The conclusion based on these two lines of evidence is that separate enzymes and different forms of cytochrome P-450 are involved in each reaction.     

NADPH-cytochrome P-450 reductase is not a component of the liver microsomal steroid 5-alpha reductase

Liver microsomal steroid 5-alpha-reduction is catalyzed by a NADPH-dependent enzyme system. The requirement of NADPH-cytochrome P-450 reductase to shuttle reduction equivalents from NADPH to steroid 5-alpha-reductase was investigated using an inhibitory antibody against NADPH-cytochrome P-450 reductase. This antibody preparation inhibited cytochrome c reduction in microsomes from female rat liver with an I50 of 0.75 mg antibody/mg of microsomal protein. Benzphetamine N-demethylation and testosterone 6-beta-hydroxylation, two cytochrome P-450-mediated oxidative reactions, were inhibited by the antibody. On the other hand, testosterone 5-alpha-reductase was not affected by the antibody. These results suggest that NADPH-cytochrome P-450 reductase is not an obligatory component of the liver microsomal steroid 5-alpha-reduction.     

Alterations of heme, cytochrome P-450, and steroid metabolism by mercury in rat adrenal

The treatment of male rats with Hg2+ resulted in significant alterations in heme and hemoprotein metabolism in the adrenal gland which, in turn, were reflected in abnormal steroidogenic activities and steroid output. Twenty-four hours after the administration of 30 mumol of HgCl2/kg (sc) the mitochondrial heme and cytochrome P-450 concentrations increased by approximately 50%. Also, Hg2+ treatment stimulated a porphyrinogenic response which included an 11-fold increase in the activity of delta-aminolevulinate synthetase. The increase in mitochondrial cytochrome P-450 content was reflected in elevated steroid 11 beta-hydroxylase and cholesterol side-chain cleavage activities. In contrast, Hg2+ treatment resulted in decreased concentrations of microsomal cytochrome P-450 (-75%) and heme (-45%). Similarly, the reduction in the microsomal cytochrome P-450 content was accompanied by reduced steroid 21 alpha-hydroxylase and benzo[alpha]pyrene hydroxylase activities. The mechanisms responsible for the loss of the microsomal cytochrome P-450 content appeared to involve a selective impairment of formation of the holocytochrome as well as an enhanced rate of heme degradation. This suggestion is made on the basis of findings that (a) the decrease in the microsomal cytochrome P-450 content was accompanied by a sevenfold increase in the activity of adrenal heme oxygenase, (b) no decrease in apocytochrome P-450 could be detected in sodium dodecyl sulfate-gel electrophoresis of the solubilized microsomal fractions stained for heme, and (c) the concentration of adrenal microsomal cytochrome b5 was significantly increased in the Hg2+-treated animals. It is suggested that Hg2+ directly caused a defect in adrenal steroid biosynthesis by inhibiting the activity of 21 alpha-hydroxylase. The apparent physiological consequences of this effect included lowered plasma levels of corticosterone and elevated concentrations of progesterone and dehydroepiandrosterone. This abnormal plasma steroid profile is indicative of a 21 alpha-hydroxylase impairment.     

Effect of spin state on reduction of cytochrome P-450 (P-450C21) from bovine adrenocortical microsomes

The effect of spin state on cytochrome P-450 reduction was studied with a reconstituted system consisting of P-450C21 and NADPH-cytochrome P-450 reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) purified from bovine adrenocortical microsomes. The absolute high spin contents of substrate-free, progesterone-bound and 17 alpha-hydroxyprogesterone-bound P-450C21 were estimated from the analysis of thermally induced difference spectra to be 25, 78 and 94% at 25 degrees C, respectively, in 50 mM potassium phosphate buffer (pH 7.2) containing 20% glycerol, 0.1 mM EDTA and 0.5% Emulgen 913. The effect of the high spin content on P-450C21 reduction by NADPH in the reconstituted system was analyzed by a steady-state method and by a stopped-flow method at 25 degrees C. The steady-state results showed that the rate of P-450C21 reduction was not affected by the high spin content of substrate-bound P-450C21 but was very slow without a steroid substrate. Biphasic reduction of P450C21 containing two first-order processes was observed in the stopped-flow experiment in the presence of either of the steroid substrates, but the reduction was very slow without the substrate. There were no significant differences in the rate and the amount of the fast phase of reduction between 17 alpha-hydroxyprogesterone-bound and progesterone-bound P-450C21. Both kinetic studies indicate that the spin state does not control the electron transfer from NADPH to P-450C21 via NADPH-cytochrome P-450 reductase but the presence of substrate is essential for the reduction of P-450C21.     

