Xenopus laevis CYP17 regulates androgen biosynthesis independent of the cofactor cytochrome b5

The enzyme CYP17 primarily regulates androgen production by mediating four reactions: conversion of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, respectively (17alpha-hydroxylase activity), followed by conversion of the 17-hydroxylated steroids to dehydroepiandrosterone and androstenedione, respectively (17,20-lyase activity). Most mammalian CYP17 isoforms have high 17alpha-hydroxylase relative to 17,20-lyase activities and preferentially mediate one of the two 17,20-lyase reactions. In contrast, Xenopus laevis CYP17 potently regulates all four reactions in the frog ovary. CYP17 isoforms generally rely on the cofactor cytochrome b(5) for the 17,20-lyase reaction, suggesting that the high lyase activity of Xenopus CYP17 might be due to a lesser dependence on b(5). The kinetics of Xenopus CYP17 expressed in yeast microsomes were therefore examined in the absence and presence of Xenopus on human b(5). Xenopus CYP17 mediated both 17,20-lyase reactions in the absence of b(5), confirming that the activity did not require b(5). However, both Xenopus and human b(5) slightly enhanced Xenopus CYP17-mediated lyase activity, indicating that the enzyme was still at least partially responsive to b(5). Surprisingly, only the human b(5) cofactor enhanced human CYP17-mediated lyase activity, implying that the human enzyme had more specific cofactor requirements than Xenopus CYP17. Studies using human/Xenopus chimeric b(5) proteins revealed that human b(5) residues 16-41 were important for the specific regulation of the lyase activity of HuCYP17, possibly serving as an interacting domain with the enzyme. CYP17 may therefore have evolved from a general producer of sex steroids in lower vertebrates to a more tightly regulated producer of both sex steroids and glucocorticoids in mammals.     

Cytochrome b5 augments 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase activity

During adrenal steroidogenesis the competition between 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3βHSD) and cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17A1) for Δ(5) steroid intermediates greatly influences steroidogenic output. Cytochrome-b(5) (Cyt-b(5)), a small electron transfer hemoprotein, known to augment the lyase activity of CYP17A1, has been shown to alter the steroidogenic outcome of this competition. In this study, the influence of Cyt-b(5) on 3βHSD activity was investigated. In COS-1 cells, Cyt-b(5) was shown to significantly increase the activity of both caprine and ovine 3βHSD towards pregnenolone, 17-OH pregnenolone and dehydroepiandrosterone in a substrate and species specific manner. Furthermore, kinetic studies revealed Cyt-b(5) to have no influence on the K(m) values while significantly increasing the V(max) values of ovine 3βHSD for all its respective substrates. In addition, the activity of ovine 3βHSD in microsomal preparations was significantly influenced by the addition of either purified Cyt-b(5) or anti-Cyt-b(5) IgG. The results presented in this study indicate that Cyt-b(5) augments 3βHSD activity and represents the first documentation of such augmentation in any species.     

A paradox: elevated 21-hydroxypregnenolone production in newborns with 21-hydroxylase deficiency

21-Hydroxypregnenolone and its metabolite 5-pregnene-3 beta, 20 alpha 21-triol have been measured in the sulfate fraction of neonatal urine. These two steroids are the major two 21-hydroxylated 5-pregnenes produced by neonates and are almost exclusively excreted as disulfates. The excretions of these steroids by normal infants and infants with 21-hydroxylase deficiency were compared. In addition to measurement of the absolute excretion, the excretion relative to the total 3 beta-hydroxy-5-ene output was also determined. The results show that 21-hydroxypregnenolone excretion is highly elevated in 21-hydroxylase deficiency (affected, mean 887 micrograms/24 h, range 453-1431 micrograms/24 h; normal, mean 117 micrograms/24 h, range 17-263 micrograms/24 h), but when compared to excretion of other delta 5 steroids the excretion is slightly low [(21-hydroxypregnenolone + 5-pregnene-3 beta, 20 alpha, 21-triol)/total 3-beta-hydroxy-5-ene steroids, 2.9% affected; 3.6% normal]. This difference was not statistically significant. There is thus no evidence that the 21-hydroxylase acting on pregnenolone is deficient in congenital adrenal hyperplasia. The explanation of the normal activity of "pregnenolone 21-hydroxylase," although not clearly defined, is probably associated with two recent findings by other workers: (a) that the human fetus has an active 21-hydroxylase distinct from the adrenal enzyme and (b) that a 21-hydroxylase structurally very different from the adrenal enzyme, with high activity towards pregnenolone (but no activity towards 17-hydroxyprogesterone), has been isolated from rabbit hepatic microsomes.     

