Differential effects of adrenocorticotropic hormone on steroid hydroxylase activities in the inner and outer zones of the guinea pig adrenal cortex.

We have studied the effects of ACTH treatment on steroid hydroxylase activities in the inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex. Animals received 5 or 10 U of ACTH daily for 6 days and enzyme activities were then assessed in isolated microsomal or mitochondrial preparations. In control animals, microsomal cytochrome P-450 concentrations were greater in the inner than outer zone, but mitochondrial P-450 levels were similar in the two zones. Microsomal 17 alpha-hydroxylase and mitochondrial 11 beta-hydroxylase activities were greater in the outer than inner zone, but microsomal 21-hydroxylase activity was greater in the inner zone. ACTH treatment decreased cytochrome P-450 concentrations in inner but not outer zone microsomes; mitochondrial P-450 levels were unaffected in both zones. ACTH caused a dose-dependent increase in inner zone 17 alpha-hydroxylase activity and decrease in 21-hydroxylase activity without affecting the activity of either enzyme in outer zone microsomes. ACTH also decreased 11 beta-hydroxylase activity in outer but not inner zone mitochondrial preparations. The net effect of ACTH treatment was to diminish the differences in steroid metabolism between the two zones. The results indicate that the effects of ACTH on steroid hydroxylase activities are both zone- and enzyme-dependent, suggesting the existence of multiple and independent regulatory mechanisms.     

Inhibition of testicular cytochrome P-450-dependent steroid biosynthesis by cis-platinum. Reversal by human chorionic gonadotropin

The treatment of rats with cis-platinum for 7 days caused a profound, and seemingly selective, decrease (70-80%) in the microsomal cytochrome P-450 levels in the testis. This decrease was accompanied by marked reductions (70-80%) in steroid 17 alpha-hydroxylase activity and in plasma testosterone concentration. The treatment of rats with human chorionic gonadotropin partially restored the cytochrome P-450 concentration and 17 alpha-hydroxylase activity and permitted the plasma testosterone level to approach control values. The effect of cis-platinum on the testicular cytochrome P-450 appeared unrelated to deficiencies in heme metabolic processes, in so far that neither was the activity of delta-aminolevulinate synthetase decreased, nor was that of heme oxygenase increased. These enzymes are rate-limiting in heme biosynthesis and degradation pathways, respectively. Also, the activities of uroporphyrinogen I synthetase, delta-aminolevulinate dehydratase, and ferrochelatase and the concentration of total porphyrins in the testis remained unchanged. The sodium dodecyl sulfate-gel electrophoresis of the microsomal preparation did not reveal a diminished level of apocytochrome; however, in this preparation, heme could not be detected in molecular weight regions corresponding to cytochrome P-450. The microsomal cytochrome b5 and the mitochondrial heme concentrations were not decreased in cis-platinum-treated rats. It is suggested that the mechanism of depletive action of cis-platinum on microsomal cytochrome P-450 involves an impairment of the effective assembly of heme and apoprotein moieties. It is further suggested that the anterior pituitary hormones control the factor(s) involved in this assembly, a process which is interrupted by cis-platinum.     

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.     

The interference of polychlorinated biphenyls (Aroclor 1254) with membrane regulation of the activities of cytochromes P-450C21 and P-450(17) alpha,lyase in guinea-pig adrenal microsomes.

Regulation of cytochromes P-450 21-hydroxylase (P-450C21) and P-450 17 alpha-hydroxylase/C17,20-lyase (P-450(17) alpha,lyase) activities and impairment of this regulation by Aroclor 1254 was studied in guinea-pig adrenal microsomes. In a membrane depleted system, a decrease in the normally predominant, P-450C21 activity and an increase in P-450(17) alpha,lyase activities was observed. The same deviations were observed in intact microsomes with increase in the reaction temperature (0-40 degrees C). Breaks in Arrhenius plots for activities of P-450C21 and P-450(17) alpha,lyase correlate with transition temperatures reported for the microsomal membrane. These results point to: (1) preference of a gel state membrane for catalytic expression of P-450C21 suggesting a clustered organization of this P-450 species with reductase; (2) preference of a fluid membrane for lyase activity suggesting a random collision mechanism for reduction of P-450(17) alpha,lyase. Aroclor 1254 introduced to reaction mixtures containing intact microsomes elicited basically the same changes as caused by depletion of the microsomal membrane or by increase in the incubation temperature. Lack of effect of Aroclor 1254 on P-450C21 and P-450(17) alpha,lyase activities in the membrane depleted system demonstrates that its interference with monooxygenase activities is mediated by the microsomal membrane. The similarities between altered cytochrome P-450 mediated activities in the presence of Aroclor 1254 and the deviations observed in the membrane depleted system or upon increase in the incubation temperature may suggest that this chemical exerts its impacts by influencing membrane fluidity.     

