Steroidal Inactivator Research Articles

Mammalian Sulfatases: Biochemistry, Disease Manifestation, and Therapy.

Sulfatases are enzymes that catalyze the removal of sulfate from biological substances, an essential process for the homeostasis of the body. They are commonly activated by the unusual amino acid formylglycine, which is formed from cysteine at the catalytic center, mediated by a formylglycine-generating enzyme as a post-translational modification. Sulfatases are expressed in various cellular compartments such as the lysosome, the endoplasmic reticulum, and the Golgi apparatus. The substrates of mammalian sulfatases are sulfolipids, glycosaminoglycans, and steroid hormones. These enzymes maintain neuronal function in both the central and the peripheral nervous system, chondrogenesis and cartilage in the connective tissue, detoxification from xenobiotics and pharmacological compounds in the liver, steroid hormone inactivation in the placenta, and the proper regulation of skin humidification. Human sulfatases comprise 17 genes, 10 of which are involved in congenital disorders, including lysosomal storage disorders, while the function of the remaining seven is still unclear. As for the genes responsible for pathogenesis, therapeutic strategies have been developed. Enzyme replacement therapy with recombinant enzyme agents and gene therapy with therapeutic transgenes delivered by viral vectors are administered to patients. In this review, the biochemical substrates, disease manifestation, and therapy for sulfatases are summarized.

Glucuronidation of Abiraterone and Its Pharmacologically Active Metabolites by UGT1A4, Influence of Polymorphic Variants and Their Potential as Inhibitors of Steroid Glucuronidation.

Abiraterone (Abi) acetate (AA) is a prodrug of Abi, a CYP17A1 inhibitor used to treat patients with advanced prostate cancer. Abi is a selective steroidal inhibitor that blocks the biosynthesis of androgens. It undergoes extensive biotransformation by steroid pathways, leading to the formation of pharmacologically active Δ4-Abi (D4A) and 5α-Abi. This study aimed to characterize the glucuronidation pathway of Abi and its two active metabolites. We show that Abi, its metabolites, and another steroidal inhibitor galeterone (Gal) undergo secondary metabolism to form glucuronides (G) in human liver microsomes with minor formation by intestine and kidney microsomal preparations. The potential clinical relevance of this pathway is supported by the detection by liquid chromatography-tandem mass spectrometry of Abi-G, D4A-G, and 5α-Abi-G in patients under AA therapy. A screening of UGT enzymes reveals that UGT1A4 is the main enzyme involved. This is supported by inhibition experiments using a selective UGT1A4 inhibitor hecogenin. A number of common and rare nonsynonymous variants significantly abrogate the UGT1A4-mediated formation of Abi-G, D4A-G, and 5α-Abi-G in vitro. We also identify Gal, Abi, and its metabolites as highly potent inhibitors of steroid inactivation by the UGT pathway with submicromolar inhibitor constant values. They reduce the glucuronidation of both the adrenal precursors and potent androgens in human liver, prostate cancer cells, and by recombinant UGTs involved in their inactivation. In conclusion, tested CYP17A1 inhibitors are metabolized through UGT1A4, and germline variations affecting this metabolic pathway may also influence drug metabolism. SIGNIFICANCE STATEMENT: The antiandrogen abiraterone (Abi) is a selective steroidal inhibitor of the cytochrome P450 17α-hydroxy/17,20-lyase, an enzyme involved in the biosynthesis of androgens. Abi is metabolized to pharmacologically active metabolites by steroidogenic enzymes. We demonstrate that Abi and its metabolites are glucuronidated in the liver and that their glucuronide derivatives are detected at variable levels in circulation of treated prostate cancer patients. UDP-glucuronosyltransferase (UGT)1A4 is the primary enzyme involved, and nonsynonymous germline variations affect this metabolic pathway in vitro, suggesting a potential influence of drug metabolism and action in patients. Their inhibitory effect on drug and steroid glucuronidation raises the possibility that these pharmacological compounds might affect the UGT-associated drug-metabolizing system and pre-receptor control of androgen metabolism in patients.