Intracellular distributian and substrate specificity of the 16 alpha-hydroxylase in the adrenal of human fetus

When [7α-³H] dehydroepiandrosterone was incubated with the adrenal homogenates of human fetus at 22 to 26 weeks gestational age, 16α-hydroxydehydroepiandrosterone and/or its sulfate was formed as the only detectable metabolite. The 16α-hydroxylase activity was concentrated in the microsomal fraction of the adrenal homogenate.[1,2-³H]Androstenedione, [4-¹⁴C] pregnenolone and [7α-³H] progesterone were also 16α-hydroxylated by incubation with the microsomal fraction. Amoung these substrates, progesterone gave the highest yield of 16α-hydroxylated products. By incubation with the microsomal fraction, formation of following steroids were also established: 6β-hydroxyandrostenedione from androstenedione; 17-hydroxypregnenolone, 17,21-dihydroxypregnenolone and dehydroepiandrosterone from pregnenolone; 17-hydroxy-progesterone, deoxycorticosterone, 11-deoxycortisol and androstenedione from progesterone.     

The role of porcine cytochrome b5A and cytochrome b5B in the regulation of cytochrome P45017A1 activities

Male pigs are routinely castrated to prevent the accumulation of testicular 16-androstene steroids, in particular 5α-androst-16-en-3-one (5α-androstenone), which contribute to an off-odour and off-flavour known as boar taint. Cytochrome P450C17 (CYP17A1) catalyses the key regulatory step in the formation of the 16-androstene steroids from pregnenolone by the andien-β synthase reaction or the synthesis of the glucocorticoid and sex steroids via 17α-hydroxylase and C17,20 lyase pathways respectively. We have expressed CYP17A1, along with cytochrome P450 reductase (POR), cytochrome b5 reductase (CYB5R3) and cytochrome b5 (CYB5) in HEK-293FT cells to investigate the importance of the two forms of porcine CYB5, CYB5A and CYB5B, in both the andien-β synthase as well as the 17α-hydroxylase and C17,20 lyase reactions. Increasing the ratio of CYB5A to CYP17A1 caused a decrease in 17α-hydroxylase (p < 0.013), a transient increase in C17,20 lyase, and an increase in andien-β synthase activity (p < 0.0001). Increasing the ratio of CYB5B to CYP17A1 also decreased 17α-hydroxylase, but did not affect the andien-β synthase activity; however, the C17,20 lyase, was significantly increased. These results demonstrate the differential effects of two forms of CYB5 on the three activities of porcine CYP17A1 and show that CYB5B does not stimulate the andien-β synthase activity of CYP17A1.     

Cytochrome b(5) forms homomeric complexes in living cells

Cytochrome b(5) (cyt-b(5)) is a ubiquitous hemoprotein also associated with microsomal cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17A1). In the steroidogenic pathway CYP17A1 catalyses the metabolism of pregnenolone, yielding both glucocorticoid and androgen precursors. While not affecting the 17α-hydroxylation of pregnenolone, cyt-b(5) augments the 17,20 lyase reaction of 17-hydroxypregnenolone, catalyzing the formation of DHEA, through direct protein-protein interactions. In this study, multimeric complex formation of cyt-b(5) and the possible regulatory role of these complexes were investigated. Cyt-b(5) was isolated from ovine liver and used to raise anti-sheep cyt-b(5) immunoglobulins. Immunochemical studies revealed that, in vivo, cyt-b(5) is primarily found in the tetrameric form. Subsequent fluorescent resonance energy transfer (FRET) studies in COS-1 cells confirmed the formation of homomeric complexes by cyt-b(5) in live cells. Site-directed mutagenesis revealed that the C-terminal linker domain of cyt-b(5) is vital for complex formation. The 17,20-lyase activity of CYP17 was augmented by truncated cyt-b(5), which is unable to form complexes when co-expressed in COS-1 cells, thereby implicating the monomeric form of cyt-b(5) as the active species. This study has shown for the first time that cyt-b(5) forms homomeric complexes in vivo, implicating complex formation as a possible regulatory mechanism in steroidogenesis.     

