Amanda C. Swart

11β-Hydroxydihydrotestosterone and 11-ketodihydrotestosterone, novel C19 steroids with androgenic activity: A putative role in castration resistant prostate cancer?

Adrenal C19 steroids, dehydroepiandrostenedione (DHEA(S)) and androstenedione (A4), play a critical role in castration resistant prostate cancer (CRPC) as they are metabolised to dihydrotestosterone (DHT), via testosterone (T), or via the alternate 5α-dione pathway, bypassing T. Adrenal 11OHA4 metabolism in CRPC is, however, unknown. We present a novel pathway for 11OHA4 metabolism in CRPC leading to the production of 11ketoT (11KT) and novel 5α-reduced C19 steroids - 11OH-5α-androstanedione, 11keto-5α-androstanedione, 11OHDHT and 11ketoDHT (11KDHT). The pathway was validated in the androgen-dependent prostate cancer cell line, LNCaP. Androgen receptor (AR) transactivation studies showed that while 11KT and 11OHDHT act as a partial AR agonists, 11KDHT is a full AR agonist exhibiting similar activity to DHT at 1nM. Our data demonstrates that, while 11OHA4 has negligible androgenic activity, its metabolism to 11KT and 11KDHT yields androgenic compounds which may be implicated, together with A4 and DHEA(S), in driving CRPC in the absence of testicular T.

The influence of Aspalathus linearis (Rooibos) and dihydrochalcones on adrenal steroidogenesis: Quantification of steroid intermediates and end products in H295R cells

The steroid hormone output of the adrenal gland is crucial in the maintenance of hormonal homeostasis, with hormonal imbalances being associated with numerous clinical conditions which include, amongst others, hypertension, metabolic syndrome, cardiovascular disease, insulin resistance and type 2 diabetes. Aspalathus linearis (Rooibos), which has been reported to aid stress-related symptoms linked to metabolic diseases, contains a wide spectrum of bioactive phenolic compounds of which aspalathin is unique. In this study the inhibitory effects of Rooibos and the dihydrochalcones, aspalathin and nothofagin, were investigated on adrenal steroidogenesis. The activities of both cytochrome P450 17α-hydroxylase/17,20 lyase and cytochrome P450 21-hydroxylase were significantly inhibited in COS-1 cells. In order to study the effect of these compounds in H295R cells, a human adrenal carcinoma cell line, a novel UPLC-MS/MS method was developed for the detection and quantification of twenty-one steroid metabolites using a single chromatographic separation. Under both basal and forskolin-stimulated conditions, the total amount of steroids produced in H295R cells significantly decreased in the presence of Rooibos, aspalathin and nothofagin. Under stimulated conditions, Rooibos decreased the total steroid output 4-fold and resulted in a significant reduction of aldosterone and cortisol precursors. Dehydroepiandrosterone-sulfate levels were unchanged, while the levels of androstenedione (A4) and 11β-hydroxyandrostenedione (11βOH-A4) were inhibited 5.5 and 2.3-fold, respectively. Quantification of 11βOH-A4 showed this metabolite to be a major product of steroidogenesis in H295R cells and we confirm, for the first time, that this steroid metabolite is the product of the hydroxylation of A4 by human cytochrome P450 11β-hydroxylase. Taken together our results demonstrate that Rooibos, aspalathin and nothofagin influence steroid hormone biosynthesis and the flux through the mineralocorticoid, glucocorticoid and androgen pathways, thus possibly contributing to the alleviation of negative effects arising from elevated glucocorticoid levels.

11β-hydroxyandrostenedione, the product of androstenedione metabolism in the adrenal, is metabolized in LNCaP cells by 5α-reductase yielding 11β-hydroxy-5α-androstanedione.