Perspective on the regulatory role of UGT2B28 as a conjugating enzyme in the progression of prostate cancer

The maintenance of steroid homeostasis in the prostate is critical, with perturbation of steroidogenesis contributing to the modulation of active ligands in the androgen pool. In this scenario, enzymes catalysing the biosynthesis, inactivation and conjugation of steroids are the key players, regulating active ligand levels and in so doing, the activation of the androgen receptor (AR). The glucuronidation of potent ligands renders them unable to bind the AR, allowing the secretion of conjugated steroids. Uridine diphosphate glucuronosyltransferase 2B type 28 (UGT2B28), one of the UGT enzymes catalyzing the glucuronidation of androgens, has recently been given a prominent role in the regulation of prostate steroidogenesis—one which stands in contrast to the accepted dogma that lower androgen levels resulting from increased conjugation are associated with decreased prostate cancer (PCa) risk and disease progression. Increased DHT and its precursors, T and androstenediol, were reported to be associated with increased UGT2B28 tumor expression levels, linked to lower PSA levels but higher Gleason scores and increased PCa risk. In addition, the complete deletion of UGT2B28, was associated with decreased T, DHT and glucuronide derivatives when compared to patients carrying both alleles. UGT2B28 is encoded by a single gene giving rise to UGT2B28 type I which catalyses androgen glucuronidation and, due to alternative splicing, also produces two distinct transcripts, UGT2B28 type II and III. Type II with its premature stop codon, is devoid of the cofactor binding domain while type III is devoid of the substrate binding domain, both catalytically inactive, truncated proteins. Increased UGT2B28 mRNA expression was reported in primary tumours, and while variable nuclear and strong cytoplasmic staining were distinctive of tumour cells, the expression levels and compartmentalization of the specific protein isoforms remain unknown. While increased expression of type I would contribute towards lowering androgen levels, increased expression of types II and III would not. The abundance of type III transcripts in multiple tissues may provide insight into a regulatory role with truncated isoforms possibly affecting androgen levels by regulating substrate and/or co-factor availability, dimerization or the formation of protein complexes with other UGTs, while protein-protein interaction may also impact cascade signaling pathways in PCa development and disease progression.

Perspective on the regulatory role of UGT2B28 as a conjugating enzyme in the progression of prostate cancer

: The maintenance of steroid homeostasis in the prostate is critical, with perturbation of steroidogenesis contributing to the modulation of active ligands in the androgen pool. In this scenario, enzymes catalysing the biosynthesis, inactivation and conjugation of steroids are the key players, regulating active ligand levels and in so doing, the activation of the androgen receptor (AR). The glucuronidation of potent ligands renders them unable to bind the AR, allowing the secretion of conjugated steroids. Uridine diphosphate glucuronosyltransferase 2B type 28 (UGT2B28), one of the UGT enzymes catalyzing the glucuronidation of androgens, has recently been given a prominent role in the regulation of prostate steroidogenesis—one which stands in contrast to the accepted dogma that lower androgen levels resulting from increased conjugation are associated with decreased prostate cancer (PCa) risk and disease progression. Increased DHT and its precursors, T and androstenediol, were reported to be associated with increased UGT2B28 tumor expression levels, linked to lower PSA levels but higher Gleason scores and increased PCa risk. In addition, the complete deletion of UGT2B28, was associated with decreased T, DHT and glucuronide derivatives when compared to patients carrying both alleles. UGT2B28 is encoded by a single gene giving rise to UGT2B28 type I which catalyses androgen glucuronidation and, due to alternative splicing, also produces two distinct transcripts, UGT2B28 type II and III. Type II with its premature stop codon, is devoid of the cofactor binding domain while type III is devoid of the substrate binding domain, both catalytically inactive, truncated proteins. Increased UGT2B28 mRNA expression was reported in primary tumours, and while variable nuclear and strong cytoplasmic staining were distinctive of tumour cells, the expression levels and compartmentalization of the specific protein isoforms remain unknown. While increased expression of type I would contribute towards lowering androgen levels, increased expression of types II and III would not. The abundance of type III transcripts in multiple tissues may provide insight into a regulatory role with truncated isoforms possibly affecting androgen levels by regulating substrate and/or co-factor availability, dimerization or the formation of protein complexes with other UGTs, while protein-protein interaction may also impact cascade signaling pathways in PCa development and disease progression.

