Metabolism of progesterone to DOC, corticosterone and 18OHDOC in cultured human melanoma cells.

We are now showing that cultured human melanoma cells can synthesize steroids such as corticosterone from progesterone or deoxycorticosterone. Corticosterone production is strongly responsive to deoxycorticosterone substrate addition (12-fold increase), but unresponsive to the adrenal stimulating factors ACTH and angiotensin II. This is the first demonstration that skin cells (malignant melanocytes) have the capability to synthesize 11-deoxycorticosterone, corticosterone, and 18-hydroxydeoxycorticosterone.     

Inhibition of hamster adrenal 11 beta/19-hydroxylase activity

Increased mineralocorticoid activity has been associated with elevated urinary levels of 19-nordeoxycorticosterone in several forms of experimental and human hypertension. Biosynthesis of 19-norsteroids involves hydroxylation of the C-19 methyl group. We synthesized the 4-hydroxy analogs of deoxycorticosterone, deoxycorticosterone acetate, progesterone, and androstenedione and evaluated them as inhibitors of deoxycorticosterone 11 beta/19-hydroxylase using hamster adrenal mitochondrial preparations. These 4-hydroxy analogs were inhibitors of this P 450 hydroxylase, with approximately 10 times weaker affinity than their respective natural substrates. 4-Hydroxydeoxycorticosterone was the most potent inhibitor evaluated in this study. The half-maximal inhibitory concentration of deoxycorticosterone hydroxylation was 5 microM, 15 microM, more than 50 microM, and 14 microM, respectively, for the above compounds.     

The influence of streptozotocin diabetes on adrenal function in male rats.

Male Wistar rats were treated with an i.v. dose of 100 mg/kg of Streptozotocin (STZ). Either 5 days or 1, 2 or 3 months after induction of diabetes, the adrenal function of these animals was studied. Short course diabetes (5 days) was accompanied by adrenal hypertrophy and high plasma corticosterone levels; during later periods the diabetic rats consistenly showed signs of adrenal hyperactivity, yet both adrenal weight and plasma corticosterone tended to be lower than in the 5 day-treated animals. Adrenal incubations with 14C-progesterone showed that 5 days and one month diabetic animals synthesized more deoxycorticosterone than controls; production of corticosterone and 18-hydroxydeoxycorticosterone was normal at all time periods studied. Synthesis of 18-hydroxycorticosterone, a compound which affects sodium metabolism, was increased in 5 day-treated rats; thereafter, the function of the zona glomerulosa seemed to be impaired in diabetic rats. These results suggest that early after induction of diabetes there is adrenal hyperfunction of the mixed type (i.e. gluco and mineralcorticoid), and that in the later periods (2-3 months), the deranged metabolism of the diabetic rat acts as a chronic stress.     

Trend analysis of serum progesterone, deoxycorticosterone, deoxycorticosterone sulfate, cortisol, corticosterone, 18-hydroxydeoxycorticosterone and estradiol in early neonates

To elucidate the origin and regulatory mechanism of deoxycorticosterone (DOC) and deoxycorticosterone sulfate during fetal life, the levels of serum DOC, DOC sulfate, progesterone, cortisol, corticosterone and 18-hydroxydeoxycorticosterone (18OH-DOC) were determined in the fraction separated on high performance liquid chromatogram (HPLC) by radioimmunoassay (RIA) using the serum from normal newborn. Elimination curves both of serum DOC and DOC sulfate showed two phases: rapidly decreasing and slowly decreasing ones. Both serum DOC and DOC sulfate correlated with progesterone (r = 0.340, p less than 0.01; r = 0.737, p less than 0.01, respectively). They also correlated with cortisol (DOC, r = 0.467, p less than 0.01; DOC sulfate, r = 0.549, p less than 0.01, respectively). Serum DOC reached normal adult levels by 16 hrs after birth. However serum DOC sulfate concentration was maintained high throughout the entire early neonatal period. On the contrary, the changes in serum cortisol, corticosterone and 18OH-DOC showed a peak surge in the initial phase after delivery. Both serum corticosterone and 18OH-DOC correlated with cortisol (r = 0.518, p less than 0.01; r = 0.410, p less than 0.01, respectively). These findings suggest that, in the fetus, serum DOC and DOC sulfate are mainly produced at extraadrenal sites isolated from normal mineralocorticoids synthesis and after birth they begin to be formed at adrenal glands.     