Phospholipases modulate the rat testicular androgen biosynthetic pathway in vitro

The role of membrane phospholipids in testicular androgen biosynthesis was investigated by monitoring the effects of phospholipase treatments on the activities of the steroid transforming enzymes. Androgen biosynthesis in untreated rat testicular microsomes was examined by monitoring the temporal appearance of pregnenolone metabolites and was found to proceed through the 4-ene route. When phospholipase A2 was included, the 5-ene steroids 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA) were formed in greater quantities, and the production of 4-ene steroids was reduced indicating that the conversion of 5-ene steroids to the 4-ene configuration was inhibited by phospholipase A2 treatment. Phospholipase C, in addition to inhibiting this step, also inhibited the conversion of C21 steroids to C19 steroids. When the enzymatic steps were measured individually, phospholipase A2 inhibited 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-Isomerase) with an ED50 of 73 mU/ml but had no effect on the activities of 17-hydroxylase, C-17, 20 lyase, or 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD). However, though phospholipase C treatment inhibited 3 beta-HSD-Isomerase, it caused less inhibition (the ED50 value was 149 mU/ml). Furthermore, 17-hydroxylase and C-17, 20 lyase activities were also inhibited by phospholipase C treatment (ED50 values were 410 and 343 mU/ml, respectively), but no effect on 17 beta-HSD was observed. The differences in the apparent phospholipid requirements of the steroidogenic enzymes provides the possibility that the metabolic fate of pregnenolone may be regulated by changes in the phospholipid composition of the microenvironment.     

Baboon cytochrome P450 17alpha-hydroxylase/17,20-lyase (CYP17).

Human cytochrome P450 17alpha-hydroxylase (CYP17) catalyses not only the 17alpha-hydroxlation of pregnenolone and progesterone and the C17,20-side chain cleavage (lyase) of 17alpha-hydroxypregnenolone, necessary for the biosynthesis of C21-glucocorticoids and C19-androgens, but also catalyses the 16alpha-hydroxylation of progesterone. In efforts to understand the complex enzymology of CYP17, structure/function relationships have been reported previously after expressing recombinant DNAs, encoding CYP17 from various species, in nonsteroidogenic mammalian or yeast cells. A major difference between species resides in the lyase activity towards the hydroxylated intermediates and in the fact that the secretion of C19-steroids take place, in some species, principally in the gonads. Because human and higher primate adrenals secrete steroids, CYP17 has been characterized in the Cape baboon, a species more closely related to humans, in an effort to gain a further understanding of the reactions catalysed by CYP17. Baboon and human CYP17 cDNA share 96% homology. Baboon CYP17 has apparent Km and V values for pregnenolone and progesterone of 0.9 micro m and 0.4 nmol.h-1.mg protein-1 and 6.5 micro m and 3.9 nmol.h-1.mg protein-1, respectively. Baboon CYP17 had a significantly higher activity for progesterone hydroxylation relative to pregnenolone. No 16alpha-hydroxylase and no lyase activity for 17alpha-hydroxyprogesterone. Sequence analyses showed that there are 28 different amino acid residues between human and baboon CYP17, primarily in helices F and G and the F-G loop.     

Unconjugated and sulfoconjugated steroids in plasma and zones of the adrenal cortex of the guinea pig

The following steroids were measured in their unconjugated and sulfoconjugated forms in plasma and in the outer and inner zones of the adrenal cortex of the guinea pig: pregnenolone, 17-hydroxypregnenolone, 21-hydroxypregnenolone, dehydroepiandrosterone and deoxycorticosterone. In plasma, pregnenolone and 21-hydroxypregnenolone were the predominant unconjugated steroids with concentrations 10-30 times higher than the other three steroids. Among the sulfoconjugated steroids, pregnenolone sulfate had a concentration 25-50 times higher than the other sulfoconjugates. For each steroid except 21-hydroxypregnenolone the sulfoconjugated form was present in a concentration 2-7 times higher than the unconjugated form. In the adrenal cortex, the content of 21-hydroxypregnenolone was significantly higher in the outer zone than in the inner zone and was present in amounts 3-100 times greater than the other unconjugated steroids in the outer zone. On the other hand, the content of pregnenolone was significantly greater in the inner zone than the outer zone, and was present in amounts 3-80 times greater than the other unconjugated steroids in the inner zone. With the exception of 21-hydroxypregnenolone and deoxycorticosterone, the steroid sulfoconjugates were significantly higher in the inner cortical zone. As in plasma, pregnenolone sulfate was the most abundant sulfoconjugated steroid. This report also describes preliminary studies concerning sulfurylated hydroxyl groups in different positions of 21-hydroxypregnenolone. The sulfoconjugate was prepared by using partially purified steroid sulfotransferase from the guinea pig adrenal. The results obtained indicated that of the total 21-hydroxypregnenolone conjugate formed, approximately 40% was the 21-sulfate and 20% the 3-sulfate, whereas 40% was non-hydrolyzable with the techniques used and was not further characterized.     

