Steroidal Inactivator Research Articles

Diet-induced alterations in hepatic progesterone (P4) catabolic enzyme activity and P4 clearance rate in lactating dairy cows

Elevated rates of steroid clearance may lead to lower reproductive success in several mammalian species. Cytochrome P450 (EC 1.14.14.1) and aldo-keto reductases (AKR; EC 1.1.1.145-151) are involved in the first phase of steroid inactivation, before second phase conjugation and excretion of the steroid metabolite. The current objectives were to determine liver blood flow (LBF), hepatic enzyme activity, and metabolic clearance rate (MCR) of progesterone (P(4)) in dairy cows consuming isoenergetic and isonitrogenous diets formulated to cause divergent insulin secretion. Insulin concentrations increased by 22% in cows fed the high cornstarch diet, and both cytochrome P450 2C and cytochrome P450 3A activities were decreased (P<0.05) by approximately 50%, while AKR1C tended (P<0.10) to be lower in cows fed the high cornstarch diet. LBF was similar between the two diets (1891+/-91 l/h). MCR of P(4) tended (P<0.10) to be lower in cows fed the high cornstarch diet (25+/-5 l/hxBW(0.75)) versus the high fiber diet (40+/-6 l/hxBW(0.75)). The half-life of P(4) was increased (P<0.05) in cows fed the high cornstarch diet (73+/-10 min) versus the high fiber diet (24+/-10 min). In summary, cows with elevated insulin concentrations and lower enzyme activity showed a decrease in P(4) clearance without any changes in LBF. This dietary relationship with hepatic enzyme activity may explain some of the observed alterations in steroid profiles during the estrous cycle or gestation of the high producing dairy cow.

A randomised study of the effects of anastrozole (A), letrozole (L) and exemestane (E) on bone turnover.

Abstract Abstract #1145 Background:&amp;#x2028; Aromatase inhibitors (AIs) reduce circulating oestrogen which increases bone turnover. L decreases circulating oestrogen levels to a greater degree than A. This study compares the effects of the non-steroidal aromatase inhibitors, A and L and the steroidal inactivator, E on bone turnover.&amp;#x2028; Patients and Methods:&amp;#x2028; This was an open, randomized pharmacodynamic study. Bone turnover markers were measured in 162 postmenopausal women with invasive ER +ve breast cancer. As part of their adjuvant hormone therapy, patients were randomized to receive either:&amp;#x2028; 16 weeks of A, 16 weeks of L or 16 weeks of E&amp;#x2028; Fasting blood and urine samples were collected at entry and after 12 and 16 weeks of each drug. Bone remodelling releases breakdown products of type 1 collagen including the terminal peptide fragments N-terminal telopeptides (NTx) and C-terminal telopeptides (CTx). An increase in these indicates bone resorption. Increases in type 1 pro-collagen peptides (N-terminal – PINP) and bone specific alkaline phosphatase (ALP) indicate bone formation. These were measured as was parathyroid hormone (PTH) which is an indirect measure of overall bone turnover.&amp;#x2028; Results:&amp;#x2028; A vs E vs L&amp;#x2028; A, E and L significantly increased the bone turnover markers - PINP, CTX and bone ALP.&amp;#x2028; E showed a greater change in bone turnover but there were no significant differences between the drugs.&amp;#x2028; &amp;#x2028; &amp;#x2028; &amp;#x2028; E resulted in a greater increase in PINP and uNTX compared to the non-steroidal AIs however these were of borderline significance.&amp;#x2028; PINP, CTX and bone ALP all have significantly higher than zero percentage changes, regardless of drug or drug type.&amp;#x2028; Conclusions:&amp;#x2028; AIs cause a significant increase in both bone breakdown and bone formation.&amp;#x2028; There is no evidence to suggest E has less effects on bone turnover than A or L. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1145.

A randomised study comparing the effects of anastrozole (A), letrozole (L), exemestane (E) and tamoxifen (T) on coagulation.

