CYP17A1 Activities Research Articles

Role of AMP-Activated Protein Kinase on Steroid Hormone Biosynthesis in Adrenal NCI-H295R Cells

Regulation of human androgen biosynthesis is poorly understood. However, detailed knowledge is needed to eventually solve disorders with androgen dysbalance. We showed that starvation growth conditions shift steroidogenesis of human adrenal NCI-H295R cells towards androgen production attributable to decreased HSD3B2 expression and activity and increased CYP17A1 phosphorylation and 17,20-lyase activity. Generally, starvation induces stress and energy deprivation that need to be counteracted to maintain proper cell functions. AMP-activated protein kinase (AMPK) is a master energy sensor that regulates cellular energy balance. AMPK regulates steroidogenesis in the gonad. Therefore, we investigated whether AMPK is also a regulator of adrenal steroidogenesis. We hypothesized that starvation uses AMPK signaling to enhance androgen production in NCI-H295R cells. We found that AMPK subunits are expressed in NCI-H295 cells, normal adrenal tissue and human as well as pig ovary cells. Starvation growth conditions decreased phosphorylation, but not activity of AMPK in NCI-H295 cells. In contrast, the AMPK activator 5-aminoimidazole-4-carboxamide (AICAR) increased AMPKα phosphorylation and increased CYP17A1-17,20 lyase activity. Compound C (an AMPK inhibitor), directly inhibited CYP17A1 activities and can therefore not be used for AMPK signaling studies in steroidogenesis. HSD3B2 activity was neither altered by AICAR nor compound C. Starvation did not affect mitochondrial respiratory chain function in NCI-H295R cells suggesting that there is no indirect energy effect on AMPK through this avenue. In summary, starvation-mediated increase of androgen production in NCI-H295 cells does not seem to be mediated by AMPK signaling. But AMPK activation can enhance androgen production through a specific increase in CYP17A1-17,20 lyase activity.

Orteronel (TAK-700), a novel non-steroidal 17,20-lyase inhibitor: Effects on steroid synthesis in human and monkey adrenal cells and serum steroid levels in cynomolgus monkeys

Surgical or pharmacologic methods to control gonadal androgen biosynthesis are effective approaches in the treatment of a variety of non-neoplastic and neoplastic diseases. For example, androgen ablation and its consequent reduction in circulating levels of testosterone is an effective therapy for advanced prostate cancers. Unfortunately, the therapeutic effectiveness of this approach is often temporary because of disease progression to the ‘castration resistant’ (CRPC) state, a situation for which there are limited treatment options. One mechanism thought to be responsible for the development of CRPC is extra-gonadal androgen synthesis and the resulting impact of these residual extra-gonadal androgens on prostate tumor cell proliferation. An important enzyme responsible for the synthesis of extra-gonadal androgens is CYP17A1 which possesses both 17,20-lyase and 17-hydroxylase catalytic activities with the 17,20-lyase activity being key in the androgen biosynthetic process. Orteronel (TAK-700), a novel, selective, and potent inhibitor of 17,20-lyase is under development as a drug to inhibit androgen synthesis. In this study, we quantified the inhibitory activity and specificity of orteronel for testicular and adrenal androgen production by evaluating its effects on CYP17A1 enzymatic activity, steroid production in monkey adrenal cells and human adrenal tumor cells, and serum levels of dehydroepiandrosterone (DHEA), cortisol, and testosterone after oral dosing in castrated and intact male cynomolgus monkeys. We report that orteronel potently suppresses androgen production in monkey adrenal cells but only weakly suppresses corticosterone and aldosterone production; the IC50 value of orteronel for cortisol was ∼3-fold higher than that for DHEA. After single oral dosing, serum levels of DHEA, cortisol, and testosterone were rapidly suppressed in intact cynomolgus monkeys. In castrated monkeys treated twice daily with orteronel, suppression of DHEA and testosterone persisted throughout the treatment period. In both in vivo models and in agreement with our in vitro data, suppression of serum cortisol levels following oral dosing was less than that seen for DHEA. In terms of human CYP17A1 and human adrenal tumor cells, orteronel inhibited 17,20-lyase activity 5.4 times more potently than 17-hydroxylase activity in cell-free enzyme assays and DHEA production 27 times more potently than cortisol production in human adrenal tumor cells, suggesting greater specificity of inhibition between 17,20-lyase and 17-hydroxylase activities in humans vs monkeys. In summary, orteronel potently inhibited the 17,20-lyase activity of monkey and human CYP17A1 and reduced serum androgen levels in vivo in monkeys. These findings suggest that orteronel may be an effective therapeutic option for diseases where androgen suppression is critical, such as androgen sensitive and CRPC.