The regulation of ovine placental steroid 17 alpha-hydroxylase and aromatase by glucocorticoid

Parturition in the pregnant sheep is preceded by an abrupt alteration in placental steroid metabolism causing a shift from progesterone to estrogen production. This change is believed to be a consequence of the prepartum rise in cortisol in the fetal circulation and involves increases in activities of the enzymes steroid 17 alpha-hydroxylase (cytochrome P-450(17)alpha), steroid C-17,20-lyase, and possibly aromatase. We have investigated the activity levels of aromatase and 17 alpha-hydroxylase in placental microsomes in late pregnancy and dexamethasone-induced labor. Over the gestational period of 118-140 days basal levels of placental aromatase were relatively constant [mean value (+/- SD) of 5.6 +/- 1.6 pmol min-1 mg microsomal protein-1 (n = 10)]. Steroid 17 alpha-hydroxylase activity was undetectable [less than 0.5 pmol min-1 mg microsomal protein-1 (n = 7)]. In six animals in labor induced with infusion of dexamethasone into the fetus, placental aromatase activity had a mean value of 14.0 +/- 2.5 pmol min-1 mg protein-1; placental steroid 17 alpha-hydroxylase, measured in four of the animals, had a mean (+/- SD) activity of 319 +/- 58 pmol min-1 mg microsomal protein-1. Immunoblotting of placental microsomal preparations with specific antibodies to cytochrome P-450(17)alpha and NADPH-cytochrome P-450-reductase indicated that the glucocorticoid-induced activity of 17 alpha-hydroxylase was associated with increased content of cytochrome P-450(17)alpha. Northern blotting with a cDNA probe for cytochrome P-450(17)alpha showed that glucocorticoid increased the levels of mRNA for the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Regulation of heme and drug metabolism activities in the brain by manganese

A novel effect of metal ions in the brain is described. Mn was found to alter heme metabolism and the cytochrome P-450-dependent mixed-function oxidase activities in rat brain. A more than 2-fold increase in benzo(alpha)pyrene hydroxylase and 7-ethoxycoumarin deethylase activities were observed in the brain of rats treated for 7 days with Mn. The increases were regionally distributed; the highest elevations were observed in the hippocampus, pons and the caudate putamen. Moreover, in rats treated with Mn for 1 or 7 days a marked depression in the activity of the mitochondrial ALA synthetase was observed. The activity of the microsomal heme oxygenase was also inhibited at 7 days, but not 1 day, after Mn treatment. These inhibitions were reflected in an initial decrease, followed by a rebound return to normal, in the concentration of cytochrome P-450 in the brain. Mn was ineffective in vitro in altering heme and drug metabolism activities. It is suggested that Mn-mediated alterations in heme metabolic activities promote changes in the composition of cytochrome P-450 species in the brain microsomal fractions, such that the relative concentrations of the molecular species which catalyse aryl hydrocarbon hydroxylase activity become selectively increased.     

Danazol inhibits human adrenal 21-and 11β-hydroxylation invitro

The effects of danazol on steroidogenesis $\text{in}$$\text{vitro}$ in the 16–20 week old human fetal adrenal were examined by studying: 1) danazol binding to adrenal microsomal and mitochondrial cytochrome P-450, and 2) enzyme kinetics of danazol inhibition of the adrenal microsomal 21-hydroxylase and the mitochondrial llβ-hydroxylase. The addition of danazol to preparations of adrenal microsomes or mitochondria elicited a type I cytochrome P-450 binding spectrum. Danazol bound to microsomal cytochrome P-450 with a high affinity apparent spectral dissociation constant (Kg) of 1 μM and with a lower affinity K's of 10 μM. Danazol bound to mitochondrial cytochrome P-450 with a Kg of 5 μM. In addition, danazol competitively inhibited the microsomal 21-hydroxylase (apparent enzymatic inhibition constant K_I = 0.8 μM) and the mitochondrial 11β-hydroxylase (K_I = 3 μM). These findings demonstrate that low concentrations of danazol directly inhibit steroidogenesis in the human fetal adrenal $\text{in}$$\text{vitro}$.     

Purification and properties of 17 alpha-hydroxylase from microsomes of pig adrenal: a second C21 side-chain cleavage system

We have purified a cytochrome P-450 from microsomes of pig adrenal glands to homogeneity (11.1 n moles heme/mg protein) as demonstrated by electrophoresis on polyacrylamide gels with sodium dodecyl SO4 and by line of identity with an antibody using double diffusion. The enzyme shows both 17 alpha-hydroxylase and C17,20-lyase activities and therefore constitutes a C21 side-chain cleavage system like that previously purified in this laboratory from neonatal pig testis. Antibody to the testicular enzyme cross-reacts (line of identity) with both enzymes. It is concluded that the adrenal enzyme is the same or very similar to the testicular enzyme, that each enzyme possesses two enzymatic activities, and that microsomes provide some regulatory device to limit the lyase activity of the enzyme in vivo. No evidence was found for the usually accepted existence of an adrenal steroid 17 alpha-hydroxylase without lyase activity.     