Abstract Abstract #1132 Background:&amp;#x2028; Aromatase inhibitors (AIs) reduce circulating oestrogens and so should have either no or a positive effect on coagulation . This study compares the effects of the non-steroidal aromatase inhibitors, A and L and the steroidal inactivator, E on coagulation.&amp;#x2028; Patients and Methods:&amp;#x2028; This was an open, randomized pharmacodynamic study. Plasma coagulation factors were measured in 120 postmenopausal women with invasive ER +ve breast cancer. As part of their adjuvant hormone therapy, patients were randomized to receive upfront therapy with either:&amp;#x2028; 16 weeks of A, 16 weeks of L or 16 weeks of E.&amp;#x2028; Fasting blood samples were collected at entry and after 12 and 16 weeks of each drug. AI patients were then switched to T and further samples measured after 8 months on T.&amp;#x2028; Plasminogen activator inhibitor (PAI) antigen, von Willebrand's Factor (vWF) antigen, antithrombin III (AT111), protein C, protein S total, protein S free, activated protein C, resistance (APCR), factor VIII and fibrinogen were measured.&amp;#x2028; Results:&amp;#x2028; Results expressed as % change from baseline.&amp;#x2028; E vs A vs L&amp;#x2028; Protein C. E caused a significant fall compared to A and L (-15.75 (-21.48, -10.02) vs -3.80 (-10.77, 3.16) vs -3.63 (-10.50, 3.25)) respectively. p = 0.008.&amp;#x2028; ATIII. E caused a significant fall from baseline (-8.55 (-13.33, -3.79))&amp;#x2028; Protein S Free. E + A caused a significant increase from baseline (E 6.90 (1.98, 11.82), A 7.36 (1.37, 13.34).&amp;#x2028; Steroidal vs non-steroidal:&amp;#x2028; Protein C. A significantly greater fall was seen with E than A+L (-15.75 (-21.45, -10.05) vs -3.71 (-8.58, 1.15)) p = 0.002.&amp;#x2028; vWF A+L caused a significant increase from baseline (6.52 (0.66, 12.38) but this was not statistically different to E 5.02 (-1.88, 11.91)).&amp;#x2028; ATIII A significant decrease from baseline was seen with A + L, -4.54 (-8.64, -0.43) but no difference from E 8.55 (13.31, -3.70) p=0.21.&amp;#x2028; Protein S Free There was a significant increase from baseline in both groups (A+L: 5.70 (1.47, 9.92), E: 6.90 (1.98, 11.81), but no difference between the groups p=0.72.&amp;#x2028; T effect:&amp;#x2028; AT111 and protein C levels fell significantly on T whereas protein S free increased on T. Prior A, E or L had no significant impact on post-tamoxifen results.&amp;#x2028; Conclusions:&amp;#x2028; No significant differences in any coagulation factors were seen between A and L&amp;#x2028; E caused a significant fall in protein C and ATIII.&amp;#x2028; The non-steroidal AIs caused a significant increase in vWF.&amp;#x2028; T caused a significant fall in protein C and ATIII and a significant increase in protein S free.&amp;#x2028; No AI significantly influenced post-tamoxifen results.&amp;#x2028; These drugs have significant effects on coagulation which helps to explain their clinical effects on thrombotic and thromboembolic disease. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1132.

The Use of Metabolising Systems for In Vitro Testing of Endocrine Disruptors

Legislation and prospective legislative proposals in for instance the USA, Europe, and Japan require, or may require that chemicals are tested for their ability to disrupt the hormonal systems of mammals. Chemicals found to test positive are considered to be endocrine active substances (EAS) and may be putative endocrine disruptors (EDs). To date, there is still little or no experience with incorporating metabolic and toxicokinetic aspects into in vitro tests for EAS. This is a situation in sharp contrast to genotoxicity testing, where in vitro tests are routinely conducted with and without metabolic capacity. Originally prepared for the Organisation of Economic Cooperation and Development (OECD), this detailed review paper reviews why in vitro assays for EAS should incorporate mammalian systems of metabolism and metabolic enzyme systems, and indicates how this could be done. The background to ED testing, the available test methods, and the role of mammalian metabolism in the activation and the inactivation of both endogenous and exogenous steroids are described. The available types of systems are compared, and the potential problems in incorporating systems in in vitro tests for EAS, and how these might be overcome, are discussed. Lastly, some recommendations for future activities are made.