Increased Activation of the Alternative “Backdoor” Pathway in Patients with 21-Hydroxylase Deficiency: Evidence from Urinary Steroid Hormone Analysis

17-Hydroxyprogesterone (17-OHP) can be converted to dihydrotestosterone (DHT) via an alternative "backdoor" route that bypasses the conventional intermediates androstenedione and testosterone. In this backdoor pathway, 17-OHP is converted to 5α-pregnane-3α,17α-diol-20-one (pdiol), which is an excellent substrate for the 17,20 lyase activity of CYP17A1 to produce androsterone. OBJECTIVE AND HYPOTHESES: The objective of this study was to obtain evidence for the presence of the backdoor pathway in patients with 21-hydroxylase deficiency (21-OHD). We compared urinary steroid hormone profiles determined by gas chromatography-mass spectrometry of 142 untreated 21-OHD patients (age range, 1 d to 25.4 yr; 51 males) with 138 control subjects. The activity of the backdoor pathway was assessed using the ratios of the urinary concentrations of pdiol to those of the metabolites of the classic Δ4 and Δ5 pathways. In contrast to etiocholanolone, which originates almost exclusively from the classic pathways, androsterone may be derived additionally from the backdoor pathway. Therefore, the androsterone to etiocholanolone ratio can be used as an indicator for the presence of the backdoor pathway. Untreated 21-OHD subjects showed increased urinary ratios of pdiol to the Δ4 and Δ5 pathway metabolites and a higher androsterone to etiocholanolone ratio. The elevated ratios of pdiol to the Δ4 and Δ5 pathway metabolites as well as the higher androsterone to etiocholanolone ratio in patients with 21-OHD indicate postnatal activity of the backdoor pathway with maximum activity during early infancy. Our data provide new insights into the pathophysiology of androgen biosynthesis of 21-OHD.

Differential regulation of the expression of cytochrome P450 aromatase, estrogen and androgen receptor subtypes in the brain–pituitary–ovarian axis of the Japanese eel ( Anguilla japonica) reveals steroid dependent and independent mechanisms

This study aimed at investigating the role of sexual steroids in the regulation of the expression of the single aromatase gene and steroid receptor subtypes in the brain–pituitary–ovarian axis of the Japanese eel. Unlike other teleosts, which possess duplicated genes for aromatase, cyp19a1a and cyp19a1b, expressed in the gonads and in the brain, respectively, eel species possess a single cyp19a1. Phylogenetic analysis indicated that eel brain/gonadal cyp19a1 branches at the basis of both teleost gonadal cyp19a1a and brain cyp19a1b clades. Female eels treated with catfish pituitary homogenate (CPH) to induce sexual maturation showed an increase in the expression of cyp19a1 and aromatase enzymatic activity in the brain and in the ovaries. Treatments with sex steroids (estradiol-17 β, E 2 or testosterone, T) revealed that the increase in cyp19a1 expression in the brain may result from E 2-specific induction. In contrast, the increase in cyp19a1 expression in the ovaries of CPH-treated eels is a result of steroid-independent control, probably from a direct effect of gonadotropins contained in the pituitary extract. Analysis of the expression of estrogen and androgen receptor subtypes, esr-α, esr-β, ar-α and ar-β, in eels treated with CPH or sex steroids revealed differential regulations. In CPH-treated eels, the expression of esr-α and ar-α was significantly increased in the brain, while the expression of ar- α and ar- β was increased in the ovaries. No change was observed in esr- β in any organ. Steroid treatments induced an upregulation by E 2 of esr- α, but not esr- β expression, in the brain, pituitary and ovaries, while no autoregulation by T of its own receptors could be observed. These results reveal both steroid-dependent and -independent mechanisms in the regulation of cyp19a1 and steroid receptor subtype expression in the eel.