Interaction of prostaglandins with adrenal microsomal cytochrome P-450 in the guinea pig.

Studies were carried out to investigate the effects of prostaglandins (PG) in vitro on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE1, PGE2, PGA1, PGF1 alpha or PGF2 alpha to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (delta A385-420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11 beta-hydroxyprogesterone. However, the affinities of prostaglandins and steroids for adrenal microsomal cytochrome P-450, as estimated by the spectral dissociation constants, were similar. Prior addition of prostaglandins to isolated adrenal microsomes did not affect steroid binding to cytochrome P-450 or the rate of steroid 21-hydroxylation. In contrast, prostaglandins inhibited adrenal metabolism of ethylmorphine and diminished the magnitude of the ethylmorphine-induced spectral change in adrenal microsomes. The results indicate that prostaglandins inhibit adrenal drug metabolism by interfering with substrate binding to cytochrome P-450. Since 21-hydroxylation was unaffected by PG, different cytochrome P-450 moieties are probably involved in adrenal drug and steroid metabolism.     

Effect of ketoconazole on placental aromatase, 3 beta-hydroxysteroid dehydrogenase-isomerase and 17 beta-hydroxysteroid dehydrogenase

Ketoconazole, an orally-active, broad spectrum mycotic agent, was shown to inhibit in vitro human placental microsomal aromatase but was without effect on 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-I) and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activities. The Km of placental aromatase for testosterone was 30 +/- 1.1 nmol/l (mean +/- SEM, n = 6). Inhibition (determined by Lineweaver-Burk plot) was non-competitive with respect to substrate with a Ki value of 3.0 +/- 1.4 mumol/l (mean +/- SEM, n = 6). Ketoconazole was without effect on the 3 beta-HSD-I and 17 beta-HSD activities when using [3H] pregnenolone and [3H] oestradiol, respectively, as substrates. Since ketoconazole is known to inhibit cytochrome P-450-dependent enzyme reactions, the results of the present study support the contention that cytochrome P-450 is involved in the aromatisation process.     

The effect of some biphenyl solubilizing and suspending agents on biphenyl 4-hydroxylase of rabbit liver microsomes

The effects of ethanol, acetone, dimethylsulfoxide (DMSO), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene sorbitan monolaurate (Tween 20), Triton X-100, and carboxymethyl cellulose (CMC) on the kinetics of biphenyl 4-hydroxylase of rabbit liver microsomes were investigated in an attempt to find a substrate-solubilizing or suspending agent (carrier) which was itself a non-effector of the mixed-function oxidase. The effects of these carriers on the activities of NADPH-cytochrome P-450 reductase, NADPH-cytochrome c reductase, and cytochrome P-450 content were also investigated.Ethanol and DMSO inhibited biphenyl 4-hydroxylase and NADPH-cytochrome P-450 reductase. Acetone inhibited the hydroxylase uncompetitively at concentrations which appeared to stimulate NADPH-cytochrome P-450 reductase. All of the detergents inhibited biphenyl 4-hydroxylase although only Triton X-100 markedly affected the reduction of cytochrome P-450. The interaction of Tween 80 with the hydroxylase gave rise to non-linear Lineweaver-Burk plots although at high concentrations of biphenyl or low concentrations of the detergent the inhibition appeared to be competitive.Biphenyl caused a 2–3-fold stimulation of NADPH-cytochrome P-450 reductase, but in the presence of Tween 80 the stimulation was absent. Since V of biphenyl 4-hydroxylase in the presence of Tween 80 was not significantly different from V in its absence it would appear that the reduction of cytochrome P-450 was not ratelimiting.Of all the carriers studied only CMC was without effect on all aspects of microsomal electron transport investigated. As far as biphenyl 4-hydroxylase is concerned, CMC appears to be the most suitable substrate carrier.     