Isolation of aldosterone synthase cytochrome P-450 from zona glomerulosa mitochondria of rat adrenal cortex

A cytochrome P-450 capable of producing aldosterone from 11-deoxycorticosterone was purified from the zona glomerulosa of rat adrenal cortex. The enzyme was present in the mitochondria of the zona glomerulosa obtained from sodium-depleted and potassium-repleted rats but scarcely detected in those from untreated rats. It was undetectable in the mitochondria of other zones of the adrenal cortex from both the treated and untreated rats. The cytochrome P-450 was distinguishable from cytochrome P-45011 beta purified from the zonae fasciculata-reticularis mitochondria of the same rats. Molecular weights of the former and the latter cytochromes P-450, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 49,500 and 51,500, respectively, and their amino acid sequences up to the 20th residue from the N terminus were different from each other at least in one position. The former catalyzed the multihydroxylation reactions of 11-deoxycorticosterone giving corticosterone, 18-hydroxydeoxycorticosterone, 18-hydroxycorticosterone, and a significant amount of aldosterone as products. On the other hand, the latter catalyzed only 11 beta- and 18-hydroxylation reactions of the same substrate to yield either corticosterone or 18-hydroxydeoxycorticosterone. Thus, at least two forms of cytochrome P-450, which catalyze the 11 beta- and 18-hydroxylations of deoxycorticosterone, exist in rat adrenal cortex, but aldosterone synthesis is catalyzed only by the one present in the zona glomerulosa mitochondria.     

Properties of an adrenal cytochrome P-450 (P-45011β) for the hydroxylations of corticosteroids

A highly purified preparation of cytochrome P-450, designated as P-450_11β, has been obtained from bovine adrenal cortex mitochondria. The P-450_11β exhibits remarkably high steroid hydroxylase activity in the reconstituted adrenal electron-donating system from NADPH via NADPH:adrenal ferredoxin oxidoreductase (EC 1.6.7.1) and adrenal ferredoxin. The turnover numbers (moles of hydroxylated product formed per minute per mole of P-450-heme) are 110 and 18 for respective 11β- and 18-hydroxylase activity when deoxycorticosterone is the substrate. The apparent K_m value is 6 μm for both reactions. The ratio, about 6:1 between the two activities, is constant under various experimental conditions including those in the presence of competitive inhibitors of hydroxylation. In addition to deoxycorticosterone, other steroids such as 11-deoxycortisol, 4-androstene-3,17-dione and testosterone are the hydroxylatable substrates. In cases in which 4-androstene-3,17-dione, a C₁₉-steroid, is the substrate, the hydroxylatable sites appear to be its respective 11β- and 19-position. The ratio between the two activities is about 4:1. In view of these results, it is concluded that one hemoprotein species, the P-450_11β, is responsible for the hydroxylase reactions of various Corticosteroids. 2-Methyl-1,2-di-3-pyridyl-1-propanone (metyrapone) inhibits the P-450_11β-catalyzed steroid hydroxylase reactions of either deoxycorticosterone at 11β- and 18-position or 4-androstene-3,17-dione at 11β- and 19-position (K_i = 0.1-0.2 μM). The P-450_scc-catalyzed cholesterol desmolase reaction is also inhibited, although weakly (K_i = 160 μM). In addition, both adrenal cytochromes appeared to differ from each other in spectral response to metyrapone.     

P-45011 beta-dependent conversion of cortisol to cortisone, and 19-hydroxyandrostenedione to 19-oxoandrostenedione

Purified bovine adrenal P-45011 beta has been shown to catalyze conversions of cortisol to cortisone (11-oxidase activity), and 19-hydroxyandrostenedione to 19-oxoandrostenedione (19-oxidase activity), in the reconstituted system consisting of NADPH, NADPH:adrenodoxin reductase, and adrenodoxin. The turnover numbers (mol of product formed/min/mol of P-450) were 1.2 for the 11-oxidase activity and 1.4 for the 19-oxidase activity. No reactions took place when any one of the electron-donating components were omitted either in the presence or in the absence of added NADP+. Likewise, rabbit antibody prepared against P-45011 beta immunoprecipitated the 11-oxidase activity with concomitant loss of deoxycorticosterone 11 beta-hydroxylase activity.     