Cytochrome b 5 augments 3β-hydroxysteroid dehydrogenase/Δ 5-Δ 4 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.

Inhibition of human and rat testicular steroidogenic enzyme activities by bisphenol A

Bisphenol A (BPA) is a potential endocrine disruptor. It has been shown that it reduces serum testosterone level in rodents after exposure. However, the mechanism is unclear. The object of the present study is to investigate the effects of BPA on human and rat steroidogenic enzymes including P450 17α-hydroxylase/17,20-lyase (CYP17A1), 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3). Human and rat testis microsomes were exposed to various concentrations of BPA (10−8–10−4M). BPA inhibited human and rat 3β-HSD, CYP17A1 and 17β-HSD3 activities. The half maximal inhibitory concentrations (IC50s) of BPA for human and rat testis 3β-HSD were 7.92±1.03 and 26.49±3.03μM (200μM pregnenolone), respectively. The IC50s for human and rat CYP17A1 (1μM progesterone) were 18.99±3.75 and 64.67±4.04μM, respectively. BPA was a weak HSD17B3 inhibitor with IC50s of about 100μM (200nM androstenedione). BPA also concentration-dependently inhibited testosterone production by rat Leydig cells. In conclusion, BPA is an inhibitor for 3β-HSD, CYP17A1 and 17β-HSD3. Human 3β-HSD and CYP17A1 are more sensitive to BPA than rat 3β-HSD and CYP17A1.

Why Human Cytochrome P450c21 Is a Progesterone 21-Hydroxylase

Human cytochrome P450c21 (steroid 21-hydroxylase, CYP21A2) catalyzes the 21-hydroxylation of progesterone (P4) and its preferred substrate 17α-hydroxyprogestrone (17OHP4). CYP21A2 activities, which are required for cortisol and aldosterone biosynthesis, involve the formation of energetically disfavored primary carbon radicals. Therefore, we hypothesized that the binding of P4 and 17OHP4 to CYP21A2 restricts access of the reactive heme-oxygen complex to the C-21 hydrogen atoms, suppressing oxygenation at kinetically more favorable sites such as C-17 and C-16, which are both hydroxylated by cytochrome P450c17 (CYP17A1). We reasoned that expansion of the CYP21A2 substrate-binding pocket would increase substrate mobility and might yield additional hydroxylation activities. We built a computer model of CYP21A2 based principally on the crystal structure of CYP2C5, which also 21-hydroxylates P4. Molecular dynamics simulations indicate that binding of the steroid nucleus perpendicular to the plane of the CYP21A2 heme ring limits access of the heme oxygen to the C-21 hydrogen atoms. Residues L107, L109, V470, I471, and V359 were found to contribute to the CYP21A2 substate-binding pocket. Mutation of V470 and I471 to alanine or glycine preserved P4 21-hydroxylase activity, and mutations of L107 or L109 were inactive. Mutations V359A and V359G, in contrast, acquired 16α-hydroxylase activity, accounting for 40% and 90% of the P4 metabolites, respectively. We conclude that P4 binds to CYP21A2 in a fundamentally different orientation than to CYP17A1 and that expansion of the CYP21A2 substrate-binding pocket allows additional substrate trajectories and metabolic switching.