Divergent Expression and Metabolic Functions of Human Glucuronosyltransferases through Alternative Splicing

Maintenance of cellular homeostasis and xenobiotic detoxification is mediated by 19 human UDP-glucuronosyltransferase enzymes (UGTs) encoded by ten genes that comprise the glucuronidation pathway. Deep RNA sequencing of major metabolic organs exposes a substantial expansion of the UGT transcriptome by alternative splicing, with variants representing 20% to 60% of canonical transcript expression. Nearly a fifth of expressed variants comprise in-frame sequences that may create distinct structural and functional features. Follow-up cell-based assays reveal biological functions for these alternative UGT proteins. Some isoforms were found to inhibit or induce inactivation of drugs and steroids in addition to perturbing global cell metabolism (energy, amino acids, nucleotides), cell adhesion, and proliferation. This work highlights the biological relevance of alternative UGT expression, which we propose increases protein diversity through the evolution of metabolic regulators from specific enzymes.

Genome-wide microarray screening for Bombyx mori genes related to transmitting the determination outcome of whether to produce diapause or nondiapause eggs.

The bivoltine silkworm Bombyx mori (Lepidoptera: Bombycidae) exhibits a maternally controlled embryonic diapause. Maternal silkworms decide whether to lay diapause or nondiapause eggs depending on environmental factors such as the temperature and photoperiod during the egg and larval stages, and then induce diapause eggs during the pupal stage. However, little is known about the molecular mechanism that conveys the outcome of whether to produce diapause or nondiapause eggs from the egg or larval stages to the pupal stage. This study used microarray analysis to investigate differentially expressed genes in the larval brains of diapause- and nondiapause-egg producers, to which bivoltine silkworms were destined by thermal or photic stimulation during the egg stage. The cytochrome P450 18a1 and Krüppel homolog 1 genes were upregulated in producers of diapause eggs compared with those of nondiapause eggs under both experimental conditions. Cytochrome P450 18a1 encodes a key enzyme for steroid hormone inactivation and Krüppel homolog 1 is an early juvenile hormone-inducible gene that mediates the repression of metamorphosis. The upregulation of these genes during the larval stage might be involved in the signaling pathway that transmits information about the diapause program from the egg stage to the pupal stage in the silkworm.

Glucocorticoid receptor is involved in the breed-dependent transcriptional regulation of 3β-hydroxysteroid dehydrogenase in the liver of preweaning piglets

BackgroundHepatic 3β-hydroxysteroid dehydrogenase (3β-HSD) plays an important role in steroid inactivation and catabolism. Serum concentrations of steroid hormones differ significantly between breeds in pigs, however the molecular mechanism regulating hepatic 3β-HSD expression in different breeds of pigs is poorly understood. In the present study, we used preweaning purebred male Large White (LW) and Erhualian (EHL) piglets as model to investigate the breed difference in the expression and regulation of 3β-HSD gene in porcine liver.ResultsThe hepatic expression of 3β-HSD mRNA was significantly lower (P < 0.01) in EHL piglets compared to that in LW piglets. Significant breed differences were detected for the hepatic expression of transcription factors such as androgen receptor (AR), glucocorticoid receptor (GR), and CCAAT/enhancer binding protein β (C/EBPβ). The nucleoprotein contents of AR (P < 0.05), GR (P < 0.01) and phospho-Ser211GR (P < 0.01) were significantly higher in the liver of EHL piglets. Chromatin immunoprecipitation (ChIP) assay demonstrated significantly lower binding of GR, but not AR or C/EBPβ, to 3β-HSD gene promoter in EHL piglets (P < 0.05). GR was not detected to interact with C/EBPβ or AR in the co-immunoprecipitation analysis.ConclusionsThese results indicate that GR binding to 3β-HSD promoter is involved in the breed-dependent 3β-HSD expression in the liver of piglets.