Substrate uptake and metabolism are preserved in hypertrophic caveolin-3 knockout hearts

Caveolin-3 (Cav3), the primary protein component of caveolae in muscle cells, regulates numerous signaling pathways including insulin receptor signaling and facilitates free fatty acid (FA) uptake by interacting with several FA transport proteins. We previously reported that Cav3 knockout mice (Cav3KO) develop cardiac hypertrophy with diminished contractile function; however, the effects of Cav3 gene ablation on cardiac substrate utilization are unknown. The present study revealed that the uptake and oxidation of FAs and glucose were normal in hypertrophic Cav3KO hearts. Real-time PCR analysis revealed normal expression of lipid metabolism genes including FA translocase (CD36) and carnitine palmitoyl transferase-1 in Cav3KO hearts. Interestingly, myocardial cAMP content was significantly increased by 42%; however, this had no effect on PKA activity in Cav3KO hearts. Microarray expression analysis revealed a marked increase in the expression of genes involved in receptor trafficking to the plasma membrane, including Rab4a and the expression of WD repeat/FYVE domain containing proteins. We observed a fourfold increase in the expression of cellular retinol binding protein-III and a 3.5-fold increase in 17beta-hydroxysteroid dehydrogenase type 11, a member of the short-chain dehydrogenase/reductase family involved in the biosynthesis and inactivation of steroid hormones. In summary, a loss of Cav3 in the heart leads to cardiac hypertrophy with normal substrate utilization. Moreover, a loss of Cav3 mRNA altered the expression of several genes not previously linked to cardiac growth and function. Thus we have identified a number of new target genes associated with the pathogenesis of cardiac hypertrophy.

Inhibition of SULT2B1b expression alters effects of 3β‐hydroxysteroids on cell proliferation and steroid hormone receptor expression in human LNCaP prostate cancer cells

Sulfation is an important steroid inactivation in human tissues. Sulfotransferase (SULT) 2B1b selectively conjugates 3beta-hydroxysteroids and is expressed in epithelial cells of normal and cancerous prostate tissues. Dehydroepiandrosterone (DHEA) and Delta(5)-androstenediol (Delta(5)-Adiol) sulfation prevents their conversion to more potent androgens and estrogens in tissues although both compounds may also be biologically active. SULT2B1b expression and activity were inhibited >85% in human LNCaP prostate adenocarcinoma cells using short interference RNA (siRNA). The effects of treating control and SULT2B1b-deficient LNCaP cells with DHEA, Delta(5)-Adiol, and 5alpha-androstane-3beta-17beta-diol (Anstane-diol) on cellular proliferation, estrogen receptors (ERs), androgen receptor (AR), and prostate specific antigen protein levels were examined. Physiological concentrations of DHEA and Delta(5)-Adiol increased proliferation of control cells and the proliferative effects were significantly increased in SULT2B1b-siRNA cells. DHEA, but not Delta(5)-Adiol increased AR levels at concentrations >/=1,000 nM in SULT2B1b-siRNA cells but not in control LNCaP cells. ER-alpha levels were not affected with any of the compounds tested. Physiological concentrations of DHEA and Delta(5)-A-diol decreased ER-beta levels in control cells and had significantly greater effects in SULT2B1b-siRNA cells. In contrast, Anstane-diol had no effect on AR or ER-alpha levels but induced more elevation of ER-beta levels in SULT2B1b-siRNA cells at concentrations >/=1,000 nM. SULT2B1b is involved in regulating prostate cell responsiveness to DHEA and Delta(5)-Adiol. Inhibition of SULT2B1b increased cell proliferation and ER-beta repression after treatment with physiological levels of DHEA and Delta(5)-Adiol indicating that SULT2B1b has an inhibitory effect on DHEA and Delta(5)-Adiol activity.

Structural and biochemical characterization of human orphan DHRS10 reveals a novel cytosolic enzyme with steroid dehydrogenase activity.