Endocrine Disrupter Bisphenol A Increases In Situ Estrogen Production in the Mouse Urogenital Sinus

The balance between androgens and estrogens is very important in the development of the prostate, and even small changes in estrogen levels, including those of estrogen-mimicking chemicals, can lead to serious changes. Bisphenol A (BPA), an endocrine-disrupting chemical, is a well-known, ubiquitous, estrogenic chemical. To investigate the effects of fetal exposure to low-dose BPA on the development of the prostate, we examined alterations of the in situ sex steroid hormonal environment in the mouse urogenital sinus (UGS). In the BPA-treated UGS, estradiol (E(2)) levels and CYP19A1 (cytochrome P450 aromatase) activity were significantly increased compared with those of the untreated and diethylstilbestrol (DES)-treated UGS. The mRNAs of steroidogenic enzymes, Cyp19a1 and Cyp11a1, and the sex-determining gene, Nr5a1, were up-regulated specifically in the BPA-treated group. The up-regulation of mRNAs was observed in the mesenchymal component of the UGS as well as in the cerebellum, heart, kidney, and ovary but not in the testis. The number of aromatase-expressing mesenchymal cells in the BPA-treated UGS was approximately twice that in the untreated and DES-treated UGS. The up-regulation of Esrrg mRNA was observed in organs for which mRNAs of steroidogenic enzymes were also up-regulated. We demonstrate here that fetal exposure to low-dose BPA has the unique action of increasing in situ E(2) levels and CYP19A1 (aromatase) activity in the mouse UGS. Our data suggest that BPA might interact with in situ steroidogenesis by altering tissue components, such as the accumulation of aromatase-expressing mesenchymal cells, in particular organs.

Upregulation of Peripubertal Rat Leydig Cell Steroidogenesis Following 24 h In Vitro and In Vivo Exposure to Atrazine

Atrazine is currently one of the most widely used herbicides in the United States and elsewhere. Here we examined 24 h in vitro and in vivo effects of atrazine on androgen production and on expression and activity of steroidogenic enzymes and regulatory proteins involved in cyclic adenosine monophosphate (cAMP)-signaling pathway in peripubertal rat Leydig cells. When in vitro added, 1-50 μM atrazine increased basal and human chorion gonadotropin-stimulated testosterone production and accumulation of cAMP in the medium of treated cells. The stimulatory action of atrazine on androgen production but not on cAMP accumulation was abolished in cells with inhibited protein kinase A. Atrazine also stimulated the expression of mRNA transcripts for steroidogenic factor-1, steroidogenic acute regulatory protein, cytochrome P450 (CYP)17A1, and 17β-hydroxysteroid dehydrogenase (HSD), as well as the activity of CYP17A1 and 17βHSD. The stimulatory effects of atrazine on cAMP accumulation and androgen production were also observed during the first 3 days of in vivo treatment (200 mg/kg body weight, by gavage) followed by a decline during further treatment. These results indicate that atrazine has a transient stimulatory action on cAMP signaling pathway in Leydig cells, leading to facilitated androgenesis.