Complete amino acid sequence of 21-hydroxylase cytochrome P-450 from porcine adrenal microsomes

Adrenal microsomal 21-hydroxylase is essential for biosynthesis or metabolism of various steroid hormones. The complete amino acid sequence of this cytochrome P-450 from pig adrenal was determined by sequence analysis on several sets of proteolytic fragments. The mature protein consists of 492 amino acid residues, corresponding to a molecular weight of 55,484. Seven out of nine total cysteine residues are localized in the amino terminal half of the molecule. The carboxyl terminal half contains only two cysteines, one of which is located at the highly conserved heme-binding region proposed in all cytochromes P-450. A structural comparison between 21-hydroxylase and 17 alpha-hydroxylase reveals that there is a preponderance of sequence homology at the carboxyl terminal region. These studies indicate that a single gene product is expressed for steroid 21-hydroxylase in porcine adrenal glands.     

Steroid interactions with cytochrome P-450 from testis microsomes

The cytochrome P-450 of gonadal microsomes is an integral component of the steroid converting enzymes, 17 alpha-hydroxylase and 17,20-lyase. Interaction of the steroid substrates with this cytochrome results in a shift in the Soret band as measured by difference spectroscopy. In these studies it is shown that in contrast to placental microsomal cytochrome P-450 which binds C19 steroids, testis microsomal cytochrome P-450 primarily binds C21 steroids. However, addition of a 17 alpha- methyl, 17 beta-acetate or a 17 beta-benzoate group to testosterone permits interaction. The addition of hydroxyl or methyl groups to other positions does not affect binding. The presence of multiple oxygen functions on C21 steroids, as in cortisol and corticosterone, precludes interaction. At least one oxygen function seems necessary for binding as 5 alpha- and 5 beta-pregnane do not bind whereas 20-deoxypregnenolone (5-pregnen-3 beta-ol) does bind. These findings indicate that factors in addition to hydrophobic interactions dictate the binding of steroid substrates to testis microsomal cytochrome P-450.     

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

Studies were carried out to investigate the effects of prostaglandins (PG) $\text{in vitro}$ on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE₁, PGE₂, PGA₁, PGF_1α or PGF_2α to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (ΔA_385–420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11β-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 cis-platinum on kidney cytochrome P-450 and heme metabolism: evidence for the regulatory role of the pituitary hormones

A novel action of the gonadotropic hormones of the adenohypophysis on the regulation of kidney heme metabolism and cytochrome P-450 concentrations is described. The treatment of rats with cis-platinum for 7 days caused a greater than twofold increase in the microsomal cytochrome P-450 and heme concentrations in the kidney. The sodium dodecyl sulfate-gel electrophoresis of the microsomal preparation revealed increased levels of both apocytochrome P-450 and heme in the molecular weight region corresponding to cytochrome P-450. In hypophysectomized rats, similar increases in heme and the cytochrome contents in the kidney were observed. Conversely, the treatment of rats with human chorionic gonadotropin (hCG) fully reversed the effect of cis-platinum on heme and cytochrome P-450 concentrations. The cellular basis of increases in concentrations of heme and the hemoprotein was explored by measuring the incorporation of [14C]glycine-labeled hemoglobin heme into the kidney microsomal heme fractions. In comparison with the control rats, the specific 14C activity of heme in microsomal fraction was not increased. Moreover, the effect of cis-platinum on kidney cytochrome P-450 appeared to be unrelated to alterations in the activities of the rate-limiting enzymes of heme biosynthesis and degradation pathways, delta-aminolevulinate synthetase, and heme oxygenase, respectively. On the other hand, ferrochelatase activity and the concentration of total porphyrins in the kidney were profoundly altered by cis-platinum treatment; a twofold increase in ferrochelatase activity and a marked reduction (40%) in the total porphyrin concentration were observed. Also, the activities of uroporphyrinogen-I synthetase and delta-aminolevulinate dehydratase were decreased in cis-platinum-treated animals. The latter effects reflect a direct inhibitory action of cis-platinum. It appears that the cis-platinum-mediated increase in the microsomal heme concentrations involves an accelerated rate of heme production as a consequence of increased ferrochelatase activity. This, in turn, could increase the production of cytochrome P-450. It is suggested that the anterior pituitary hormones control the concentration of the cytochrome P-450 in the kidney, and this process may be interrupted by cis-platinum.     