Luteinizing hormone and cyclic AMP-mediated induction of microsomal cytochrome P-450 enzymes in cultured mouse Leydig cells

Treatment of mouse Leydig cell cultures with luteinizing hormone (LH) or with 8-bromo-cAMP (8-Br-cAMP) for 5 days elicited a dose- and time-dependent increase in the microsomal cytochrome P-450 enzyme activities. 17 alpha-Hydroxylase and C17-20 lyase as well as a parallel increase in testosterone production. Reduction of the oxygen tension from 19 to 1% resulted in a greater increase in enzyme activity. Induction of microsomal cytochrome P-450 activities was 35 to 50% greater with 8-Br-cAMP than with LH and the increase in C17-20 lyase activity was 4-fold greater than that of 17 alpha-hydroxylase. Maximal induction of P-450 enzyme activities was observed between 3 and 5 days of continual treatment with 8-Br-cAMP or LH. Removal of 8-Br-cAMP from the culture medium inhibited any further increase in C17-20 lyase activity and testosterone production. The role of protein synthesis in the induction process was investigated by incubating Leydig cell cultures with and without cycloheximide between 24 and 48 h of treatment with 8-Br-cAMP. Cycloheximide completely inhibited the induction of C17-20 lyase activity and the increase in testosterone production. After removal of the inhibitor, cultures responded in a manner that paralleled induction in cultures that had not been treated with cycloheximide. In both cases, a 24-h lag period occurred prior to an increase in cytochrome P-450 activity. These data suggest that the increase in microsomal cytochrome P-450 activities represents an increase in enzyme synthesis and, furthermore, that reduction of oxygen tension decreases degradation of newly synthesized Leydig cell microsomal cytochrome P-450 activities as recently reported (Quinn, P.G., and Payne, A.H. (1984) J. Biol. Chem. 259, 4130-4135).     

Neonatal imprinting and hepatic cytochrome P-450 II. Partial purification of a sex-dependent and neonatally imprinted form(s) of cytochrome P-450

A new form of cytochrome P-450 was partially purified from hepatic microsomes of neonatally imprinted rats (adult male and adult male castrated at four weeks of age). This new form of cytochrome P-450 appears to have an apparent molecular weight of approximately 50,000 daltons as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It appears that this form of cytochrome P-450 is either absent or present in low concentrations in cytochrome P-450 preparations isolated from neonatally nonimprinted rats (adult female and adult male castrated at birth). Reconstitution of testosterone hydroxylase and benzphetamine N-demethylase activities of this partially purified cytochrome P-450 revealed that the presence of testosterone 16α-hydroxylase activity, an imprintable microsomal enzyme, was in parallel with the imprinting status of the animals; a significantly higher activity was detected in the neonatally imprinted than that of the nonimprinted animals. This was in contrast to the nonimprintable benzphetamine N-demethylase, testosterone 7α-and 6β-hydroxylase activities which exhibited no correlation with the imprinting status of the animals. We have prepared antisera from rabbits using the partially purified cytochrome P-450 preparations from adult male rats as antigens. These antisera inhibited microsomal testosterone 16α- and 7α-hydroxylase activities in a concentration-dependent manner, without impairing 6β-hydroxylase activity. These data suggest that the partially purified cytochrome P-450 from adult male rats consists of both imprintable (16α-) and nonimprintable (7α-) testosterone hydroxylase activities. The antisera formed immunoprecipitant lines in the Ouchterlony double diffusion plates with partially purified cytochrome P-450 from both neonatally imprinted and nonimprinted adult rats. The immunoprecipitant lines, as stained by coomassie blue, suggest the homology of the cytochrome P-450 preparations from neonatally imprinted and nonimprinted rats. Immunoabsorption of the antisera against neonatally nonimprinted, partially purified cytochrome P-450 completely removed the immunoprecipitant lines without appreciably impairing the inhibitory effects of antisera on the microsomal testosterone 16α-and 7α-hydroxylase activities. In contrast, immunoabsorption of the antisera against partially purified cytochrome P-450 from adult male rats (imprinted) abolished completely both the immunoprecipitant lines and the inhibition on microsomal testosterone hydroxylation reaction (16α and 7α). The inhibitory actin of antisera on testosterone hydroxyulation was also abolished upon boiling the antisera at 100°C for 5 minutes. The biochemical and immunochemical data in this study suggest that the neonatally imprintable form or forms of hepatic microsomal cytochrome P-450 accounts for a small fraction of the bulk of total cytochrome P-450. However, the existence of this form of cytochrome P-450 is regulated by gonadal hormones during the neonatal period and accounts for the major imprintable sex difference in drug and steroid metabolism in adulthood.