Adrenal Mitochondrial Cytochrome P-450 Research Articles

Inactivation of adrenal cytochromes P450 by 1-aminobenzotriazole: Divergence of in vivo and in vitro actions

Recent investigations demonstrated that administration of 1-aminobenzotriazole (ABT) to rats caused adrenal gland enlargement. Studies were done to pursue the mechanism(s) involved. Preliminary experiments revealed that the adrenal enlargement caused by ABT was associated with a decline in plasma corticosterone concentrations, suggesting inhibition of adrenal steroidogenesis. Indeed, a single injection of ABT (25 or 50 mg/kg body weight) to rats caused concentration-dependent declines (60–80%) in adrenal mitochondrial and microsomal cytochrome P450 (P450) concentrations. The decreases in adrenal P450 levels exceeded those in hepatic microsomes. Accompanying the declines in adrenal P450 concentrations were decreases in steroid hydroxylase activities. Mitochondrial 11β-hydroxylase and cholesterol side-chain cleavage activities and microsomal 21-hydroxylase activity were diminished markedly (60–90%) by ABT treatment. In contrast, activity of adrenal 3β-hydroxysteroid dehydrogenase-isomerase was not affected by ABT, indicating specificity for P450-dependent reactions. Incubation of adrenal microsomes or mitochondria in vitro with ABT plus an NADPH-generating system had no effect on P450 concentrations or on steroid hydroxylase activities. Similar incubations with hepatic microsomes caused declines in P450 levels and in the rates of P450-mediated xenobiotic metabolism. The results demonstrate that ABT is a potent inhibitor of adrenal steroid hydroxylases in vivo, but the in vitro studies indicate that the mechanism of action differs from that on other P450 isozymes. The absence of inhibitor effects in vitro suggests that an extra-adrenal metabolite of ABT is responsible for the in vivo inactivation of steroidogenic enzymes.

Adrenal mitochondrial metabolism of spironolactone: Absence of metabolic activation

Previous investigations have established that spironolactone (SL) administration to guinea pigs decreases adrenal mitochondrial and microsonnal cytochrome P-450 content, and that the latter requires microsomal activation of the drug. Studies were carried out to determine if adrenal mitochondrial metabolism (activation) of SL was similarly involved in the effects of the drug on mitochondrial cytochrome P-450 destruction. Incubation of guinea pig adrenal mitochondria with SL in the absence of NADPH resulted in the formation of 7α-thio-SL as the only metabolite. In the presence of an NADPH-generating system, an unknown polar metabolite was also produced. The mass spectrum of the unknown compound suggested that it was a hydroxylated derivative of SL. Incubation of mitochondrial preparations with 7α-thio-SL also resulted in the formation of a polar metabolite, but the latter had a different HPLC retention time than that of the SL metabolite. Formation of the polar SL metabolite was prevented by metyrapone, an 11β-hydroxylase inhibitor, and was greatest in mitochondria from the adrenal zone having the highest 11β-hydroxylase activity. Steroid substrates for 11β-hydroxylation inhibited the production of the SL metabolite. Mitochondrial incubations with SL or with 7α-thio-SL in the presence or absence of an NADPH-generating system did not affect cytochrome P-450 concentrations. The results indicate that, unlike the microsomal effects of SL, local activation of SL is not responsible for the destruction of adrenal mitochondrial cytochromes P-450. The major adrenal mitochondrial metabolites of SL appear to be 11β-hydroxy-SL and 7β-thio-SL.

ACTH control of cholesterol side-chain cleavage at adrenal mitochondrial cytochrome P-450 scc. Regulation of intramitochondrial cholesterol transfer