To this day, a significant proportion of the human genome remains devoid of functional characterization. In this study, we present evidence that the previously functionally uncharacterized product of the human DHRS10 gene is endowed with 17beta-HSD (17beta-hydroxysteroid dehydrogenase) activity. 17beta-HSD enzymes are primarily involved in the metabolism of steroids at the C-17 position and also of other substrates such as fatty acids, prostaglandins and xenobiotics. In vitro, DHRS10 converts NAD+ into NADH in the presence of oestradiol, testosterone and 5-androstene-3beta,17beta-diol. Furthermore, the product of oestradiol oxidation, oestrone, was identified in intact cells transfected with a construct plasmid encoding the DHRS10 protein. In situ fluorescence hybridization studies have revealed the cytoplasmic localization of DHRS10. Along with tissue expression data, this suggests a role for DHRS10 in the local inactivation of steroids in the central nervous system and placenta. The crystal structure of the DHRS10 apoenzyme exhibits secondary structure of the SDR (short-chain dehydrogenase/reductase) family: a Rossmann-fold with variable loops surrounding the active site. It also reveals a broad and deep active site cleft into which NAD+ and oestradiol can be docked in a catalytically competent orientation.

Improved Homogeneous Proximity-Based Screening Assay of Potential Inhibitors of 17β-Hydroxysteroid Dehydrogenases

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) are substrate- and tissue-specific isoenzymes that regulate activation and inactivation of steroid hormones. Up-regulation and downregulation in expression of 17beta-HSDs are linked to onset of many steroid-dependent diseases, such as colon, prostate, and breast cancer; thus 17beta-HSDs are potential drug screening targets. Currently their enzymatic activities are usually measured using laborious chromatographic separations followed by radioactive detection of substrate and product. We have previously reported the use of a homogeneous luminescence resonance energy transfer-based immunoassay for 17beta-estradiol in screening of potential inhibitors of 17beta-HSD type 1 (17beta-HSD-1). By replacing the previously used cell-based enzyme reactions with recombinant enzyme reactions the sensitivity of the screening assay improved considerably. In addition, the single assay was able to detect the influence of a tested compound not only on 17beta-HSD-1 but also on 17beta-HSD type 2 (17beta-HSD-2), catalyzing the opposite reaction. The screening results of the tested molecules obtained from the optimized immunoassay were very similar when compared with the results of high performance liquid chromatography separation analysis. The Z factors were 0.79 and 0.83 for 17beta-HSD-1 and 17beta-HSD-2 assays, respectively. Thus the immunoassay measuring samples converted with the recombinant enzymes was a very suitable method for primary high throughput screening, and it could be used also in further characterization of potential drugs.

NSABP B-42: A Clinical Trial to Determine the Efficacy of Five Years of Letrozole Compared with Placebo in Patients Completing Five Years of Hormonal Therapy Consisting of an Aromatase Inhibitor (AI) or Tamoxifen Followed by an AI in Prolonging Disease-Free Survival in Postmenopausal Women with Hormone Receptor—Positive Breast Cancer

Aromatase Inhibitors as Adjuvant Therapy Adjuvant hormonal therapy significantly improves disease-free and overall survival in patients with stage I, II, or III breast cancer and hormone receptor–positive tumors.1,2 For some time, 5 years of adjuvant tamoxifen has been the gold standard. However, recent results from studies evaluating the third generation aromatase inhibitors (AIs) as adjuvant therapy in postmenopausal women have challenged the primacy of tamoxifen for this group of patients.3-14 In patients with advanced-stage breast cancer, the third generation nonsteroidal AIs anastrozole and letrozole as well as the steroidal inactivator exemestane have been found superior to megestrol acetate as second-line therapy15-21 and at least equivalent (and possibly superior) to tamoxifen as first-line therapy.21-25 Based on these results, several clinical trials have evaluated or are currently evaluating AIs as adjuvant therapy in postmenopausal patients with early-stage breast cancer. In the adjuvant setting, AIs have demonstrated activity in 3 distinct clinical scenarios. In the first, an AI was compared with tamoxifen as initial adjuvant hormonal therapy in patients with resected operable breast cancer.3-5 The ATAC (Arimidex, Tamoxifen, Alone or in Combination) trial demonstrated that 5 years of anastrozole significantly improved disease-free survival (DFS) when compared with 5 years of tamoxifen.3,4 More recently, the BIG (Breast Intergroup) 1-98 trial also demonstrated similar benefit for 5 years of letrozole compared with 5 years of tamoxifen.5 In the second scenario, an AI was compared with tamoxifen in patients who had already received 2-3 years of adjuvant tamoxifen.6-10 In 3 randomized trials (the IES [Intergroup Exemestane Study] trial,6,7 the ABCSG [Austrian Breast and Colorectal Cancer Study Group]–8/ARNO [ArimidexNSABP B-42: A Clinical Trial to Determine the Efficacy of Five Years of Letrozole Compared with Placebo in Patients Completing Five Years of Hormonal Therapy Consisting of an Aromatase Inhibitor (AI) or Tamoxifen Followed by an AI in Prolonging Disease-Free Survival in Postmenopausal Women with Hormone Receptor–Positive Breast Cancer