Serum 11-deoxycorticosterone levels in adrenal-regeneration hypertension under conditions of quiescence and stress.

A time course study to measure adrenal cortical function was undertaken for the period prior to the development of hypertension until the onset of hypertension in the adrenal-regeneration hypertension (ARH) model. Quiescent rat kills were used so that all adrenal cortical parameters investigated would reflect basal or resting levels for controls. Thus a more accurate determination of the differences between control and experimental animals could be made. A radioimmunoassay procedure for deoxycorticosterone was developed to measure this steroid in individual rat serum samples. Elevated serum deoxycorticosterone levels were observed in rats with regenerating adrenals when they were killed under quiescent conditions. This agreed with our recently reported in vitro finding of restoration of cholesterol side chain cleavage activity while 11beta-hydroxylase activity remained imparied 25 days after adrenal enucleation. When rats were killed after ether stress, deoxycorticosterone levels were elevated in both control rats and in rats with regenerating adrenals but the difference was not significant. In contrast, after ether stress serum corticosterone levels were lower in rats with regenerating adrenals than in controls. These studies, in conjunction with our previous in vitro findings, point to the importance of deoxycorticosterone in the pathogenesis of adrenal regeneration hypertension and help to explain the anomalous corticosteroid secretion rate data found in this experimental hypertension model.     

Selective 19-hydroxylase inhibition by an aromatase inhibitor, 4-hydroxyandrostenedione

19-Nor-deoxycorticosterone is a newly recognized mineralocorticoid which has been associated with some forms of genetic, experimental, and human hypertension. To further examine this relationship, specific inhibitors of 19-nor-deoxycorticosterone biosynthesis must be developed. Since 19-hydroxylation is the pivotal step in both 19-nor-deoxycorticosterone biosynthesis and aromatization of androgens to estrogens, we evaluated an aromatase inhibitor, 4-hydroxyandrost-4-ene-3,17-dione on the inhibition of 19-hydroxylation in both rat and human adrenal mitochondria in vitro and 19-nor-deoxycorticosterone production and blood pressure in spontaneously hypertensive rats in vivo. Adrenal mitochondria from 48 male Sprague-Dawley rats and 1 patient with an aldosterone-producing adenoma were incubated in the presence of deoxycorticosterone substrate both with and without 4-hydroxyandrost-4-ene-3,17-dione. 4-Hydroxyandrost-4-ene-3,17-dione produced significant inhibition of 19-hydroxy-deoxycorticosterone production in both rat and human adrenal mitochondria, with a smaller and not significant inhibition of corticosterone and 18-hydroxy-corticosterone. 4-Hydroxyandrost-4-ene-3,17-dione given subcutaneously to spontaneously hypertensive rats lowered 19-nor-deoxycorticosterone by 69% and completely abolished hypertension compared to Wistar-Kyoto controls. These data demonstrate that 4-hydroxyandrost-4-ene-3,17-dione is a specific inhibitor of 19-hydroxylase, that it lowers 19-nor-deoxycorticosterone production and prevents hypertension in the spontaneously hypertensive rat. These studies reinforce the possible pathogenic significance of 19-nor-deoxycorticosterone in hypertension in spontaneously hypertensive rats.     

Cytochrome P-450 from mitochondria of bovine adrenal cortex: comparison of cholesterol side-chain cleavage P-450 with steroid 11beta-hydroxylation P-450 and immunochemical cross-reactivity between adrenal mitochondrial and liver microsomal cytochromes P-450.

Adrenocortical mitochondrial cytochrome P-450 specific to the cholesterol side-chain cleavage (desmolase) reaction differs from that for the 11beta-hydroxylation reaction of deoxycorticosterone. The former cytochrome appears to be more loosely bound to the inner membrane than the latter. Upon ageing at 0 degrees C or by aerobic treatment with ferrous ions, the desmolase P-450 was more stable than the 11beta-hydroxylase P-450. By utilizing artificial hydroxylating agents such as cumene hydroperoxide, H2O2, and sodium periodate, the hydroxylation reaction of deoxycorticosterone to corticosterone in the absence of NADPH was observed to a comparable extent with the reaction in the presence of adrenodoxin reductase, adrenodoxin and NADPH. However, the hydroxylation reaction of cholesterol to pregnenolone was not supported by these artificial agents. Immunochemical cross-reactivity of bovine adrenal desmolase P-450 with rabbit liver microsomal P-450LM4 was also investigated. We found a weak but significant cross-reactivity between the adrenal mitochondrial P-450 and liver microsomal P-450LM4, indicating to some extent a homology between adrenal and liver cytochromes P-450.     