Rat adrenal mitochondria exhibit a linear 2-fold accumulation of cholesterol for 20 min following either in vivo ether stress or ACTH administration, providing cholesterol metabolism is inhibited by aminoglutethimide (AMG). Additional cycloheximide (CX) pretreatment only slightly decreases this increase, but the location of accumulation shifts from the inner membrane to the outer membrane, implying a decreased cholesterol transfer from outer to inner membrane. Although the capacity of outer mitochondrial membranes was saturated after a 10-min treatment with CX, a 20-min treatment resulted in further retention of cholesterol in intact mitchondria that was not recovered in the isolated membranes. An additional pool of loosely bound cholesterol is proposed for CX mitochondria. These studies provide evidence that the CX-sensitive step of adrenal steroidogenesis attributed to loss of a labile ACTH regulatory protein (Pedersen R.C. and Brownie A.C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1882–1886) involves cholesterol transfer from the outer to the inner mitochondrial membrane. ACTH also enhances the PI and PE content of the outer membranes by a CX-sensitive mechanism that may contribute to intramitochondrial cholesterol transport. CX treatment does not affect cholesterol uptake by the inner membrane from phospholipid vesicles. The initial rate of endogenous metabolism in isolated inner membranes is insensitive to pretreatment (2 nmol/nmol P-450/min). The duration of this linear rate was increased 4-fold by AMG treatment while this increase was prevented by CX treatment. The kinetics indicate differences in inner membrane reactive cholesterol levels. Inner membranes also contained a fraction of unreactive cholesterol that is insensitive to pretreatment. Cholesterol- P-450 scc complex formation for all pretreatments fits a single hyperbolic function of the reactive cholesterol content of the inner mitochondrial membrane ( K d = 0.025 mol cholesterol/mol phospholipid), and is activated over 5-fold upon mitochondrial disruption. All changes in inner membranes caused by CX can, therefore, be attributed solely to the restricted cholesterol access in vivo.

Biphasic 19-nor-deoxycorticosterone excretion after adrenal enucleation is associated with altered adrenal mitochondrial cytochrome P450 11 beta-, 18-, and 19-hydroxylase activities.

After adrenal enucleation (AE) rats avidly retain sodium (early phase), but after 7-10 days they lose this sodium avidity (late phase). Although increased production of a mineralocorticoid, 19-nor-deoxycorticosterone (19-Nor-DOC), has been implicated, 19-Nor-DOC levels during the early and late phases of AE have not been systematically measured. Furthermore it is not known why 19-Nor-DOC production should increase during a time when production of 11 beta- and 18-hydroxylated corticosteroids are decreased in AE. The purpose of this study was to examine the 11 beta, 18-, and 19-hydroxylase pathways in the early and late phases of AE. The results demonstrate increased urinary 19-Nor-DOC and decreased 18-OH-DOC and corticosterone excretion in the early phase of AE at a time when adrenal mitochondrial 11 beta- and 18-hydroxylase activities were decreased but 19-hydroxylase activity was unchanged. During the late phase of AE, urinary 19-Nor-DOC had decreased and 18-OH-DOC and corticosterone had increased to levels indistinguishable from those in sham controls. This reduction in 19-Nor-DOC was associated with a decrease in 19-hydroxylase activity in AE. Since the 11 beta, 18-, and 19-hydroxylases have a common substrate (DOC), it is possible that differential flux of DOC through these pathways could account for the changes in steroid production in AE. These data suggest that the increased 19-Nor-DOC excretion in AE may be due to alterations in enzyme activity leading to a shunting of DOC into the 19-Nor-DOC pathway. In addition, the synchronicity of 19-Nor-DOC with sodium excretion suggests that it has an important role in the pathogenesis of the sodium retention in AE.

Inhibition of steroidogenic cytochrome P-450 enzymes in rat testis by ketoconazole and related imidazole anti-fungal drugs

Ketoconazole, an imidazole antifungal drug, has previously been shown to diminish testosterone and cortisol production in patients as well as rat and mouse cells in vitro. Inhibition of adrenal mitochondrial cytochrome P-450 enzymes was demonstrated. In this study we tested several imidazole antifungal drugs and examined the individual steps in testicular steroidogenesis to determine which enzymes in the androgen pathway were blocked. In addition, we studied 25-hydroxyvitamin D 24-hydroxylase activity in cultured pig kidney cells (LLC-PK 1) to assess a mitochondrial P-450 enzyme in another organ. All imidazoles tested inhibited both total testosterone production and 24-hydroxylase activity but the relative potencies differed. We next studied the individual testicular enzymatic steps between cholesterol and testosterone. Ketoconazole inhibited cholesterol-side-chain-cleavage enzyme (mitochondrial) and C-17,20 lyase (microsomal). The three inhibited enzymes (two testicular and one renal) are all P-450 cytochromes. Testicular 17-hydroxylase, also a P-450 cytochrome, was not inhibited even at high doses of ketoconazole. This is an interesting finding because the testicular hydroxylase and lyase have been shown to be a single protein. Non-cytochrome P-450 enzymes in the androgen pathway were not inhibited. The results demonstrate that several imidazole antifungal drugs all inhibit both microsomal and mitochondrial cytochrome P-450 enzymes in multiple organs.