Expression profile analysis of silkworm P450 family genes after phoxim induction

Silkworm (Bombyx mori) is an important economic insect and a model species for Lepidopteran. Each year, O,O-diethyl O-(alpha-cyanobenzylideneamino) phosphorothioate (phoxim) pesticide poisoning in China results in huge economic losses in sericulture. Silkworm fat body is the main organ for nutrient storage, energy supply, intermediary metabolism, and detoxification. Microarray analysis of silkworm Cytochrome P450 detoxification enzyme genes revealed that all tested P450 4 (CYP4) family genes are expressed in the fat body. Quantitative Real-time PCR (QRT-PCR) was used to detect the expression of CYP4 family genes in silkworm fat body 0, 24, 48, and 72 h after phoxim exposure. The expression levels of silkworm molting hormone synthesis-related genes started to change 24 h after phoxim exposure, with those of CYP302A1, CYP306A1, and CYP314A1 being elevated by 1.38-, 1.33-, and 2.10-fold, respectively. The CYP18A1 gene that participates in steroid hormone inactivation and the CYP15C1 gene that participates in the epoxidation during the synthesis of juvenile hormone (JH) from methyl farnesoate (MF) were increased by 3.85- and 7.82-fold, respectively. Phylogenetic analysis indicated that these endogenous hormone metabolism-related genes belong to CYP mito clan and clan 2, and that phoxim exposure may affect silkworm development and metamorphosis. The CYP4, CYP6, and CYP9 families all showed some degrees of increases in gene expression; among them, CYP49A1, CYP4L6, CYP6AB4, CYP9G3, CYP9A19, and CYP9A22's transcription levels were significantly upregulated to 12.77-, 2.64-, 2.42-, 4.06-, 3.32-, and 2.98-fold, respectively, of the control levels. In the fat body, CYP49A1, CYP6AB4, CYP9A19, and CYP9A22 were constantly expressed at high levels after 24, 48, and 72 h of phoxim treatments; according to phylogenetic analysis, these genes belong to detoxification-related clan 3 and clan 4 CYP families. These genes may participate in the metabolism of phoxim in silkworm fat body. The results obtained in this study provide a basis for future in-depth investigations of insect P450 family genes in metabolic detoxification.

CYP18A1 regulates tissue-specific steroid hormone inactivation in Bombyx mori

Insect development and metamorphosis are regulated by two major hormones, juvenile hormone and ecdysteroids. Despite being the key regulator of insect developmental transitions, the metabolic pathway of the primary steroid hormone, 20-hydroxyecdysone (20E), especially its inactivation pathway, is still not completely elucidated. A cytochrome P450 enzyme, CYP18A1, has been shown to play key roles in insect steroid hormone inactivation through 26-hydroxylation. Here, we identified two CYP18 (BmCYP18A1 and BmCYP18B1) orthologs in the lepidopteran model insect, Bombyx mori. Interestingly, BmCYP18A1 gene is predominantly expressed in the middle silk gland (MSG) while BmCYP18B1 expresses ubiquitously in B. mori. BmCYP18A1 is induced by 20E in vitro, suggesting its role in 20E metabolism. Using the binary Gal4/UAS transgenic system, we ectopically overexpressed BmCYP18A1 in a MSG-specific manner with a Sericin1-Gal4 (Ser-Gal4) driver or in a ubiquitous manner with an Actin3-Gal4 (A3-Gal4) driver. Ectopic overexpression of BmCYP18A1 in MSG or in all tissues resulted in developmental arrestment of transgenic animals during the final instar larval stage. The 20E titers in the transgenic animals expressing BmCYP18A1 were lower compared to the levels in the control animals. Although the biological significance of MSG-specific expression of BmCYP18A1 is unclear, our results provide the first evidence that BmCYP18A1, which is conserved in most arthropods, is involved in a tissue-specific steroid hormone inactivation in B. mori.

Hepatic steroid metabolizing enzyme activity during early, mid, and late bovine pregnancy