11β-Hydroxyandrostenedione (11OHA4), which is unique to the adrenal, was first isolated from human adrenal tissue in the fifties. It was later shown in the sixties that 11β-hydroxytestosterone (11OHT) was also produced by the human adrenal. Attention has shifted back to these adrenal androgens once more, as improved analytical techniques have enabled more accurate detection of steroid hormones. In this paper, we investigated the origin of these metabolites as well as their subsequent metabolism and examined a possible physiological role for 11OHA4 in prostate cancer cells. In H295R cells treated with forskolin and trilostane, etomidate, a reported cytochrome P450 11β-hydroxylase (CYP11B1) inhibitor, blocked the production of corticosterone, cortisol, 11OHA4 and 11OHT. The metabolism of androstenedione and testosterone by CYP11B1 and aldosterone synthase (CYP11B2) was assayed. Androstenedione was converted by CYP11B1, while the conversion by CYP11B2 was negligible. Both enzymes readily converted testosterone. The metabolism of these 11β-hydroxylated metabolites by 11β-hydroxysteroid dehydrogenase (11βHSD) types 1 and 2 was subsequently investigated. 11βHSD2 catalyzed the conversion of both 11OHA4 and 11OHT to their respective keto-steroids, while 11βHSD1 catalyzed the conversion of 11-ketoandrostenedione and 11-ketotestosterone to their respective hydroxy-steroids in Chinese hamster ovary cells. Investigating a functional role, steroid 5α-reductase types 1 and 2 converted 11OHA4 to 11β-hydroxy-5α-androstanedione (11OH-5α-dione), identified by accurate mass detection. UPLC-MS/MS analyses of 11OHA4 metabolism in LNCaP androgen-dependent prostate cancer cells, identified the 5α-reduced metabolite as well as 11-ketoandrostenedione and 11-ketotestosterone, with the latter indicating conversion by 17β-hydroxysteroid dehydrogenase. Downstream metabolism by 11βHSD2 and by 5α-reductase may therefore indicate a physiological role for 11OHA4 and/or 11OH-5α-dione in normal and prostate cancer cells.

The effect of Sutherlandia frutescens on steroidogenesis: confirming indigenous wisdom.

Sutherlandia frutescens (Cancer bush), a Southern African indigenous plant, is traditionally used to treat stress related maladies linked to the endocrine system. Extracts of the shrub were used to investigate the claimed stress‐relieving properties of the shrub. Dysregulation of the stress response is associated with elevated glucocorticoid levels. A model of chronic intermittent immobilization stress was investigated in 40 adult male Wistar rats to determine the effect of Sutherlandia. Immobilization stress resulted in increased corticosterone levels in the control group while rats receiving Sutherlandia extract showed significantly decreased corticosterone levels (P < 0.005). Since the biosynthesis of glucocorticoids in the adrenals is catalyzed by the cytochrome P450‐dependent enzymes, the influence of Sutherlandia extracts on adrenal steroidogenesis was determined in ovine adrenocortical microsomes and mitochondria, using spectral binding and enzyme conversion assays. Water extracts showed inhibition of substrate binding to cytochrome P450 21‐hydroxylase (CYP21) by 38% and cytochrome P450 11β‐hydroxylase (CYP11B1) by 60%. The conversion of progesterone and pregnenolone was inhibited by 34% and 30%, respectively. Subsequent extractions with chloroform and methanol showed inhibition of substrate binding and conversion with hydrophobic compounds exhibiting a greater inhibitory effect on deoxycorticosterone binding to CYP11B1 (30%) and on progesterone binding to CYP21 (50%). The inhibition of binding of pregnenolone to CYP17 by the chloroform extract was 62%, with negligible inhibition by the methanol extract. The chloroform extract showed a greater inhibitory effect than the methanol extract on progesterone and pregnenolone metabolism (20%–50%).

The influence of Sutherlandia frutescens on adrenal steroidogenic cytochrome P450 enzymes.