Identification of phospholipids capable of modulating the activities of some enzymes involved in androgen and 16-androstene biosynthesis in the immature pig testis.

Testicular steroidogenic enzymes in the microsomal fraction from immature pigs were investigated for the effects of phospholipids of known structure on androgen and 16-androstene biosynthesis. Untreated (control) microsomes metabolized pregnenolone to 17-hydroxypregnenolone, DHA and small quantities of progesterone, 17-hydroxyprogesterone, androstenedione and testosterone; and to 5,16-androstadien-3 beta-ol (andien-beta) and 4,16-androstadienone (dienone) in the 16-androstene pathway. Phosphatidyl(P)-serine, P-glycerol, P-ethanolamine, P-inositol, P-choline and phosphatidic acid did not significantly alter the 17-hydroxylase/C-17,20 lyase or "andien-beta-synthetase" activities. Thus, the C21 side-chain cleavage reactions appeared not to be dependent upon phospholipids for optimal activity. The conversion of pregnenolone to 4-ene steroids (progesterone, 17-hydroxyprogesterone, androstenedione and testosterone) was inhibited by dilinoleoyl-phosphatidyl-choline, but other phospholipids tested were without effect. On the other hand, the conversion of andien-beta to dienone was inhibited by P-serine, P-inositol and P-cholines with short saturated or long polyunsaturated acyl chains. Therefore, the presence of these phospholipids in pregnenolone incubations had different consequences for 3 beta-hydroxysteroid dehydrogenase-isomerase activities. It is concluded that substrate specific 3 beta-HSD-isomerases exist for androgen and 16-androstene biosynthesis and that phospholipids may play an intrinsic role in their catalytic activity.     

Phospholipases modulate immature pig testicular androgen and 16-androstene biosynthetic pathways in vitro.

The role of membrane phospholipids in porcine testicular androgen and 16-androstene biosynthesis was examined by monitoring the effects of phospholipase treatments on the activities of the steroid transforming enzymes. Untreated (control) microsomes from immature pig testes converted pregnenolone to 17-hydroxypregnenolone and DHA to 5,16-androstadien-3 beta-ol (andien-beta) and 4,16-androstadien-3-one (dienone) in the 16-androstene pathway, these metabolites accounting for most (65%) of the pregnenolone converted. The 4-ene steroids in the androgen pathway (progesterone, 17-hydroxyprogesterone, androstenedione and testosterone) totalled less than 10% of the pregnenolone metabolites. No estrogens or 5 alpha-reduced metabolites were detected. Treatment with phospholipase A2 or C, decreased the conversion of pregnenolone to 4-ene-3-oxo steroids but did not decrease the quantities of 5-ene-3 beta-hydroxysteroids. Confirmation of these findings was obtained by measuring the individual enzymatic steps. Phospholipases A2 and C significantly reduced the conversion of DHA to androstenedione and andien-beta to dienone but did not affect 17-hydroxylase or 'andien-beta-synthetase'. However, when the C-17, 20 lyase step was measured alone, phospholipase C decreased the quantity of androstenedione produced indicating that the side-chain cleavage reaction may involve a lipid component. The different effects of phospholipases on these enzymes suggests that pregnenolone metabolism may be regulated by alterations in the membrane microenvironment.     