Potassium intake and aldosterone biosynthesis: The role of cytochrome P-450

K + Repletion for 48 h of rats previously kept on a low K + diet for 2 weeks specifically increased the conversion of corticosterone into aldosterone and 18-hydroxycorticosterone by incubated capsular fractions of rat adrenal tissue. This increase in the activity of the final steps of aldosterone biosynthesis was not accompanied by an increase in capsular adrenal mitochondrial cytochrome P-450 concentration. By contrast, an increased corticosterone-induced absorbance change (BI) was consistently found in capsular adrenal mitochondria upon K + repletion. In addition, a type I-like absorbance change was induced with 18-hydroxy-11-deoxycorticosterone but not with 18-hydroxycorticosterone. Therefore, K + repletion of K + depleted rats specifically increased the binding of corticosterone and possibly 18-hydroxy-11-deoxy-corticosterone to the 18-methyl oxydase enzyme complex. Whether this increased binding was due to an increase in enzyme protein concentration or due to a better availability of the substrate to the enzyme, could not be decided from these experiments.

Relation of Canrenone to the Actions of Spironolactone on Adrenal Cytochrome P-450-Dependent Enzymes*

Studies were carried out to determine the actions of spironolactone (SL) and its metabolite, canrenone, on adrenal mixed function oxidases in the guinea pig. Administration of SL (25 mg/kg day for 3 days) to adult male guinea pigs decreased adrenal mitochondrial and microsomal cytochrome P-450 concentrations. The activities of various cytochrome P-450- dependent enzymes, including mitochondrial 11β-hydroxylase and microsomal 21-hydroxylase, benzo(a)-pyrene (BP) hydroxylase and ethylmorphine (EM) demethylase, were also reduced by SL treatment. Canrenone, when given at the same dose, had no effect on mitochondrial or microsomal cytochrome P-450 levels. Pretreatment with canrenone decreased 11β- and 21- hydroxylase activities but did not affect BP hydroxylation or EM demethylation. Addition of SL or canrenone to isolated adrenal mitochondria or microsomes produced typical type I difference spectra. Both compounds inhibited the binding of steroid substrates to cytochrome P-450 in isolated mitochondria and mic...

Cytochrome P-450 of adrenal mitochondria. Steroid binding sites on two distinguishable forms of rat adrenal mitochondrial cytochrome P-450sec.

Cytochrome P-450 of adrenal mitochondria. Steroid binding sites on two distinguishable forms of rat adrenal mitochondrial cytochrome P-450sec.

Site of Action of Growth Hormone on Adrenocortical Steroidogenesis in Rats

Studies were carried out to define the mechanism of action of growth hormone on adrenocortical steroidogenesis in hypophysectomized female rats. ACTH administration for 7 days increased corticosterone secretion in vivo and corticosterone production by adrenal tissue in vitro. Adrenal mitochondrial and microsomal cytochrome P-450 concentrations as well as the activities of cytochrome P-450-dependent enzymes (cholesterol sidechain cleavage, 11beta-hydroxylase, 21-hydroxylase) were also increased by ACTH. Administration of bovine growth hormone alone to hypophysectomized rats had no effect on any of the parameters evaluated. However, when given in combination with ACTH, growth hormone synergistically enhanced the effects of ACTH on cholesterol sidechain cleavage activity and corticosterone secretion. The magnitude of the pregnenolone-induced difference spectrum in adrenal mitochondria, indicative of cholesterol binding to cytochrome P-450, was also increased by growth hormone, but neither cytochrome P-450 content nor the activities of other steroidogenic enzymes were affected. The results indicate that growth hormone interacts with ACTH to promote corticosterone secretion by increasing the association of cholesterol with adrenal mitochondrial cytochrome P-450, thereby increasing the activity of cholesterol sidechain cleavage, the rate-limiting step in steroidogenesis.