Functional genome analysis indicates loss of 17beta-hydroxysteroid dehydrogenase type 2 enzyme in the zebrafish

Among the family of 17beta-hydroxysteroid dehydrogenases, the type 2 (17beta-HSD 2) is the main enzyme responsible for inactivation of estrogens and androgens, catalyzing the oxidation of the C17 hydroxyl group. 17beta-HSD 2 has been studied only in mammals, its occurrence and function in other vertebrates hardly known. We investigated the presence of homologs in non-mammalian species and found sequences of 17beta-HSD 2 and its closest homolog 11beta-HSD 2 in zebrafish ( Danio rerio), Takifugu rubripes, Tetraodon nigroviridis, Xenopus tropicalis and chicken databases. Furthermore, we cloned zebrafish 17beta-HSD 2 from ovarian tissue and found high expression also in the testis of adult fish and throughout embryogenesis. The enzyme, though, is inactive likely due to a non-sense N-terminal region including a dysfunctional cofactor binding motif. Replacement of the affected part by the corresponding human 17beta-HSD 2 sequence fully restored enzymatic activity. Comparison of all retrieved 17beta-HSD 2 sequences indicates that this functional loss may have occurred only in zebrafish, where steroid inactivation at position C17 seems to pursue without the protein studied. The closely related 11beta-HSD 2 is unlikely to substitute for 17beta-HSD 2 since in our hands it did not catalyze the respective oxidation of testosterone or estradiol.

Exemestane (Exe) as primary treatment in hormonosensible early breast cancer of the elderly: Preliminary results of a phase II multicenter open Italian study

10689 Background: In the late 90s two large phase III trials (GRETA &amp; CRC) showed that tamoxifen as sole treatment was a possible alternative to surgery in old women. Two Phase III neoadjuvant trials showed recently that letrozole is superior and anastrozole is not inferior to tamoxifen.A few phase II and a small fase III trials showed interesting results also with the steroidal inactivator exemestane. Methods: We are running a fase II open trial in early breast cancer patients aged over 70 with receptor rich tumors, treated with exemestane 25 mgs/day for 6 months before surgery. Primary endpoints are clinical response, breast conservative surgery (BCS) rate and safety. Results: Of the planned 100 patients we report preliminary results on 50 patients (mean age 81 , range 70–92) evaluable for clinical and mammographic response. 33/50 had a partial response (66%), 2/50 PD on treatment. Clinical basal mean size of tumors was 4.4 cms (4,81 in responders) and 2,67 cms the final in responders patients. Eight patients refused surgery, 12 are too early, 23/30 underwent BCS where 17 of them (74%) were unfit for BCS at basal evaluation. No remarcable exemestane related toxicity was recorded. Conclusions: As expected from published data of phase III trials in metastatic and adjuvant setting, exemestane appears to be very active also in the neoadjuvant setting. Probably there is no more need of large phase III trials in order to consider exemestane in the preoperative setting in rich receptor tumors. In old and frail patients this could also be considered as sole treatment. [Table: see text]

Is C‐26 hydroxylation an evolutionarily conserved steroid inactivation mechanism?

Sterols are essential components of virtually all higher eukaryotic organisms, though the exact identity of the dominating sterol varies between species, from the C-27 of cholesterol in vertebrates to the C-28 and C-29 sterols of plants and invertebrates. In addition to their role as structural components of cell membranes these sterols are also converted into a variety of biologically active hormones. This conversion generally involves modifications of the basic structure of the sterol by dealkylation, hydroxylation and/or isomerization. Recent studies have demonstrated that irreversible inactivation of both plant and insect hormones is achieved by a specific C-26 hydroxylation. The concept of sterol deactivation by 26-hydroxylation appears to be an example of an evolutionarily conserved mechanism that has persisted despite the widely varying requirements for sterols in the species where it has been detected.