C21 steroid side chain cleavage enzyme from porcine adrenal microsomes. Purification and characterization of the 17 alpha-hydroxylase/C17,20-lyase cytochrome P-450

The properties and the purity of a cytochrome P-450 (17 alpha-hydroxylase) from porcine adrenal microsomes have been examined following a report that the corresponding enzyme from bovine adrenocortical microsomes is inactive as a 17 alpha-hydroxylase and fails to show a high spin spectrum on addition of substrate, once the enzyme has been purified (Bumpus, J. A., and Dus, K. M. (1982) J. Biol. Chem. 257, 12696-12704). The purity of the porcine enzyme was demonstrated by electrophoresis on polyacrylamide with sodium dodecyl sulfate, immunoelectrophoresis, and NH2-terminal amino acid sequence (16 residues). The pure enzyme shows Mr = 54,000, heme content of greater than 0.8 nmol/nmol of protein, and absorption spectra typical of cytochrome P-450. The enzyme is active with both delta 4 (progesterone) and delta 5 (pregnenolone) substrates as a 17 alpha-hydroxylase and with the corresponding 17 alpha-hydroxysteroids as a C17,20-lyase. All four substrates produce typical type I spectra with the enzyme (so-called high spin form). We conclude that: 1) porcine adrenal microsomes contain a 17 alpha-hydroxylase/C17,20-lyase which is a single protein molecule readily purified to an enzymatically active form; 2) the C17,20-lyase activity is largely suppressed in the microsomes; and 3) the enzyme closely resembles that found in testicular microsomes. We propose that this enzyme be referred to as the adrenal C21 steroid side chain cleavage enzyme.     

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

The effect of spironolactone administration on the content of adrenal microsomal cytochrome P-450 and on the activity of adrenal 17α-hydroxylase was examined in male cortisol and corticosterone-producing animals. Decreases in the content of microsomal cytochrome P-450 and in the activity of the 17α-hydroxylase after spironolactone treatment occur only in those animals which predominantly produce cortisol rather than corticosterone and which 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 to 80% loss of microsomal cytochrome P-450 with a concomitant decrease in the activity of the microsomal 17α-hydroxylase. In contrast to its effect in cortisol-producing animals, the administration of spironolactone caused either an increase or slight alteration in the concentration of adrenal microsomal cytochrome P-450 in corticosterone-producing animals such as the rat and the rabbit.     

Increased rat testicular heme oxygenase activity associated with depressed microsomal heme and cytochrome P-450 levels after repeated administration of human chorionic gonadotropin

Repeated administration of human chorionic gonadotropin to rats results in a maximal depression of testicular microsomal heme and cytochrome P-450 levels at 24 h, followed by increases that plateau at pretreatment levels by day six. Associated with the depressed levels of microsomal heme and cytochrome P-450 is an increase of testicular microsomal heme oxygenase activity at 12-24 h. Testicular mitochondrial delta-aminolevulinic acid synthase activity was increased at 24 h, and remained elevated throughout the 9-day treatment period. Pretreatment with 1,4,6-androstatrien-3,17-dione, an aromatase inhibitor, failed to prevent the depression of testicular microsomal heme or cytochrome P-450 or increased heme oxygenase activity caused by repeated administration of human chorionic gonadotropin, and administration of estradiol benzoate failed to alter testicular microsomal heme oxygenase activity suggesting that these parameters were not related to altered testicular estrogen content caused by increased aromatase activity. These results suggest that increased testicular heme oxygenase activity is associated with decreased microsomal heme and cytochrome P-450 content during human chorionic gonadotropin-induced desensitization.     

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.     

Changes in adrenocortical monooxygenase activities in alloxan-diabetic rabbits

Studies were carried out to determine if diabetes mellitus influenced the activities of adrenal steroidogenic enzymes. Adult male rabbits were made diabetic by an i.v. infusion of alloxan (100 mg/kg) and were killed 1 or 2 months later. Mitochondrial cytochrome P-450 concentrations were not affected by diabetes but steroid 11 beta-hydroxylase activity was greater in the diabetics than in controls after both 1 and 2 months. The type I spectral change produced by 11-deoxycorticosterone, the substrate for 11 beta-hydroxylation, was also greater in mitochondria from diabetics. By contrast, mitochondrial cholesterol side-chain cleavage activity was similar in controls and diabetics. Microsomal cytochrome P-450 concentrations were unaffected by diabetes but 21-hydroxylase activity was significantly lower in adrenal microsomes from diabetics than from controls. The results indicate that alloxan-induced diabetes alters adrenocortical steroid metabolism which may contribute to changes in the pattern of steroid secretion noted by other investigators.