Inhibition of cholesterol side-chain cleavage by 22-azacholesterol

The side-chain of cholesterol is oxidatively cleaved by a cytochrome P-450-dependent enzyme system present in mammalian adrenal mitochondria. The reaction was postulated to proceed via one or more hydroxylated intermediates. Inhibition of the reaction by the potent inhibitor 22-azacholesterol did not result in a build-up of the postulated intermediates: (20S)-20-hydroxycholesterol, (22R)-22-hydroxycholesterol, (20R,22R)-20,22-dihydroxycholesterol or (20S)-cholesterol hydroperoxide. Spectral studies indicate a modified type II spectrum for the reaction of 22-azacholesterol with adrenal mitochondrial cytochrome P-450. In addition, spectral and electron spin resonance studies indicate a common interaction of cholesterol and 22-azacholesterol with the cytochrome. Although this study does not resolve the question of the intermediacy of the oxygenated derivatives in the cleavage reaction, it indicates that the binding of 22-azacholesterol depends on its steroid structure rather than on its amine character. A discussion of the proximity of the heme to the substrate binding site is also presented.

Spironolactone and cytochrome P-450: Impairment of steroid 21-hydroxylation in the adrenal cortex

The effect of spironolactone administration on the activities of adrenal 21-hydroxylases was examined in male cortisol- and corticosterone-producing animals. Decreases in the activities of the 21-hydroxylases after spironolactone treatment occur only in those animals that predominantly produce cortisol rather than corticosterone and that have a high activity of adrenal steroid 17α-hydroxylase. The administration of spironolactone to cortisol-producing animals, namely, the guinea pig and the dog, caused a 50–75% loss in the activities of adrenal 21-hydroxylases with a concomitant decrease in the content of microsomal cytochrome P-450 and microsomal heme and in the activity of microsomal 17α-hydroxylase. Spironolactone treatment was also found to decrease the content of adrenal mitochondrial cytochrome P-450 in male guinea pigs but not male dogs. In contrast to its effect in cortisol-producing animals, the administration of spironolactone caused an increase in the activities of the microsomal 21-hydroxylases in the adrenals of corticosterone-producing animals such as the rat and the rabbit.

Cytochrome P-450 of adrenal mitochondria. Spin states as detected by difference spectroscopy.

Adrenal mitochondrial cytochrome P-450 which functions in cholesterol side chain cleavage (P-450scc) exhibited type I (lambdamax 385, lambdamin 420 nm) and inverse type I (lambdamin 385, lambdamax 420 nm) difference spectra with several steroids. The magnitude and type of response were dependent on the particular steroid and on the extent to which cholesterol was bound to the cytochrome in the intact mitochondrion. the inverse type I difference spectrum induced by 3beta-hydroxy-pregn-5-ene-20-one (pregnenolone) was dependent on the proportion of high spin cholesterol-cytochrome P-450scc complexes. With rat adrenal mitochondria cholest-5-ene-3beta, 20alpha-diol (20alpha-hydroxycholesterol) invariably induced a smaller inverse type I response and, under conditions where cytochrome P-450scc was nearly free of cholesterol, even produced a small type I response. Two distinct steroid binding sites on cytochrome P-450scc were detected by, respectively, the slow type I response to cholest-5-ene-3beta, 25-diol (25-hydroxycholesterol) and the rapid type I response to a subsequent addition of cholest-5-ene-3beta, 20alpha, 22 R-triol (20alpha, 22R-dihydroxycholesterol). The relative proportions of the spectral responses to these steroids were dependent on the previous extent of adrenal activation by adrenocorticotropic hormone (ACTH), because this stimulatory process altered the combination of mitochondrial cholesterol with cytochrome P-450scc. It is proposed that the two steroid binding sites on cytochrome P-450scc interact with steroids in the following way: site I binds cholesterol, 25-hydroxycholesterol, and 20alpha, 22R-dihydroxycholesterol with formation of a partially high spin cytochrome; site II binds both pregnenolone and 20alpha-OH cholesterol resulting in a low spin cytochrome. Interactions between sites I and II are not competitive, and occupancy of site II ensures a low spin state irrespective of the occupancy of site I. A second mode of interaction by 20alpha, 22R-dihydroxycholesterol stabilizes a high spin cytochrome and is competitive with site II binding by 20alpha-hydroxycholesterol or pregnenolone. Formation of a maximally high spin cytochrome follows occupancy by 20alpha, 22R-dihydroxycholesterol at both sites.

Cytochrome P-450 of adrenal mitochondria. In vitro and in vivo changes in spin states.