A steroid metabolomic approach to 17α-hydroxylase/17,20 lyase deficiency

The CYP17A1 gene encodes 17OHase and 17,20-lyase activities. We have previously reported an E305G mutation in CYP17A1 that was found by expression studies to cause only isolated 17,20-lyase deficiency. Further clinical investigations and urinary steroid examination have revealed that this mutation has in fact an extensive effect on 17OHase activity. The aim of the present study was to analyze the metabolic patterns of our patients using a novel application of a mathematical approach. Implementing an unbiased systems biology metabolomics approach, together with methods adapted from gene expression data analysis, we investigated the steroid metabolomic network relevant for 17,20-lyase deficiency of patients with the CYP17A1 gene mutation E305G. Six homozygote patients with a CYP17A1 gene E305G mutation and eight unaffected controls. We used gas chromatography–mass spectrometry (GC–MS) to quantitatively assess 31 urinary steroid metabolites. We applied statistical methods adopted from transcriptomics to identify differential metabolites and differential metabolite ratios, and a heatmap to visualize the results. We assigned Threshold Number of Misclassifications (TNoM) scores to metabolite ratios. Implementation of the unbiased approach revealed alteration in the metabolic balance of unsuspected enzyme systems in our patients. Of the 166 metabolite ratios that reached the discriminatory TNoM score of 0 or 1, 138 discriminated between the groups on the basis of CYP17A1 activity, three indicated inhibition of 3βHSD2 activity, ten indicated inhibition of 3βHSD2/21OHase/11OHase activity, and four enhanced 11βHSD2 activity. The unbiased all-inclusive metabolomic approach powerfully discriminated between the patients and the control groups on the basis of CYP17A1 activity. Other adrenal and extra-adrenal enzymes were affected by this mutation. We present an informatics tool that suggests disease-specific profiles and opens interesting research avenues.

1α,25-Dihydroxyvitamin D3 affects hormone production and expression of steroidogenic enzymes in human adrenocortical NCI-H295R cells

The current study presents data indicating that 1α,25-dihydroxyvitamin D3 affects the production of hormones and expression of crucial steroidogenic enzymes in the human adrenocortical cell line NCI-H295R. This cell line is widely used as a model for adrenal steroidogenesis. Treatment of the cells with 1α,25-dihydroxyvitamin D3 suppressed the levels of corticosterone, aldosterone, DHEA, DHEA-sulfate and androstenedione in the culture medium. In order to study the mechanisms behind this suppression of hormone production, we investigated the effects of 1α,25-dihydroxyvitamin D3 on important genes and enzymes controlling the biosynthesis of adrenal hormones. The mRNA levels were decreased for CYP21A2 while they were increased for CYP11A1 and CYP17A1. No significant changes were observed in mRNA for CYP11B1, CYP11B2 or 3β-hydroxysteroid dehydrogenase (3βHSD). In similarity with the effects on mRNA levels, also the endogenous enzyme activity of CYP21A2 decreased after treatment with 1α,25-dihydroxyvitamin D3. Interestingly, the two CYP17A1-mediated activities were influenced reciprocally — the 17α-hydroxylase activity increased whereas the 17,20-lyase activity decreased. The current data indicate that the 1α,25-dihydroxyvitamin D3-mediated decrease in corticosterone and androgen production is due to suppression of the 21-hydroxylase activity by CYP21A2 and the 17,20-lyase activity by CYP17A1, respectively. In conclusion, the current study reports novel findings on 1α,25-dihydroxyvitamin D3-mediated effects on hormone production and regulation of genes and enzymes involved in steroidogenesis in the adrenocortical NCI-H295R cell line, a model for human adrenal cortex.

Abstract LB-105: Single nucleotide polymorphisms (SNPs) in the CYP19A1 gene are strongly associated with CYP1A2 expression: First evidence for epistasis in the CYP superfamily