Down-regulation of dehydroepiandrosterone sulfotransferase gene in human hepatocellular carcinoma

Differential display (DD) PCR cloning of differentially expressed genes in hepatocellular carcinoma (HCC) and adjacent unaffected tissue demonstrated preferential down-regulation of a vital sex steroid precursor (dehydroepiandrosterone sulfotransferase; DHEA-ST; SULT2A1) in HCC. SULT2A1 mRNA and/or protein expression in HCC were markedly reduced in 61 of 120 (50.8%) primary unicentric HCCs. The down-regulation was more frequent in grade III versus grade I HCC (68.1% versus 32.1%, P = 0.0025), and in stage 3 versus stage 1 HCC (62.7% versus 29.2%, P = 0.007). The lowered expression in tumor cells of SULT2A1 in HCC tissues involved in metabolism and/or inactivation of sex steroids is consistent with a regulatory role of the SULT2A1 gene product in the development and/or tumor cell differentiation and progression of human HCC. This suggestion is partly supported by our observations that the down-regulated SULT2A1 gene expression correlated with a higher grade and stage of HCC.

Cellular localization of mRNA expression of enzymes involved in the formation and inactivation of hormonal steroids in the mouse prostate.

It is well documented that several tissues, including the prostate, are actively involved in the local formation and inactivation of hormonal steroids. To identify the cell types involved in the formation and inactivation of androgens and estrogens in the ventral lobe prostate, we have localized by in situ hybridization (ISH) a large number of steroidogenic as well as steroid-inactivating enzyme mRNAs in the adult mouse prostate. In parallel studies, we also measured enzyme mRNA levels by quantitative real-time PCR (RT-PCR) in ventral lobe prostates. From the results obtained with quantitative RT-PCR, it appears that, with a few exceptions, the enzyme with low mRNA expression could not be detected by ISH. The following enzymes have been localized by ISH: 17beta-hydroxysteroid dehydrogenase (17beta-HSD) types 1, 2, 3, 4, 7, 8, 9, 10, and 11; 5alpha-reductase type 2; 5beta reductase type 1; P450 7alpha hydroxylase; estrogen sulfotransferase type 1; 11beta-HSD types 1 and 2; and UDP-glucuronosyltransferase 1A6. All of these mRNAs are expressed in the epithelial cells of prostatic acini. Several enzyme mRNAs were also localized in stromal cells. Types 1, 7, and 10 17beta-HSD, estrogen sulfotransferase type 1, and 11beta-HSD types 1 and 2 were found only in epithelial cells. The present results indicate that both epithelial and stromal cells in the mouse prostate play a role in local formation and inactivation of hormonal steroids.

Cellular Localization of mRNA Expression of Enzymes Involved in the Formation and Inactivation of Hormonal Steroids in the Mouse Prostate

Cellular Localization of mRNA Expression of Enzymes Involved in the Formation and Inactivation of Hormonal Steroids in the Mouse Prostate

First evidence of endocrine disruption in feral carp from the Ebro River

Feral carps ( Cyprinus carpio) were collected in spring 2001 from five sites along the lower course of Ebro River (Spain) with the aim of investigating the existence of endocrine-disrupting effects. Several findings (low gonadosomatic index (GSI), plasmatic vitellogenin (VTG), depressed levels of testosterone, and histological alterations in gonads) detected in male carps downstream of Zaragoza's sewage treatment plant (STP) strongly suggest that the concentration of sewage effluent in the area is a major causal factor leading to the detected estrogenic effects. Important alterations (viz. delayed maturation in females, indications of arrested spermatogenesis in males) were detected in carps from Flix, a heavily industrialized area. Low ovarian P-450 aromatase and reduced glucuronidation of testosterone and estradiol in males were observed in Zaragoza and Canal Imperial de Aragón—an agricultural area—which suggest decreased estrogen synthesis, and possibly, reduced sex hormone excretion in those organisms. These results were related to some in vitro assays aimed to assess the interference of model compounds (atrazin, vinclozolin, diuron, pp'-DDE, dicofol, triphenyltin, nonylphenol, and fenarimol) with the glucuronidation of testosterone and estradiol by liver microsomal fractions. The fungicide fenarimol (10–20 μM) and nonylphenol (50 μM) were found to significantly inhibit (20%) both activities at relatively low doses. Overall, this work provides the first evidence of the existence of significant alterations of the endocrine system of carps from the medium-low course of the Ebro River and demonstrates the ability of several chemicals to modulate the inactivation of endogenous steroids.