19-Hydroxylated steroids, new metabolites produced by the Y1 adrenal cell line

The expression of 19-hydroxylase activity in the Y1 adrenal cell line is reported here for the first time. Two new metabolites from the incubation of deoxycorticosterone (DOC) with these cells, 19-hydroxy-20 alpha-dihydroDOC and 19-hydroxy-20 alpha-dihydrocorticosterone, have been identified. The most important of the two is the 11 beta,19-dihydroxylated metabolite, which is produced in smaller amounts than 18-hydroxy-20 alpha-dihydrocorticosterone. A third 19-hydroxylated metabolite was identified as 19-hydroxy-20 alpha-dihydroprogesterone, produced from the cholesterol in the serum supplemented medium. These results show that the cytochrome P-450(11)beta of this cell line expresses 19-hydroxylase activity in addition to 11 beta- and 18-hydroxylase activities, as do those of other species.     

19-Hydroxylation of 18-hydroxy-11-deoxycorticosterone by adrenal mitochondria prepared from various animal species

We have recently reported that bovine adrenocortical cytochrome P-45011 beta catalyzes 19-hydroxylation of 18-hydroxy-11-deoxycorticosterone (18(OH)DOC) in addition to 11 beta-hydroxylation of the steroid. In this report, we examine the presence of these two activities in 18(OH)DOC and 11 beta- and 18-hydroxylation activities on deoxycorticosterone (DOC) among the adrenal mitochondria prepared from man, ox, pig, rabbit, guinea-pig and rat. The results indicate that these animals could be classified into three groups with respect of these hydroxylation activities. Mitochondria of the first group comprising ox and pig showed rather high 19- and 11 beta-hydroxylation activities on 18(OH)DOC compared to the hydroxylation activities on DOC. Mitochondria prepared from the second group which comprised rabbit, guinea-pig and man showed low 19-hydroxylation activity on 18(OH)DOC, whereas the 11 beta-hydroxylation of 18(OH)DOC well occurred in these species. The last group comprising rat had very low activity both of 11 beta- and 19-hydroxylations when 18(OH)DOC was used as the substrate, whereas both 11 beta- and 18-hydroxylations of DOC were high in rat adrenal mitochondria. No significant difference of these activities could be found between zona glomerulosa cells and zonae fasciculata-reticularis cells of bovine adrenal cortex, and between adrenal mitochondria from spontaneously hypertensive rat and those from WKY normotensive rat.     

Novel Endocrine Disrupter Effects of Classic and Atypical Antipsychotic Agents and Divalproex: Induction of Adrenal Hyperandrogenism,Reversible with Metformin or Rosiglitazone