The objective was to examine hepatic steroid inactivating enzymes throughout gestation and determine the effect of early to mid-gestation maternal nutrient restriction followed by realimentation on the activity of these enzymes. On day 30 of gestation, cows were assigned to dietary treatments: control (CON; 100% National Research Council; n = 18) and restricted (RES; 60% National Research Council; n = 30). On day 85, cows were slaughtered (CON, n = 6 and RES, n = 6), remained on control (CC, n = 12) and restricted (RR, n = 12), or were realimented to control (RC, n = 11). On day 140, cows were slaughtered (CC, n = 6; RR, n = 6; RC, n = 5), remained on control (CCC, n = 6; RCC, n = 5), or were realimented to control (RRC, n = 6). On day 254, all remaining cows were slaughtered. Jugular blood samples were collected before the slaughter for steroid analysis. At slaughter, maternal liver samples were collected for hepatic enzyme activity analysis. Activity of cytochrome P450 3A decreased (P = 0.05) from mid- to late-gestation. Peroxisome proliferator-activated receptor alpha DNA binding activity was increased (P < 0.01) on day 140 and 254 of gestation vs day 85. Concentrations of estradiol-17β (E2) increased (P < 0.01) as gestation proceeded, whereas progesterone concentrations (P4) tended to increase (P = 0.06) from mid- to late-gestation. Activity of cytochrome P450 1A and 2C were decreased (P < 0.05) in nutrient restricted cows vs control, whereas concentrations of E2 were increased (P < 0.05) in nutrient restricted cows vs control. A longer period of nutrient realimentation from mid- to late-gestation increased (P < 0.05) aldo-keto reductase 1C activity and decreased (P < 0.05) P4 concentrations compared with the shorter period of nutrient realimentation. In addition, significant negative correlations were observed for cytochrome P450 3A activity vs E2 (r2 = −0.30; P < 0.05) and aldo-keto reductase 1C activity vs P4 (r2 = −0.29; P < 0.05). The present study implicates hepatic steroid inactivation in the partial modulation of peripheral concentrations of E2 and P4 during gestation.

Cloning, characterization and functional expression of Taenia solium 17 beta-hydroxysteroid dehydrogenase

The 17β-hydroxysteroid dehydrogenases (17β-HSD) are key enzymes involved in the formation (reduction) and inactivation (oxidation) of sex steroids. Several types have been found in vertebrates including fish, as well as in invertebrates like Caenorhabditis elegans, Ciona intestinalis and Haliotis diversicolor supertexta. To date limited information is available about this enzyme in parasites. We showed previously that Taenia solium cysticerci are able to synthesize sex steroid hormones in vitro when precursors are provided in the culture medium. Here, we identified a T. solium 17β-HSD through in silico blast searches in the T. solium genome database. This coding sequence was amplified by RT-PCR and cloned into the pcDNA 3.1(+) expression vector. The full length cDNA contains 957bp, corresponding to an open reading frame coding for 319 aa. The highest identity (84%) at the protein level was found with the Echinococcus multilocularis 17β-HSD although significant similarities were also found with other invertebrate and vertebrate 17β-HSD sequences. The T. solium Tsol-17βHSD belongs to the short-chain dehydrogenase/reductase (SDR) protein superfamily.HEK293T cells transiently transfected with Tsol17β-HSD induced expression of Tsol17β-HSD that transformed 3H-androstenedione into testosterone. In contrast, 3H-estrone was not significantly transformed into estradiol. In conclusion, T. solium cysticerci express a 17β-HSD that catalyzes the androgen reduction. The enzyme belongs to the short chain dehydrogenases/reductase family and shares motifs and activity with the type 3 enzyme of some other species.

N-Benzoyl anthranilic acid derivatives as selective inhibitors of aldo–keto reductase AKR1C3

Human aldo–keto reductases AKR1C1–AKR1C3 are involved in the biosynthesis and inactivation of steroid hormones and prostaglandins and thus represent attractive targets for the development of new drugs. We synthesized a series of N-benzoyl anthranilic acid derivatives and tested their inhibitory activity on AKR1C enzymes. Our data show that these derivatives inhibit AKR1C1–AKR1C3 isoforms with low micromolar potency. In addition, five selective inhibitors of AKR1C3 were identified. The most promising inhibitors were compounds 10 and 13, with IC50 values of 0.31μM and 0.35μM for AKR1C3, respectively.

P3-15-02: The Change of Bone Turnover Markers during Neoadjuvant Anastrozole Versus Exemestane: A Randomized Single-Center Study.