AIM OF THE STUDY The aim of this study was to investigate whether Sutherlandia frutescens, subsp. microphylla (family: Fabaceae/Leguminosa), which is traditionally used to treat symptoms of chronic stress generally associated with increased circulating glucocorticoids, influences the biosynthesis of these glucocorticoids. METHODS We investigated the interaction of Sutherlandia frutescens with cytochrome P450 enzymes, CYP17 and CYP21, which catalyse key reactions in glucocorticoid biosynthesis. The binding of progesterone and pregnenolone to these enzymes and their metabolism were assayed in the presence of extracts and the bioactive compounds, l-canavanine, pinitol, GABA, flavonoids and triterpenoid glucosides present in the shrub. RESULTS While the aqueous and methanol extracts inhibited the type I progesterone-induced difference spectrum (p<0.05), inhibition of pregnenolone binding (p=0.25) was negligible, with the aqueous extract exhibiting greater inhibition. The triterpenoid fraction inhibited both the type I pregnenolone- and progesterone-induced difference spectra and elicited a type II difference spectrum in the absence of substrate. Both pregnenolone and progesterone metabolism were inhibited by the aqueous extract, the inhibition of CYP21 being greater than that of CYP17, influencing the flux through glucocorticoid precursor pathways. CONCLUSION This attenuation of adrenal P450 enzymes may thus demonstrate a possible mechanism by which Sutherlandia frutescens reduces glucocorticoid levels and alleviates symptoms associated with stress.

Advances in the analytical methodologies: Profiling steroids in familiar pathways-challenging dogmas.

The comprehensive evaluation of the adrenal steroidogenic pathway, given its complexity, requires methodology beyond the standard techniques currently employed. Advances in LC-MS/MS, coupled with in vitro cell models that produce all the steroid metabolites of the mineralo-, glucocorticoid and androgen arms, present a powerful approach for the comprehensive evaluation of adrenal steroidogenesis in response to compounds of interest including bioactives, drug treatments and endocrine disrupting compounds. UHPLC-MS/MS analysis of steroid panels in forskolin, angiotensin II and K(+) stimulated H295R cells provides a snapshot of their effect on intermediates and end products of adrenal steroidogenesis. The impact of full steroid panel evaluations by LC- and GC-MS/MS extends to clinical profiling with the characterization of normal pediatric steroid reference ranges in sexual development and of disease-specific profiles improving diagnosis and sub classification. Comprehensive analyses of steroid profiles may potentially improve patient outcomes together with the application of treatments specifically suited to clinical subgroups. LC-MS/MS and GC-MS/MS applications in the analyses of comprehensive steroid panels are demonstrated in clinical conditions such as congenital adrenal hyperplasia in newborns requiring accurate diagnoses and in predicting metabolic risk in polycystic ovary syndrome patients. Most notable perhaps is the impact of LC-MS/MS evaluations on our understanding of the basic biochemistry of steroidogenesis with the detection of the long forgotten adrenal steroid, 11β-hydroxyandrostenedione, at significant levels. The characterization of its metabolism to androgen receptor ligands in the LNCaP prostate cancel cell model, specifically within the context of recurring prostate cancer, lends new perspectives to old dogmas. We demonstrate that UHPLC-MS/MS has enabled the analyses of novel metabolites of the enzymes, SRD5A, 11βHSD and 17βHSD, in LNCaP cells. Undoubtedly, the continuous advances in the analytical methodologies used for steroid profiling and quantification will give impetus to the unraveling of the remaining enigmas, old and new, of both hormone biosynthesis and metabolism.