Abstract CYP1A2 is a member of the cytochromes P450 enzymes responsible for the oxidative metabolism of a wide array of endo- and xenobiotics, and has been demonstrated to be the major isoform responsible for oxidation of estrone and estradiol. To date, SNPs identified in this gene do not adequately explain the 60-80-fold variation in activity observed in Caucasians. We hypothesized that CYP1A2 could be a target of epistatic regulation (i.e., gene-gene interactions where SNPs in one gene influence the expression of another). CYP1A2 and CYP19A1 both lie on chromosome 15, and since CYP19A1 (aromatase) is responsible for the production of estrogens, and CYP1A2 is the primary catabolic P450, we hypothesized that these genes could be coordinately regulated, and SNPs influencing levels of CYP19A1 could also be related to expression levels of CYP1A2. Two TagSNPs (rs749292 A<G and rs727479 A<C) in CYP19A1 were genotyped using Beckman Single Base Extension Kit and TaqMan MGB probe, respectively. Levels of CYP1A2 protein in human liver microsomes from 100 donors were assessed by Western blotting and correlations between CYP19A1 SNPs and CYP1A2 protein amounts were determined. CYP1A2 expression was significantly higher in specimens with CYP19A1 (rs727479) AA homozygous compared with CC genotype (P<0.001), a SNP associated with increased CYP19A1 activity. CYP1A2 expression also is related CYP19A1 (rs749292) polymorphism and AA genotype carriers have higher CYP1A2 expression compared with GG genotype carriers. Moreover, in silico analyses shows the presence of over 300 putative estrogen-responsive elements upstream of the CYP1A2 transcription start sit. SNPs in CYP19A1 are significantly associated with CYP1A2 protein expression. This is the first evidence of epistasis in the P450 enzymes and suggests a role of estrogens in the regulation of CYP1A2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-105.

Restoration of mutant cytochrome P450 reductase activity by external flavin

Cytochrome P450 oxidoreductase (POR) supplies electrons from NADPH to steroid and drug metabolizing reactions catalyzed by the cytochrome P450s located in endoplasmic reticulum. Mutations in human POR cause a wide spectrum of disease ranging from disordered steroidogenesis to sexual differentiation. Previously we and others have shown that POR mutations can lead to reduced activities of steroidogenic P450s CYP17A1, CYP19A1 and CYP21A1. Here we are reporting that mutations in the FMN binding domain of POR may reduce CYP3A4 activity, potentially influencing drug and steroid metabolism; and the loss of CYP3A4 activity may be correlated to the reduction of cytochrome b 5 by POR. Computational molecular docking experiments with a FMN free structural model of POR revealed that an external FMN could be docked in close proximity to the FAD moiety and receive electrons donated by NADPH. Using FMN supplemented assays we have demonstrated restoration of the defective POR activity in vitro.

A biphasic effect of the fungicide prochloraz on aldosterone, but not cortisol, secretion in human adrenal H295R cells—Underlying mechanisms

The widely used imidazole fungicide prochloraz displays anti-androgenic effects partly via inhibition of testicular steroidogenesis and testosterone secretion. Adrenal steroidogenesis and hormone secretion may also be a target of this endocrine disruptor. Herein, we demonstrate a dose-dependent inhibition of cortisol secretion and a biphasic effect on aldosterone secretion, with a 2-fold stimulation at low concentrations and a strong inhibition at high concentrations, following prochloraz treatment (0–10μM) of human adrenocortical H295R cells. Analysis of the dose-dependent effects of prochloraz on the secretion of steroidogenic intermediates suggested that the observed effects on cortisol and aldosterone secretion might be mediated by inhibition of the steroidogenic steps catalysed by CYP17A1 and CYP21A2. The inhibition of CYP17A1 was reflected on the level of expression of steroidogenic genes as analysed by quantitative RT-PCR. In addition, analysis of enzyme activity showed a dose-dependent inhibitory effect of prochloraz on the activity of CYP17A1 and CYP21A2, but not CYP11B1. We have demonstrated specific effects of prochloraz on adrenal steroidogenic pathways and hormone secretion via inhibition of steroidogenic CYP enzymes. The disruption of adrenal hormone secretion may result in altered endocrine homeostasis and affect human health.