ObjectiveTo ascertain an association between the a priori known insulin resistance caused by antipsychotic agents and divalproex and adrenal hyperandrogenism and to determine whether the associated hyperandrogenism is reversible with insulin sensitizers.MethodsWe studied 26 consecutive psychiatric inpatients (22 women and 4 men) receiving the aforementioned medications, who were referred to us for a consultation. They ranged in age from 19 to 79 years and had a mean body mass index (SEM) of 32.35 ± 1.26 kg/m². Between 8 AM and 9 AM, blood samples were collected for 17-hydroxyprogesterone, 17-hydroxypregnenolone, androstenedione, dehydroepiandrosterone (DHEA), DHEA sulfate, 11-deoxycortisol, luteinizing hormone and follicle-stimulating hormone (in reproductive age women), estrone, estradiol (in reproductive age women), free testosterone (in women), deoxycorticosterone, and sex hormone-binding globulin (SHBG), which were measured by radioimmunoassay, after chromatography if necessary. For intact, premenopausal women, measurement of the abnormal steroid metabolite or SHBG level was repeated during prednisone therapy (5 mg at bedtime) to document the likely adrenal origin of the abnormality. Men, women who had undergone bilateral oophorectomy, and postmenopausal women had hyperandrogenism of adrenal origin by default. Clinical features included central obesity, acanthosis, hirsutism, alopecia, type 2 diabetes mellitus, and oligomenorrhea.ResultsWe found reversed estrone/estradiol ratios in 4 patients, decreased SHBG in 4, increased 17-hydroxypregnenolone in 8, increased 17-hydroxyprogesterone in 2, increased deoxycorticosterone in 2, increased DHEA sulfate in 1, increased 11-deoxycortisol in 4, increased androstenedione in 1, and reversed ratios of luteinizing hormone to follicle-stimulating hormone in 2. The biochemical abnormalities were corrected in 8 of 8 patients receiving metformin and in 2 of 2 patients receiving rosiglitazone.ConclusionInsulin resistance caused by antipsychotic agents and divalproex is associated with adrenal hyperandrogenism. Metformin and rosiglitazone correct the biochemical abnormalities detected without compromising their psychotropic effect. Adrenal androgen synthesis may be increased by hyperinsulinemia-induced hyperphosphorylation of P450c17α, resulting in an increase in its 17,20-lyase activity, which magnifies the effects of any distal steroidogenic enzyme defects. Treatment with metformin or rosiglitazone prevents excess adrenal androgen synthesis. (Endocr Pract. 2007; 13:601-608)     

Transaminative pathway of glutamate oxidation in adrenal-cortex mitochondria

Mitochondria isolated from adrenal cortex of beef do oxidize glutamate if the amino group acceptor-oxaloacetate (or its precursor-malate) is present in the incubation medium. The glutamate (plus oxaloacetate) oxidation was enhanced by ADP or deoxycorticosterone, indicating that this respiration can support both oxidative phosphorylation and 11 beta-hydroxylation of deoxycorticosterone to corticosterone. Avenaciolide (inhibitor of glutamate entry into the mitochondria), aminooxyacetate (inhibitor of aspartate aminotransferase activity) and arsenite (inhibitor of 2-oxoglutarate dehydrogenase) when introduced into the incubation media before respirating substrates, inhibited the ability of ADP or deoxycorticosterone to stimulate the rate of glutamate (plus oxaloacetate) oxidation.     

Adrenal P-450scc catalyzes deoxycorticosterone 6 beta-hydroxylase reaction

Purified bovine P-450scc, the cholesterol side-chain cleaving P-450 in adrenal cortex mitochondria, was found to catalyze a deoxycorticosterone 6 beta-hydroxylase reaction. A turnover number (moles of product formed/min/mol of P-450) of 12 was found similar to that for cholesterol side chain cleavage activity. Conversion was dose-dependent in terms of P-450scc and no reaction took place when any one of the required electron donating components such as NADPH, NADPH-adrenodoxin reductase, or adrenodoxin was omitted. These results confirm and extend earlier observations that 21-hydroxypregnenolone is transformed into both deoxycorticosterone and 6 beta-hydroxydeoxycorticosterone by incubation of adrenal gland slices.     

The effects of adrenal hormones, endotoxin and turpentine on serum components of the plaice (Pleuronectes platessa L.)

1. Within 24 hr of injection into plaice, cortisol, deoxycorticosterone, adrenalin or endotoxin cause an increase (P less than 0.001) in circulating C-reactive protein (CRP). Turpentine and soluble dexamethasone have no effect. 2. The increase in CRP with endotoxin is not enhanced with adrenalin or deoxycorticosterone, and in conjunction with cortisol the increase is additive. 3. Changes in CRP are independent of the amounts of serum amyloid P-component or total protein. 4. Turpentine, cortisol and adrenalin cause a rapid increase in circulating glucose. 5. It is concluded that some adrenal hormones stimulate the CRP acute phase response in plaice, without an apparent provoking agent.     