Cytochrome b5: Novel roles in steroidogenesis

Cytochrome b(5) (cyt-b(5)) is essential for the regulation of steroidogenesis and as such has been implicated in a number of clinical conditions. It is well documented that this small hemoprotein augments the 17,20-lyase activity of cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1). Studies have revealed that this augmentation is accomplished by cyt-b(5) enhancing the interaction between cytochrome P450 reductase (POR) and CYP17A1. In this paper we present evidence that cyt-b(5) induces a conformational change in CYP17A1, in addition to facilitating the interaction between CYP17A1 and POR. We also review the recently published finding that cyt-b(5) allosterically augments the activity of 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3βHSD), a non cytochrome P450 enzyme, by increasing the enzymes affinity for its cofactor, NAD(+). The physiological importance of this finding, in terms of understanding adrenal androstenedione production, is examined. Finally, evidence that cyt-b(5) is able to form homomeric complexes in living cells is presented and discussed.

The Identification of Two CYP17 Alleles in the South African Angora Goat

South African Angora goats (Capra hircus) are susceptible to cold stress, due to the inability of the adrenal cortex to produce sufficient levels of cortisol. Two CYP17 isoforms were identified, cloned and characterized in this study. Sequence analysis revealed three amino acid differences between the two CYP17 isoforms, which resulted in a significant difference in 17,20 lyase activity of the expressed enzymes in both the presence and absence of cytochrome b5. Furthermore, cotransfections with 3βHSD revealed that one CYP17 isoform strongly favours the Δ5 steroid pathway. Our data implicates CYP17 as the primary cause of the observed hypoadrenocorticoidism in the South African Angora goat.

Ovine steroid 17α-hydroxylase cytochrome P450: characteristics of the hydroxylase and lyase activities of the adrenal cortex enzyme

The steroid 17-hydroxylase cytochrome P450 (CYP17) found in mammalian adrenal and gonadal tissues typically exhibits not only steroid 17-hydroxylase activity but also C-17,20-lyase activity. These two reactions, catalyzed by CYP17, allow for the biosynthesis of the glucocorticoids in the adrenal cortex, as a result of the 17-hydroxylase activity, and for the biosynthesis of androgenic C(19) steroids in the adrenal cortex and gonads as a result of the additional lyase activity. A major difference between species with regard to adrenal steroidogenesis resides in the lyase activity of CYP17 toward the hydroxylated intermediates and in the fact that the secretion of C(19) steroids takes place, in some species, exclusively in the gonads. Ovine CYP17 expressed in HEK 293 cells converts progesterone to 17-hydroxyprogesterone and pregnenolone to dehydroepiandrosterone via 17-hydroxypregnenolone. In ovine adrenal microsomes, minimal if any lyase activity was observed toward either progesterone or pregnenolone. Others have demonstrated the involvement of cytochrome b(5) in the augmentation of CYP17 lyase activity. Although the presence of cytochrome b(5) in ovine adrenocortical microsomes was established, ovine adrenal microsomes did not convert pregnenolone or 17-hydroxypregnenolone to dehydroepiandrosterone. Furthermore the addition of purified ovine cytochrome b(5) to ovine adrenal microsomes did not promote lyase activity. We conclude that, in the ovine adrenal cortex, factors other than cytochrome b(5) influence the lyase activity of ovine CYP17.

11β-hydroxyandrostenedione: Downstream metabolism by 11βHSD, 17βHSD and SRD5A produces novel substrates in familiar pathways

11β-Hydroxyandrostenedione (11OHA4), a major C19 steroid produced by the adrenal, was first reported in the 1950s. Initially the subject of numerous studies, interest dwindled due to the apparent lack of physiological function and, by the end of the century, 11OHA4 was no longer considered as an adrenal C19 steroid. Our recent studies, however, showed that 11OHA4 is the precursor to novel active androgens which include 11-ketodihydrotestosterone (11KDHT) which has been implicated in prostate cancer, thereby renewing interest in 11OHA4. In this paper we review the biosynthesis and downstream metabolism of 11OHA4. We discuss the extra-adrenal biosynthesis of 11OHA4 in humans and in other species, highlighting the well-documented role of 11OHA4 in the testes of male fish in which the steroid functions as an active androgen. Finally, we discuss the physiological relevance of 11OHA4 metabolism in castration resistant prostate cancer and outline future prospects.