Steroid-induced difference spectra have been used to examine the combination of cholesterol with adrenal mitochondrial cytochrome P-450 which participates in cholesterol side chain cleavage (P-450scc) and the depletion of cholesterol from the cytochrome which results from turnover of the enzyme system. Type I difference spectra-induced by cholest-5-ene-3beta, 25-diol (25-hydroxycholesterol) and cholest-5-ene-3beta, 20 alpha, 22R-triol (20alpha, 22R dihydroxycholesterol) have been used to quantitate binding of cholesterol to two sites (I and II) on cytochrome P-450scc. The action of adrenocorticotropic hormone (ACTH) in vivo and the action of calcium or phosphate ions on isolated mitochondria stimulate the combination of cholesterol with site I but not site II. Cholesterol derived from lecithin-cholesterol micelles, however, binds to both sites. Malate-induced cholesterol depletion occurred at a comparable rate to the transfer of cholesterol from lecithin-cholesterol micelles. However, a residual proportion of cholesterol-cytochrome P-450scc complexes remained, even after 10 min of exposure to malate, and was of similar magnitude in mitochondria from both cycloheximide-treated and stressed rats. It is suggested that this reflects a less reactive form of cholesterol-cytochrome complex. Steroid-induced difference spectra indicate that sites I and II on cytochrome P-450scc are similarly depleted after metabolism of mitochondrial cholesterol in vitro and after inhibition of the action of ACTH in vivo. Anaerobiosis of adrenal cells after excision of the accumulation of cholesterol at cytochrome P-450cc. When anaerobiosis was prevented, cytochrome P-450scc in the freshly isolated mitochondria was apparently essentially free of complexed cholesterol, irrespective of the extent of ACTH action. For 30 min after suspension of the mitochondria in 0.25 M sucrose at 4 degrees, cholesterol combines with cytochrome P-450scc. The extent of this process was not affected by the presence of cycloheximide during ether stress treatment of the rats. It is concluded that there are at least two pools of mitochondrial cholesterol with access to cytochrome P-450scc but that ACTH stimulates only the pool which most readily interacts with the cytochrome.

The effect of bee venom on serum corticosterone levels and adrenal mitochondrial cytochrome P-450 in intact and hypophysectomized rats

The effect of bee venom on serum corticosterone levels and adrenal mitochondrial cytochrome P-450 in intact and hypophysectomized rats

Inhibition of cholesterol side-chain cleavage by azacholesterols

Abstract The side-chain of cholesterol is oxidatively cleaved by a cytochrome P-450-dependent enzyme system present in mammalian adrenal mitochondria. The reaction was postulated to proceed via one or more hydroxylated intermediates. Inhibition of the reaction by the potent inhibitor 22-azacholesterol did not result in a build-up of the postulated intermediates: (20S)-20-hydroxycholesterol, (22R)-22-hydroxycholesterol, (20R,22R)-20,22-dihydroxycholesterol or (20S)-cholesterol hydroperoxide. Spectral studies indicate a modified type II spectrum for the reaction of 22-azacholesterol with adrenal mitochondrial cytochrome P-450. In addition, spectral and electron spin resonance studies indicate a common interaction of cholesterol and 22-azacholesterol with the cytochrome. Although this study does not resolve the question of the intermediacy of the oxygenated derivatives in the cleavage reaction, it indicates that the binding of 22-azacholesterol depends on its steroid structure rather than on its amine character. A discussion of the proximity of the heme to the substrate binding site is also presented.

Effect of androgens on adrenal cortical 11β-hydroxylation

The effects of the chronic administration of testosterone, androstenedione and DHA upon adrenal cortical function in the rat were compared. Only testosterone induced hypertensive disease and decreased 11β- and 18-hydroxylase activity in vitro. Significant decreases in adrenal mitochondrial cytochrome P-450 levels were apparent in all androgen-treated groups. Testosterone administration also resulted in a significant increment in the concentration of cytochromes a + a 3. The DOC-induced difference spectrum was reduced in all androgen-treated groups when expressed per mg of protein but only in mitochondria from testosterone-treated rats when calculated per nanomole of cytochrome P-450. The results suggest that the inhibition of the 11β- and 18-hydroxylation of DOC by testosterone is the result of competitive binding to cytochrome P-450 and not the induced decrease in cytochrome P-450 levels per se.