Corticosteroid regulation of gonadotropin secretion and induction of ovulation in the rat

In the human polycystic ovarian syndrome, glucocorticoids have been demonstrated to have beneficial effects in inducing ovulation in a number of cases. These beneficial effects were assumed to be due to suppression of adrenal overproduction of androgens. However, the possibility exists that glucocorticoids may directly regulate gonadotropin secretion and thereby improve menstrual rhythm and ovulatory activity. Herein, we report that the corticoid, deoxycorticosterone, and the synthetic glucocorticoid, triamcinolone acetonide, like progesterone (P4), are able to induce luteinizing hormone and follicle-stimulating hormone surges and facilitate ovulation in the pregnant mare serum gonadotropin-primed rat. This effect is not shared by cortisol. Prolactin release was also stimulated by deoxycorticosterone, cortisol, and progesterone, but not by triamcinolone acetonide. Similar to progesterone, triamcinolone acetonide and deoxycorticosterone administration caused a loss of fluid retention in the uterus. This effect of triamcinolone acetonide and deoxycorticosterone may be related to progesterone action as opposed to anti-inflammatory action since cortisol had no effect on uterine fluid retention. These findings raise the possibility that the beneficial effects seen with glucocorticoids in inducing ovulation in polycystic ovarian syndrome may be due in part to their direct effects upon the release of gonadotropins.     

Common characteristics of the cytochrome P-450 system involved in 18- and 11 beta-hydroxylation of deoxycorticosterone in rat adrenals.

18- and 11beta-Hydroxylation of deoxycorticosterone and side chain cleavage of cholesterol were studied in mitochondria and submitochondrial reconstituted systems prepared from rat and bovine adrenals. A mass fragmentographic technique was used that allows determination of hydroxylation of both exogenous and endogenous cholesterol. The following results were obtained. (1) Treatment of rats with excess potassium chloride in drinking fluid increased mitochondrial cytochrome P-450 as well as 18- and 11beta-hydroxylase activity in the adrenals. Cholesterol side chain cleavage was not affected. In the presence of excess adrenodoxin and adrenodoxin reductase, cytochrome P-450 isolated from potassium chloride-treated rats had higher 18- and 11beta-hydroxylase activity per nmol than cytochrome P-450 isolated from control rats. The stimulatory effects on 18- and 11beta-hydroxylation were of similar magnitude. (2) Long-term treatment with ACTH increased cholesterol side chain cleavage in the adrenals but had no effect on 18- and 11beta-hydroxylase activity. The amount of cytochrome P-450 in the adrenals was not affected by the treatment. It was shown with isolated mitochondrial cytochrome P-450 in the presence of excess adrenodoxin and adrenodoxin reductase that the effect of ACTH was due to increase of side chain cleavage activity per nmol cytochrome P-450. Side chain cleavage of exogenous cholesterol was affected more than that of endogenous cholesterol. (3) Gel chromatography of soluble cytochrome P-450 prepared from rat and bovine adrenal mitochondria yielded chromatographic fractions having either a high 18- and 11beta-hydroxylase activity and a low cholesterol side chain cleavage activity or the reverse. The ratio between 18- and 11beta-hydroxylase activity was approximately constant, provided the origin of cytochrome P-450 was the same. (4) Addition of progesterone to incubations of deoxycorticosterone with soluble or insoluble rat adrenal cytochrome P-450 competitively inhibited 18- and 11beta-hydroxylation of deoxycorticosterone to the same degree. Addition of deoxycorticosterone competitively inhibited 11beta-hydroxylation of progesterone with the same system. Progesterone was not 18-hydroxylated by the system. From the results obtained, it is concluded that 18- and 11beta-hydroxylation have similar properties and that the binding site for deoxycorticosterone is similar or identical in the two hydroxylations. The possibility that the same specific type of cytochrome P-450 is responsible for both 18- and 11beta-hydroxylation of deoxycorticosterone is discussed.     

Conversion of progesterone to corticosteroids by the midterm fetal adrenal and kidney

Midterm fetal adrenal and kidney tissue homogenates were incubated with 3H-progesterone (1 microM) and its conversion to te 3H-corticosteroids metabolites studied. Cortisol (36.3%) and corticosterone (4.7%) were isolated from the adrenal, and 11-deoxycortisol (32.5%) and deoxycorticosterone (21.1%) from the kidney. The results of these incubations confirmed the presence of 17- and 21-hydroxylase activities in both fetal tissues, and that of 11 beta-hydroxylase activity only in fetal adrenal tissue. We conclude that during pregnancy when progesterone levels are high, biosynthesis by the fetal kidney of 11-deoxycortisol, the most abundant corticosteroid formed by this tissue in this investigation, might provide to the fetal adrenal an important precursor for cortisol biosynthesis within the fetal compartment.