Transforming growth factor-β1 inhibits luteinization and promotes apoptosis in bovine granulosa cells

We have previously shown that TGFB1 inhibits estradiol (E(2)) and progesterone (P(4)) biosynthesis in FSH-stimulated bovine granulosa cells by selective inhibition of steroidogenic enzymes. The objective of this study was to assess the effects of TGFB1 on E(2) and P(4) steroidogenesis in bovine granulosa cells cultured in the absence of FSH and to measure the effects of TGFB1 on cell proliferation and apoptosis in the presence and absence of FSH. Bovine granulosa cells from 2 to 5 mm follicles were cultured in serum-free medium for 2-6 days. In the absence of FSH, the secretion of P(4) increased with time in culture (P<0.05). Addition of TGFB1 for 6 days decreased P(4) secretion and mRNA levels of the P(4) synthesis-associated genes STAR, CYP11A1, HSD3B1, and GSTA (P<0.05). In the absence of FSH, the secretion of E(2) decreased and addition of TGFB1 for 6 days partially reversed this decline and stimulated E(2) biosynthesis, CYP19A1 and HSD17B1 mRNA levels and CYP19A1 activity (P<0.05). Conversely, TGFB1 did not affect HSD17B7 expression and HSD17B-reducing activity. TGFB1 decreased the proportion of cells in the G0/G1 and S+G2/M phases in FSH-stimulated and unstimulated granulosa cells (P<0.05). Furthermore, in the presence or absence of FSH, TGFB1 increased the proportion of cells in apoptosis measured by propidium iodide staining and flow cytometry and confirmed by increased levels of cleaved caspase-3 (P<0.05). Our results therefore indicate that TGFB1 inhibits luteinization in cultured bovine granulosa cells while maintaining an estrogenic phenotype, and this effect was associated with increased apoptosis.

A novel communication role for CYP17A1 in the progression of castration‐resistant prostate cancer

CYP17A1 is currently a target for total androgen blockade in advanced prostate cancer (CaP) patients. After castration, or removal of testicular androgens, CYP17A1 can act as a rate-limiting enzyme in androgen synthesis from cholesterol or other adrenal precursors within the tumor microenvironment ultimately contributing to disease progression. Herein we provide evidence that CYP17A1 could also be a mediator of cell-to-cell communication within the CaP tumor microenvironment. CYP17A1 expression was evaluated by immunohistochemical analysis of human tumor sections and Western blot analysis of CaP patients' serum and exosome isolates. CYP17A1 activity assays were conducted in human serum (and positive control human liver and kidney microsomes) using progesterone as a precursor and an LC-MS endpoint. These studies revealed that the expression pattern of CYP17A1 is typical of a secretory protein as it is localized to the luminal pole of the cells in exocrine secretory mode. CYP17A1 is expressed in human serum and in fact is elevated in the serum of CaP patients as compared to healthy controls. Serum CYP17A1 activity could not be confirmed, however, verification of CYP17A1 expression in exosomes suggests a role in cell-to-cell communication within the tumor microenvironment. CYP17A1 is a crucial enzyme for de novo androgen synthesis within the tumor microenvironment after removal of testicular androgens by castration. We provide evidence for a novel role for CYP17A1 in serum and further reiterate the importance of targeting this enzyme in CaP progression.

Cytochrome P450 Oxidoreductase Deficiency with Antley-Bixler Syndrome: Steroidogenic Capacities

For patients with cytochrome P450 oxidoreductase deficiency (PORD), steroid replacement is recommended at times of stress. However, it is unknown how hormones respond to actual physical stress in these patients. We report a female infant with PORD accompanied by the Antley-Bixler syndrome phenotype. Her urinary steroid profile revealed defective CYP17A1 and CYP21A2 activities, and an adrenocorticotropin (ACTH) stimulation test showed potential adrenal insufficiency. Hormonal responses to actual physical stress were as follows: Vigorous crying during blood sampling rarely affected the serum cortisol level. Acute viral gastroenteritis led to marked increases in blood ACTH and 17alpha-hydroxyprogesterone levels in proportion to the severity of the illness. The serum cortisol level also responded to this stress, but the response might have been blunted. Regarding peri-operative steroid replacement, intravenous hydrocortisone administration even at a dose of 6 mg/kg, which is lower than that recommended for congenital adrenal hyperplasia in Japan, proved to be excessive.

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.