Abstract Background: Non-steroidal aromatase inhibitors including anastrozole and letrozole have been reported to increase the rate of bone turnover, accelerate loss of bone, and increase the incidence of fractures. Although steroidal inactivator exemestane has similar inhibitory effect on the aromatization, animal studies have shown a weak but potentially important anabolic effect of exemestane metabolites, which might lead to decreased bone resorption. This randomized trial was conducted to compare the effect of exemestane and anastorozole on bone turnover markers. Patients and Methods: Fifty-two postmenopausal women with ER-positive, HER2−negative, invasive, nonmetastatic, and operable breast cancer were randomly assigned to neoadjuvant exemestane (25 mg daily) or anastrozole (1 mg daily) for 4 months. The primary endpoint was change in bone turnover markers including resorption markers urinary and serum N-telopeptide (NTX) and the formation markers serum bone alkaline phosphatase (BAP). The secondary endpoint was tumor objective response (OR) assessed by both caliper and ultrasound. Comparisons were also made of breast conservation rate and adverse events. Results: The changes in serum NTX from baseline to week 16 were not statistically different between anastrozole and exemestane. The changes in urinary NTX from baseline to week 16 were also not statistically different between both groups .BAP did not show any significant increase in the exemestane group (10.8%; p=0.21), whereas the increase in BAP from baseline to week 16 in the anastrozole group was 15.0% and marginally significant (p=0.05). There were no significant differences in OR in the intent-to-treat population between patients receiving anastrozole or exemestane (45.8% vs. 39.2%; p=0.63). The OR was similar between the patients with a baseline Ki67 index of ≥15% and &amp;lt;15% in exemestane group (37.5% vs. 41.2%; p=0.86). In anastrozole group the OR in patients with a baseline Ki67 index of ≥15% and &amp;lt;15% was 44.4% and 53.8%, respectively(p=0.66). Breast conservation rate was similar between anastrozole and exemestane (62.5% vs. 67.9%; p=0.68). Treatment was well tolerated and much the same for both groups. Conclusions: There is no significant differences of the change in bone turnover markers between anastrozole and exemestane. These results indicate exemestane had no anabolic effects which would lead to fewer adverse effects on bone. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-15-02.

Structure-activity relationships for hydroxylated polychlorinated biphenyls as inhibitors of the sulfation of dehydroepiandrosterone catalyzed by human hydroxysteroid sulfotransferase SULT2A1.

Polychlorinated biphenyls (PCBs) are persistent worldwide pollutants that are of concern due to their bioaccumulation and health effects. Metabolic oxidation of PCBs results in the formation of hydroxylated metabolites (OHPCBs). Among their biological effects, OHPCBs have been shown to alter the metabolism of endocrine hormones, including inhibition of mammalian cytosolic sulfotransferases (SULTs) that are responsible for the inactivation of thyroid hormones and phenolic steroids (i.e., hSULT1A1, hSULT1B1, and hSULT1E1). OHPCBs also interact with a human hydroxysteroid sulfotransferase that plays a role in the sulfation of endogenous alcohol-containing steroid hormones and bile acids (i.e., hSULT2A1). The objectives of our current study were to examine the effects of a series of OHPCB congeners on the activity of hSULT2A1 and to develop a three-dimensional quantitative structure-activity relationship (3D-QSAR) model for OHPCBs as inhibitors of the enzyme. A total of 15 OHPCBs were examined, and the sulfation of 1 μM [(3)H] dehydroepiandrosterone (DHEA) was utilized as a model reaction catalyzed by the enzyme. All 15 OHPCBs inhibited the sulfation of DHEA, with IC(50) values ranging from 0.6 μM to 96 μM, and eight of these OHPCBs were also substrates for the enzyme. Comparative molecular field analysis (CoMFA) provided a predictive 3D-QSAR model with a q(2) value of 0.697 and an r(2) value of 0.949. The OHPCBs that had the highest potency as inhibitors of DHEA sulfation were those with a 3, 5-dichloro-4-hydroxy substitution pattern on the biphenyl ring system, and these congeners were also substrates for sulfation catalyzed by hSULT2A1.

Human type 2 17beta-hydroxysteroid dehydrogenase in umbilical vein and artery endothelial cells: differential inactivation of sex steroids according to the vessel type

The human placenta produces high amounts of estradiol. 17β-hydroxysteroid dehydrogenase type 2 (17βHSD2) is expressed by placental endothelial cells and was proposed to regulate sex hormone levels. Previous results obtained in term placenta suggested that 17βHSD2 expression and activity differ among umbilical cord vessels. In this study, 17βHSD2 expression level and enzymatic activity, and estrogen receptor α and β expression levels, were measured in endothelial cell cultures from umbilical arteries (HUAEC) and vein (HUVEC) using real-time quantitative PCR, western blot, and radiolabeled steroids. 17βHSD2-specific activities were also measured in proximal and distal segments of freshly isolated umbilical cord arteries and vein. 17βHSD2 mRNA level and activity were higher in HUAEC than in HUVEC. Activity was higher in umbilical arteries than in the umbilical vein. In arteries, enzymatic activity was higher near the placenta, suggesting a gradient of expression. No difference was found in ERα expression, whereas ERβ was expressed at a higher level in HUAEC than in HUVEC. Expression profiles of estrogen receptors and 17βHSD2 suggest a vessel type-specific response to estrogens. Our data support a differential modulation of biologically active sex steroid levels according to the vessel type in the foeto-placental unit, with apparent higher inactivation in the arterial system.