Breast cancer prevention—clinical trials strategies involving aromatase inhibitors

Estrogens and their metabolites have been implicated in both the initiation and the prevention of breast cancer. The reduction in breast cancer incidence seen in the tamoxifen arms of the four prospective trials to date has established the proof of principle that antagonizing estrogen is a potential means of reducing breast cancer risk. However, the areas to improve on these results include: (a) enhanced efficacy, (b) reduction in the incidence of receptor-negative tumors, (c) improved overall and endocrinological side effects, and (d) improved function on end-organs other than the breast. The aromatase inhibitors offer the potential to achieve these goals in part in the following ways: (a) greater reduction in risk of disease as evidenced by superior efficacy in advanced breast cancer and by inhibition of both initiation and promotion of breast cancer, (b) reduction in receptor-negative tumors by synergy with COX-2 inhibitors resulting in growth factor inhibition, anti-angiogenesis and inhibition of tumor-associated aromatase expression, (c) fewer vasomotor and urogenital abnormalities, and (d) reduced thromboembolism and cardiovascular complications and satisfactory effects on bone metabolism. Important differences may exist between non-steroidal reversible inhibitors and steroidal irreversible inactivators in particular related to the androgenic/anabolic effects of the steroidal inactivators. Pilot studies of aromatase inhibitors described elsewhere in this session have begun in healthy women with dense mammography, or a high-risk genetic and/or histocytopathologic profile, to determine potential efficacy, as well as effects on end-organ function. A number of phase three trials with aromatase inhibitors are also underway or in planning. Among these are the BRCA 1 and 2 study of exemestane versus placebo in unaffected postmenopausal carriers, the International Breast Intervention Study 2 (IBIS 2) of anastrozole versus placebo in women with a high-risk profile, and the National Cancer Institute of Canada’s Clinical Trial Group (NCIC CTG) study of exemestane with or without celecoxib versus placebo in women at risk of the disease. For premenopausal women, combination strategies of gonadotrophin agonists and aromatase inhibitors are being investigated. The potential of using low doses of aromatase inhibitors to lower “in breast” estrogen levels without unduly perturbing plasma concentrations is also being explored. The potential of the aromatase gene functioning as an oncogene within the breast may be tied to breast density which in turn may represent both a selection tool for elevated risk and an intermediate marker of prevention. The strong link between postmenopausal estrogen levels and breast cancer risk suggests the possibility that plasma estrogen levels may be a useful intermediate marker of prevention. The aromatase inhibitors offer us the first ever tool to render women virtually free of estrogen and are potentially an exciting tool for the prevention of breast cancer.

Isolation and characterization of the monkey UGT2B30 gene that encodes a uridine diphosphate-glucuronosyltransferase enzyme active on mineralocorticoid, glucocorticoid, androgen and oestrogen hormones.