Side-chain cleavage of C21 steroids by testicular microsomal cytochrome P-450 (17 alpha-hydroxylase/lyase): involvement of heme

The two steps in the side-chain cleavage of C21 steroids to give C19 steroids (i.e. 17 alpha-hydroxylation and C17,20 lyase activity) were examined using a highly purified cytochrome P-450 from microsomes of neonatal pig testis to determine the photochemical action spectra for the two reactions. Photochemical action spectra, using either 4-ene (progesterone) or 5-ene (pregnenolone) substrates, showed maximal reversal of inhibition by CO with light of 451 nm. Evidently the heme of cytochrome P-450 is involved in both 17 alpha-hydroxylation and in C17,20-lyase activity as in the case of the side-chain cleavage of cholesterol. Mechanisms proposed to account for enzymatic cleavage of the alpha-ketol side-chain of C21 steroids (C17,20 lyase activity) must be consistent with these findings.     

Structures of Human Steroidogenic Cytochrome P450 17A1 with Substrates

The human cytochrome P450 17A1 (CYP17A1) enzyme operates at a key juncture of human steroidogenesis, controlling the levels of mineralocorticoids influencing blood pressure, glucocorticoids involved in immune and stress responses, and androgens and estrogens involved in development and homeostasis of reproductive tissues. Understanding CYP17A1 multifunctional biochemistry is thus integral to treating prostate and breast cancer, subfertility, blood pressure, and other diseases. CYP17A1 structures with all four physiologically relevant steroid substrates suggest answers to four fundamental aspects of CYP17A1 function. First, all substrates bind in a similar overall orientation, rising ∼60° with respect to the heme. Second, both hydroxylase substrates pregnenolone and progesterone hydrogen bond to Asn²⁰² in orientations consistent with production of 17α-hydroxy major metabolites, but functional and structural evidence for an A105L mutation suggests that a minor conformation may yield the minor 16α-hydroxyprogesterone metabolite. Third, substrate specificity of the subsequent 17,20-lyase reaction may be explained by variation in substrate height above the heme. Although 17α-hydroxyprogesterone is only observed farther from the catalytic iron, 17α-hydroxypregnenolone is also observed closer to the heme. In conjunction with spectroscopic evidence, this suggests that only 17α-hydroxypregnenolone approaches and interacts with the proximal oxygen of the catalytic iron-peroxy intermediate, yielding efficient production of dehydroepiandrosterone as the key intermediate in human testosterone and estrogen synthesis. Fourth, differential positioning of 17α-hydroxypregnenolone offers a mechanism whereby allosteric binding of cytochrome b₅ might selectively enhance the lyase reaction. In aggregate, these structures provide a structural basis for understanding multiple key reactions at the heart of human steroidogenesis.     

Developmental changes of serum steroids produced by cytochrome P450c17 in rat

Serum levels of 17-hydroxypregnenolone, dehydroepiandrosterone, 17-hydroxyprogesterone, and androstenedione were measured during the postnatal development of rats 1-14 weeks of age. A significant decrease in the serum levels of these steroids with increasing age was observed, using multiple regression analysis: 17-hydroxypregnenolone (beta= -1.56, S.E.= 0.25, P < 0.00001), dehydroepiandrosterone (beta= -0.43, S.E.= 0.07, P < 0.00001), 17-hydroxyprogesterone (beta= -2.51, S.E.= 0.45, P < 0.00001), and androstenedione (beta= -1.63, S.E.= 0.33, P < 0.00001). A sex-related difference was not found. The observed decline in the serum levels of the steroids was directly proportional to the previously reported decrease in mRNA expression and enzyme activity of cytochrome P450c17 in the rat liver. Yet, despite this decrease to undetectable levels in liver after 7-8 weeks, significant amounts of 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, and androstenedione were still observed in the rat serum. This may partly be due to the mRNA expression of cytochrome P450c17 in tissues other than the liver, such as the testis and/or duodenum, after 4 weeks of age. Serum levels of pregnenolone, progesterone, and corticosterone in the developing rats were also examined.     