Species-specific regulation of PXR/CAR/ER-target genes in the mouse and rat liver elicited by o, p'-DDT

BackgroundDichlorodiphenyltrichloroethane (DDT) is a persistent estrogenic organochlorine pesticide that is a rodent hepatic tumor promoter, with inconclusive carcinogenicity in humans. We have previously reported that o, p'-DDT elicits primarily PXR/CAR-mediated activity, rather than ER-mediated hepatic responses, and suggested that CAR-mediated effects, as opposed to ER-mediated effects, may be more important in tumor promotion in the rat liver. To further characterize species-specific hepatic responses, gene expression analysis, with complementary histopathology and tissue level analyses were investigated in immature, ovariectomized C57BL/6 mice treated with 300 mg/kg o, p'-DDT, and compared to Sprague-Dawley rat data.ResultsRats and mice exhibited negligible histopathology with rapid o, p'-DDT metabolism. Gene expression profiles were also similar, exhibiting PXR/CAR regulation with the characteristic induction of Cyp2b10 and Cyp3a11. However, PXR-specific target genes such as Apoa4 or Insig2 exhibited more pronounced induction compared to CAR-specific genes in the mouse. In addition, mouse Car mRNA levels decreased, possibly contributing to the preferential activation of mouse PXR. ER-regulated genes Cyp17a1 and Cyp7b1 were also induced, suggesting o, p'-DDT also elicits ER-mediated gene expression in the mouse, while ER-mediated effects were negligible in the rat, possibly due to the inhibitory effects of CAR on ER activities. In addition, o, p'-DDT induced Gadd45a, Gadd45b and Cdkn1, suggesting DNA damage may be an additional risk factor. Furthermore, elevated blood DHEA-S levels at 12 h after treatment in the mouse may also contribute to the endocrine-related effects of o, p'-DDT.ConclusionAlthough DDT is known to cause rodent hepatic tumors, the marked species differences in PXR/CAR structure, expression patterns and ligand preference as well as significant species-specific differences in steroidogenesis, especially CYP17A1 expression and activity, confound the extrapolation of these results to humans. Nevertheless, the identification of potential modes of action as well as species-specific responses may assist in the selection and further development of more appropriate models for assessing the toxicity of DDT to humans and wildlife.

Adrenocortical Hormone Abnormalities in Men with Chronic Prostatitis/Chronic Pelvic Pain Syndrome

To identify adrenocortical hormone abnormalities as indicators of endocrine dysfunction in chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). We simultaneously measured the serum concentrations of 12 steroids in patients with CP/CPPS and controls, using isotope dilution liquid chromatography, followed by atmospheric pressure photospray ionization and tandem mass spectrometry. We evaluated 27 patients with CP/CPPS and 29 age-matched asymptomatic healthy controls. In the mineralocorticoid pathway, progesterone was significantly greater, and the corticosterone and aldosterone concentrations were significantly lower, in the patients with CP/CPPS than in the controls. In the glucocorticoid pathway, 11-deoxycortisol was significantly lower and the cortisol concentrations were not different between the patients and controls. In the sex steroid pathway, the androstenedione and testosterone concentrations were significantly greater in those with CP/CPPS than in the controls. The estradiol, dehydroepiandrosterone, and dehydroepiandrosterone sulfate concentrations were not different between the patients and controls. The National Institutes of Health-Chronic Prostatitis Symptom Index total and pain domain scores correlated positively with the 17-hydroxyprogesterone and aldosterone (P <0.001) and negatively with the cortisol (P <0.001) concentrations. Our results suggest reduced activity of CYP21A2 (P450c21), the enzyme that converts progesterone to corticosterone and 17-hydroxyprogesterone to 11-deoxycortisol. Furthermore, these results provide insights into the biologic basis of CP/CPPS. Follow-up studies should explore the possibility that patients with CP/CPPS meet the diagnostic criteria for nonclassic congenital adrenal hyperplasia and whether the hormonal findings improve or worsen in parallel with symptom severity.