Effect of testosterone on the rat adrenal cortical 11β-hydroxylation system

The influence of testosterone on the adrenal mitochondrial steroid hydroxylation system was investigated in the female Sprague-Dawley rat. After the injection of animals with testosterone for 10 days, it was possible to demonstrate impairment of progesterone metabolism in adrenal homogenates. The formation of corticosterone and 18-hydroxy-11-deoxycorticosterone was lower than in controls and there was an accumulation of 11-deoxycorticosterone (DOC). Adrenal mitochondria isolated from testosterone-treated rats showed low rates of conversion of DOC-4- 14C to corticosterone and 18-hydroxy-DOC, confirming the low activity of steroid 11β- and 18-hydroxylases. Rates of corticosterone formation from unlabeled DOC were also lower in adrenal mitochondria from testosterone-treated animals. Accompanying these low steroid hydroxylation activities in isolated mitochondria were decreased levels of adrenal mitochondrial cytochrome P-450. In addition, spectral studies revealed that the DOC-induced spectral change was considerably less in the adrenal mitochondria from testosterone-treated rats. The results described in this communication are consistent with the concept that, after injection into the rat, testosterone reaches the adrenal cortex and there interacts with the mitochondrial cytochrome P-450 in such a way that the natural substrate, DOC, is less able to be bound and hydroxylated. This hypothesis was confirmed by the addition of testosterone to rat adrenal mitochondria in vitro and the observation of inhibition of both DOC-induced spectral changes and steroid hydroxylation.

PH-Dependent changes in the spin state of cytochrome P450 from adrenal mitochondria

Cytochrome P450 in the adrenal cortex participates as a mixed function oxidase in several steps in the biosynthesis of corticosterone, of which side chain cleavage (XC) of cholesterol [l] and 1 l/3-hydroxylation of 1 1-deoxycorticosterone (1 I-DOC) occur in the mitochondria [2]. These two oxidations are specifically inhibited by aminoglutethimide and metapyrone [3] respectively, suggesting that different enzymes are involved in these oxidations. Recently the cytochrome P450 of adrenal mitochondria has been separated into two fractions which, with the same reducing system, specifically carry out 1 lh high spin (sm = 7.49) and low spin (sm = 2.45, g = 2.26, sm = 1.91) [5]. In addition, steroid-induced changes in the above EPR spectra and in the absorption spectra of the separated cytochrome P450 fractions [4] indicate that low spin 1 l/3-hydroxylase cytochrome P450 changes to a high spin state binding 1 l-DOC, while high spin SCCP450 is converted to a low spin state by pregnenolone. In this paper we show that the spin state and ligand binding properties of the SCC-cytochrome P450, but not 1 l/3-hydroxylase cytochrome P450, are very sensitive to pH.

Testosterone-induced hypertension in the rat.

The effect of ACTH upon the inhibition of corticosteroidogenesis produced by treating rats with several synthetic adrogens has been evaluated. In experiments lasting about 2 weeks in which the simultaneous treatment with ACTH prevented the fall in adrenal weight due to the androgen therapy, corticosteroidogenesis measured in adrenal homogenates was still greatly reduced. Associated with the reduced production of corticosterone from progesterone there was a marked fall in the level of adrenal mitochondrial cytochrome P-450. The fall in cytochrome P-450 was not prevented by simultaneous treatment with ACTH. Treatment of rats with all 3 androgens, 17α-methylandrostenediol, 17α-methyltestosterone and 11β-hydroxy- 17α-methyltestosterone, brought about the decrease in cytochrome P-450 levels, whereas when metyrapone, a nonsteroidal competitive inhibitor of steroid 11β-hydroxylase, was given to rats for 6 weeks no change occurred in the level of adrenal mitochondrial cytochrome P-450. These data suggest that thi...

Some studies on the effect of androgens on adrenal cortical function of rats.

The effect of ACTH upon the inhibition of corticosteroidogenesis produced by treating rats with several synthetic adrogens has been evaluated. In experiments lasting about 2 weeks in which the simultaneous treatment with ACTH prevented the fall in adrenal weight due to the androgen therapy, corticosteroidogenesis measured in adrenal homogenates was still greatly reduced. Associated with the reduced production of corticosterone from progesterone there was a marked fall in the level of adrenal mitochondrial cytochrome P-450. The fall in cytochrome P-450 was not prevented by simultaneous treatment with ACTH. Treatment of rats with all 3 androgens, 17α-methylandrostenediol, 17α-methyltestosterone and 11β-hydroxy- 17α-methyltestosterone, brought about the decrease in cytochrome P-450 levels, whereas when metyrapone, a nonsteroidal competitive inhibitor of steroid 11β-hydroxylase, was given to rats for 6 weeks no change occurred in the level of adrenal mitochondrial cytochrome P-450. These data suggest that thi...