Further evidence that there is more than one adrenal 21-hydroxylase system

The 21-hydroxylase activity of microsomes isolated from bovine adrenal cortex have been assayed using [21-3H]17-hydroxypregnenolone and [1,2-3H]17-hydroxyprogesterone as substrates. When the assays are performed in the presence of an NADH regenerating system, to inhibit steroid 3 beta-hydroxy isomerase-dehydrogenase activity, the microsomes oxidize the 3 beta-hydroxy-5-ene steroid at a rate of 0.37 nmol/min.nmol cytochrome P-450 and the 3-keto-4-ene steroid at a rate of 6.4 nmol/min.nmol. When the microsomes are solubilized with Triton CF-54 they lose the ability to oxidize the 3-hydroxy-5-ene steroid, while the specific activity of the microsomes for the 3-keto-4-ene steroid is enhanced 3-fold. In contrast, when the microsomes are solubilized with sodium cholate, their specific activity towards the 4-ene steroid is decreased by 50% while the specific activity for a low concentration of the 5-ene steroid, 1 microM, is unchanged. In addition, when the oxidations of the labeled steroids (at 1 microM) by the microsomes, are examined in the presence of unlabeled 17-hydroxyprogesterone (at 20 microM) the oxidation of the 3-keto-4-ene steroid is inhibited by 92% while the oxidation of the 3 beta-hydroxy-5-ene steroid is only inhibited by 20%. These results all suggest that there are at least two 21-hydroxylases in bovine adrenal tissue, one of which can utilize the 3-keto-4-ene steroids only, the other of which, in addition, can utilize the 3 beta-hydroxy-5-ene steroids as substrates.     

The role of cytochrome b5 in adrenal microsomal steroidogenesis.

The role of cytochrome b5 in adrenal microsomal steroidogenesis was studied in guinea pig adrenal microsomes and also in the liposomal system containing purified cytochrome P-450s and NADPH-cytochrome P-450 reductase. Preincubation of the microsomes with anti-cytochrome b5 immunoglobulin decreased both 17 alpha- and 21-hydroxylase activity in the microsomes. In liposomes containing NADPH-cytochrome P-450 reductase and P-450C21 or P-450(17) alpha,lyase, addition of a small amount of cytochrome b5 stimulated the hydroxylase activity while a large amount of cytochrome b5 suppressed the hydroxylase activity. The effect of cytochrome b5 on the rates of the first electron transfer to P-450C21 in liposome membranes was determined from stopped flow measurements and that of the second electron transfer was estimated from the oxygenated difference spectra in the steady state. It was indicated that a small amount of cytochrome b5 activated the hydroxylase activity by supplying additional second electrons to oxygenated P-450C21 in the liposomes while a large amount of cytochrome b5 might suppress the activity through the interferences in the interaction between the reductase and P-450C21.     

Synthesis of halogenated pregnanes, mechanistic probes of steroid hydroxylases CYP17A1 and CYP21A2

The human steroidogenic cytochromes P450 CYP17A1 (P450c17, 17α-hydroxylase/17,20-lyase) and CYP21A2 (P450c21, 21-hydroxylase) are required for the biosynthesis of androgens, glucocorticoids, and mineralocorticoids. Both enzymes hydroxylate progesterone at adjacent, distal carbon atoms and show limited tolerance for substrate modification. Halogenated substrate analogs have been employed for many years to probe cytochrome P450 catalysis and to block sites of reactivity, particularly for potential drugs. Consequently, we developed efficient synthetic approaches to introducing one or more halogen atom to the 17- and 21-positions of progesterone and pregnenolone. In particular, novel 21,21,21-tribromoprogesterone and 21,21,21-trichloroprogesterone were synthesized using the nucleophilic addition of either bromoform or chloroform anion onto an aldehyde precursor as the key step to introduce the trihalomethyl moieties. When incubated with microsomes from yeast expressing human CYP21A2 or CYP17A1 with P450-oxidoreductase, CYP21A2 metabolized 17-fluoroprogesterone to a single product, whereas incubations with CYP17A1 gave no products. Halogenated steroids provide a robust system for exploring the substrate tolerance and catalytic plasticity of human steroid hydroxylases.     