Genome-wide copy number variation (CNV) in patients with autoimmune Addison's disease

BackgroundAddison's disease (AD) is caused by an autoimmune destruction of the adrenal cortex. The pathogenesis is multi-factorial, involving genetic components and hitherto unknown environmental factors. The aim of the present study was to investigate if gene dosage in the form of copy number variation (CNV) could add to the repertoire of genetic susceptibility to autoimmune AD.MethodsA genome-wide study using the Affymetrix GeneChip® Genome-Wide Human SNP Array 6.0 was conducted in 26 patients with AD. CNVs in selected genes were further investigated in a larger material of patients with autoimmune AD (n = 352) and healthy controls (n = 353) by duplex Taqman real-time polymerase chain reaction assays.ResultsWe found that low copy number of UGT2B28 was significantly more frequent in AD patients compared to controls; conversely high copy number of ADAM3A was associated with AD.ConclusionsWe have identified two novel CNV associations to ADAM3A and UGT2B28 in AD. The mechanism by which this susceptibility is conferred is at present unclear, but may involve steroid inactivation (UGT2B28) and T cell maturation (ADAM3A). Characterization of these proteins may unravel novel information on the pathogenesis of autoimmunity.

Deletions of the Androgen-MetabolizingUGT2BGenes Have an Effect on Circulating Steroid Levels and Biochemical Recurrence after Radical Prostatectomy in Localized Prostate Cancer

The prognostic relevance of inherited variations in hormone-related genes in the context of prostate cancer (PCa) progression has not been well studied. Of these, UDP-glucuronosyltransferase (UGT) gene products lead to inactivation of steroids. Our objective was to determine whether polymorphisms in five UGT genes, involved in steroid metabolism, are associated with the risk of biochemical recurrence after radical prostatectomy (RP) and to examine their relationship with hormonal exposure. The study included 526 Caucasian and 320 Asian men who underwent RP for clinically localized PCa. The relationship between genotypes and biochemical recurrence were assessed with multivariate Cox proportional hazard models. Plasma steroids were measured using specific and sensitive mass spectrometry-based methods. The presence of at least two deleted copies of UGT2B17 and UGT2B28 genes resulted in a hazard ratio of 2.26 (95% confidence interval = 1.41-3.61; P = 0.0007) for Caucasians and 2.16 (95% confidence interval = 1.24-3.73; P = 0.006) for Asians. A positive association was observed only between UGT2B17 deletion and the Gleason score in Asians, whereas no other interaction was shown with prostate-specific antigen, Gleason score, and TNM (tumor node metastasis) staging. Patients carrying UGT2B17 deletions and those with three deleted UGT2B copies had significantly lower androgen glucuronides, in support of an altered androgen metabolism. This study is the first to recognize the prognostic significance of common deletions in steroid inactivation pathways in localized PCa after RP. Alteration of circulating steroid levels associated with UGT2B gene deletions further support the notion that such inherited genomic deletions have the potential to modify hormonal exposure and risk of recurrence.

Steroid Hormone Inactivation Is Required during the Juvenile-Adult Transition in Drosophila

Steroid hormones are systemic signaling molecules that regulate juvenile-adult transitions in both insects and mammals. In insects, pulses of the steroid hormone 20-hydroxyecdysone (20E) are generated by increased biosynthesis followed by inactivation/clearance. Although mechanisms that control 20E synthesis have received considerable recent attention, the physiological significance of 20E inactivation remains largely unknown. We show that the cytochrome P450 Cyp18a1 lowers 20E titer during the Drosophila prepupal to pupal transition. Furthermore, this reduction of 20E levels is a prerequisite to induce βFTZ-F1, a key factor in the genetic hierarchy that controls early metamorphosis. Resupplying βFTZ-F1 rescues Cyp18a1-deficient prepupae. Because Cyp18a1 is 20E-inducible, it appears that the increased production of steroid is responsible for its eventual decline, thereby generating the regulatory pulse required for proper temporal progression of metamorphosis. The coupling of hormone clearance to βFTZ-F1 expression suggests a general mechanism by which transient signaling drives unidirectional progression through a multistep process.