The present study reports the genomic organization and the characterization of a novel cynomolgus monkey UDP-glucuronosyltransferase (UGT) enzyme, UGT2B30. UGT enzymes are microsomal proteins that catalyse the transfer of the glucuronosyl group from UDP-glucuronic acid (UDPGA) to a wide variety of lipophilic compounds, namely hormonal steroids. The 15 kb UGT2B30 gene amplified by PCR showed a genomic organization similar to those encoding UGT2B human enzymes. The cDNA encoding UGT2B30 was isolated from a cynomolgus monkey prostate cDNA library, and the deduced amino acid sequence showed an identity of 94% with UGT2B19, a monkey isoform previously characterized. Stable expression of UGT2B30 protein in human kidney 293 (HK293) cells was assessed by Western-blot analysis and its conjugating activity was screened using 39 potential substrates. The UGT2B30 enzyme is active on many compounds of different classes, including testosterone, dihydrotestosterone, 5alpha-androstane-3alpha,17beta-diol, androsterone, oestradiol, tetrahydroaldosterone and tetrahydrocortisone, with glucuronidation efficiencies (V(max)/K(m) ratios) ranging from 0.6 to 8.8 microl x min(-1) x mg of protein(-1). Reverse-transcriptase-PCR analysis revealed that the UGT2B30 transcript is expressed in several tissues, including prostate, testis, mammary gland, kidney, adrenals and intestine. The relative activity of UGT2B30 in comparison with other simian UGT2B isoforms, as well as its large variety of substrates, strongly suggest that this enzyme is essential to inactivation of several steroids.

Analysis and characteristics of multiple types of human 17β-hydroxysteroid dehydrogenase

Androgens and estrogens are not only synthesized in the gonads but also in peripheral target tissues. Accordingly, recent molecular cloning has allowed us to identify multiple types of 17β-hydroxysteroid dehydrogenases (17β-HSD), the key and exclusive enzymes involved in the formation and inactivation of sex steroids. However, only one form, namely, type 3 17β-HSD, is responsible for pseudohermaphroditism in deficient boys. To date, seven human 17β-HSDs have been isolated and characterized. Although they catalyze substrates having a similar structure, 17β-HSDs have very low homology. In intact cells in culture, these enzymes catalyze the reaction in a unidirectional way — types 1, 3, 5 and 7 catalyze the reductive reaction, while types 2, 4 and 8 catalyze the oxidative reaction. It is noteworthy that rat type 6 17β-HSD also catalyzes the reaction in the oxidative direction. In this report, we analyze the different characteristics of the multiple types of human 17β-HSD.

Genomic structure of rat 3α-hydroxysteroid/dihydrodiol dehydrogenase (3α-HSD/DD, AKR1C9)

Rat liver 3α-hydroxysteroid/dihydrodiol dehydrogenase (3α-HSD/DD) is a member of the aldo-keto reductase (AKR) superfamily. It is involved in the inactivation of steroid hormones and the metabolic activation of polycyclic aromatic hydrocarbons (PAH) by converting trans-dihydrodiols into reactive and redox-active o-quinones. The structure of the 5′-flanking region of the gene and factors involved in the constitutive and regulated expression of this gene have been reported [H.-K. Lin, T.M. Penning, Cloning, sequencing, and functional analysis of the 5′-flanking region of the rat 3α-hydroxysteroid/dihydrodiol dehydrogenase gene, Cancer Res. 55 (1995) 4105–4113]. We now describe the complete genomic structure of the rat type 1 3α-HSD/DD gene. Charon 4A and P1 genomic clones contained at least three rat genes (type 1, type 2 and type 3 3α-HSD/DD) each of which encoded for the same open reading frame (ORF) but differed in their exon–intron organization. 5′-RACE confirmed that the type 1 3α-HSD/DD gene encodes for the dominant transcript in rat liver and it was the regulation of this gene that was previously studied. The rat type 1 3α-HSD/DD gene is 30 kb in length and consists of nine exons and eight introns. Exon 9 encodes +931 to 966 bp of the ORF and the 1292 bp 3′-UTR implicated in mRNA stability. This genomic structure is nearly identical to the homologous human genes, type 1 3α-HSD (chlordecone reductase/DD4, AKR1C4), type 2 3α-HSD (AKR1C3) and type 3 3α-HSD (bile-acid binding protein, AKR1C2) genes. Three different cDNA's containing identical ORFs for 3α-HSD have been reported suggesting that all three genes may be expressed in rat liver. Using 5′ primers corresponding to the 5′-UTR's of the three different cDNA's only one PCR fragment was obtained and corresponded to the type 1 3α-HSD/DD gene. These data suggested that the type 2 and type 3 3α-HSD/DD genes are not abundantly expressed in rat liver. It is unknown whether the type 2 and type 3 3α-HSD/DD genes represent pseudo-genes or whether they represent genes that are differentially expressed in other rat tissues.