An adrenocortical tumor secreting weak mineralocorticoids

An adrenocortical carcinoma (15.5 g) secreting excessive amounts of steroids with weak mineralocorticoid activity in a 25-year-old woman was studied with particular reference to its in vivo and in vitro secretions of steroids. Severe hypertension, occasional low serum potassium and suppressed PRA were the major clinical findings, and were improved with removal of the tumor. In the preoperative stage, plasma levels of 11-deoxycorticosterone, 18-hydroxy-11-deoxycorticosterone, corticosterone and 18-hydroxycorticosterone were all increased. However, the plasma level of aldosterone was repeatedly normal. Although plasma levels of pregnenolone, 17-hydroxypregnenolone, progesterone and 17-hydroxyprogesterone were very high, those of other late step steroids, i.e. 11-deoxycortisol, cortisol, dehydroepiandrosterone, androstenedione and testosterone were almost normal. From these findings, a major etiological role of weak mineralocorticoids such as 11-deoxycorticosterone, 18-hydroxycorticosterone and corticosterone in her hypertension was suggested. Pregnenolone and 17-hydroxypregnenolone in tumor tissue were increased, but 11-deoxycorticosterone, corticosterone, aldosterone, cortisol and adrenal androgens such as dehydroepiandrosterone, androstenedione and testosterone were below normal or low normal. In vitro production of 11-deoxycorticosterone, aldosterone or cortisol by the tumor tissue slices was very low and scarcely responded to synthetic ACTH.     

Cytochrome P-450 C21scc: One enzyme with two actions: Hydroxylase and lyase

Testis, adrenal, ovary and placenta contain a microsomal cytochrome P-450 that is capable of converting progesterone to androstenedione and pregnenolone to dehydroepi-androsterone. This conversion requires 17-hydroxylation followed by C_17,20-lyase activity which are both catalyzed by this one protein. Gene cloning and Northern blotting reveal that, at least in man, the same gene is responsible for both testicular and adrenal enzymes. The enzyme was first purified from neonatal pig testis. Both the testicular and adrenal enzymes show a marked preference for the 5-ene substrate (pregnenolone) in keeping with the extensive use of the 5-ene pathway in that species. Affinity alkylation with 17-bromoacetoxyprogesterone reveals a conserved cysteine at the active site of the enzyme and confirms the conclusion that a single enzyme catalyzes both reactions. Under some circumstances the enzyme catalyzes only 17-hydroxylation to permit the formation of the C₂₁ steroid cortisol. The regulation of lyase activity, i.e. the determination of the extent to which the second activity is expressed, results from the availability of P-450 reductase. No doubt the greater concentration of this protein in testicular as opposed to adrenal microsomes (× 3.5) is responsible for the production of androgens in the testis and cortisol in the adrenal. Testicular cytochrome b₅ also specifically stimulates lyase activity and also causes the porcine enzyme to catalyze a new reaction, i.e. Δ¹⁶-synthetase, resulting in synthesis of the important pheromone androsta-4,16-dien-3 one from progesterone.     

Effects of cadmium in vitro on microsomal steroid metabolism in the inner and outer zones of the guinea pig adrenal cortex

Studies were carried out to evaluate the effects of cadmium in vitro on microsomal steroid metabolism in the inner (zona reticularis) and outer (zona fasciculata and zona glomerulosa) zones of the guinea pig adrenal cortex. Microsomes from the inner zone have greater 21-hydroxylase than 17α-hydroxylase activity, resulting in the conversion of progesterone primarily to 11-deoxycorticosterone and of 17α-hydroxy progesterone principally to its 21-hydroxylated metabolite, 11-deoxycortisol. Microsomes from the outer zones, by contrast, have far greater 17α-hydroxylase and C_17,20-lyase activities than 21-hydroxylase activity. As a result, progesterone is converted primarily to its 17-hydroxylated metabolite, 17α-hydroxyprogesterone; and 17α-hydroxyprogesterone is converted principally to δ⁴-androstenedione, with only small amounts of 21-hydroxylated metabolites being produced. Addition of cadmium to incubations with inner zone microsomes causes concentration-dependent decreases in 21-hydroxylation and increases in 17α-hydroxylase and C_17,20-lyase activities, resulting in a pattern of steroid metabolism similar to that in normal outer zone microsomes. Cadmium similarly decreases 21-hydroxylation by outer zone microsomes but has no effect on the formation of 17-hydroxylated metabolites or on androgen (Δ⁴-androstenedione) production. In neither inner nor outer zone microsomes did cadmium affect cytochrome P-450 concentrations, steroid interactions with cytochrome(s) P-450, or NADPH–cytochrome P-450 reductase activities. The results indicate that cadmium produces both quantitative and qualitative changes in adrenal microsomal steroid metabolism and that the nature of the changes differs in the inner and outer adrenocortical zones. In inner zone microsomes, there appears to be a reciprocal relationship between 21-hydroxylase and 17α-hydroxylase/C_17,20-lyase activities which may influence the physiological function(s) of that zone.