CYP18A1, a key enzyme of Drosophila steroid hormone inactivation, is essential for metamorphosis

Ecdysteroids are steroid hormones, which coordinate major developmental transitions in insects. Both the rises and falls in circulating levels of active hormones are important for coordinating molting and metamorphosis, making both ecdysteroid biosynthesis and inactivation of physiological relevance. We demonstrate that Drosophila melanogaster Cyp18a1 encodes a cytochrome P450 enzyme (CYP) with 26-hydroxylase activity, a prominent step in ecdysteroid catabolism. A clear ortholog of Cyp18a1 exists in most insects and crustaceans. When Cyp18a1 is transfected in Drosophila S2 cells, extensive conversion of 20-hydroxyecdysone (20E) into 20-hydroxyecdysonoic acid is observed. This is a multi-step process, which involves the formation of 20,26-dihydroxyecdysone as an intermediate. In Drosophila larvae, Cyp18a1 is expressed in many target tissues of 20E. We examined the consequences of Cyp18a1 inactivation on Drosophila development. Null alleles generated by excision of a P element and RNAi knockdown of Cyp18a1 both result in pupal lethality, possibly as a consequence of impaired ecdysteroid degradation. Our data suggest that the inactivation of 20E is essential for proper development and that CYP18A1 is a key enzyme in this process.

Effect of cytochrome P450 and aldo-keto reductase inhibitors on progesterone inactivation in primary bovine hepatic cell cultures

Progesterone is required for maintenance of pregnancy, and peripheral concentrations of progesterone are affected by both production and inactivation. Hepatic cytochrome P450 (EC 1.14.14.1) and aldo-keto reductase (EC 1.1.1.145–151) enzymes play a pivotal role in the first step of steroid inactivation, which involves the addition of hydroxyl groups to various sites of the cyclopentanoperhydrophenanthrene nucleus. The current objective was to discern the proportional involvement of hepatic progesterone inactivating enzymes on progesterone decay using specific enzyme inhibitors. Ticlopidine, diltiazem, curcumin, dicumarol, and naproxen were used because of their selective inhibition of cytochrome P450s, aldo-keto reductases, and glucuronosyltransferases. Liver biopsies were collected from 6 lactating Holstein dairy cows, and cells were dissociated using a nonperfusion technique. Confluent wells were preincubated for 4h with enzyme inhibitor and then challenged with progesterone for 1h. Cell viability was unaffected by inhibitor treatment and averaged 84±1%. In control wells, 50% of the progesterone had been inactivated after a 1-h challenge with 5ng/mL of progesterone. Preincubation with curcumin, ticlopidine, or naproxen caused the greatest reduction in progesterone inactivation compared with controls and averaged 77, 39, or 37%, respectively. Hydroxylation of 4-nitrophenol to 4-nitrocatechol in intact cells was inhibited by approximately 65% after treatment with curcumin or ticlopidine. Glucuronidation of phenol red or 4-nitrocatechol in intact cells was inhibited by treatment with curcumin, dicumarol, or naproxen. In cytoplasmic preparations, aldo-keto reductase 1C activity was inhibited by curcumin, dicumarol, or naproxen treatment. Microsomal cytochrome P450 2C activity was inhibited by treatment with curcumin or ticlopidine, whereas cytochrome P450 3A activity was inhibited by treatment with curcumin or diltiazem. The contribution of cytochrome P450 2C and cytochrome P450 3A enzymes to progesterone inactivation in bovine hepatic cell cultures was 40 and 15%, respectively. Depending on the inhibitor used, it would appear that the aldo-keto reductase enzymes contribute approximately 40% to the observed progesterone inactivation, although a portion of this inactivation may be attributed to the loss of glucuronosyltransferase activity. Future work focusing on decreasing the activity of these enzymes in vivo could lead to an increase in the bioavailability of progesterone.