Direct inhibition of human and rat 11ß-hydroxysteroid dehydrogenase 2 by per- and polyfluoroalkyl substances: Structure-activity relationship and in silico docking analysis.

Per- and polyfluoroalkyl substances (PFAS) are persistent in the environment and may disrupt the endocrine system. Our previous study showed that perfluorooctanoic acid (PFOA, C8) and perfluorooctanesulfonic acid (PFOS, C8S) can inhibit 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) activity leading to an active glucocorticoid accumulation. In this study, we extended investigation for 17 PFAS, including carboxylic and sulfonic acids, with different carbon-chain lengths, to determine their inhibitory potency and structure-activity relationship in human placental and rat renal 11ß-HSD2. C8-C14 PFAS at 100 µM significantly inhibited human 11ß-HSD2 with a potency as C10 (half-maximal inhibitory concentration, IC50, 9.19 µM) > C11 (15.09 µM) > C12 (18.43 µM) > C9 (20.93 µM) > C13 (124 µM) > C14 (147.3 µM) > other C4-C7 carboxylic acids, and C8S > C7S = C10S > other sulfonic acids. For rat 11ß-HSD2, only C9 and C10 and C7S and C8S PFAS exhibited significant inhibitory effects. PFAS are primarily mixed/competitive inhibitors of human 11ß-HSD2. Preincubation and simultaneous incubation with the reducing agent dithiothreitol significantly increased human 11ß-HSD2 but not rat 11ß-HSD2, and preincubation but not simultaneous incubation with dithiothreitol partially reversed C10-mediated inhibition on human 11ß-HSD2. Docking analysis showed that all PFAS bound to the steroid-binding site and carbon-chain length determined the potency of inhibition, with the optimal molecular length (12.6 Å) for potent inhibitors PFDA and PFOS, which is comparable to the molecular length (12.7 Å) of the substrate cortisol. The length between 8.9 and 17.2 Å is the probable threshold molecular length to inhibit human 11ß-HSD2. In conclusion, the carbon-chain length determines the inhibitory effect of PFAS on human and rat 11ß-HSD2, and the inhibitory potency of long-chain PFAS on human and rat 11ß-HSD2 showed V-shaped pattern. Long-chain PFAS may partially act on the cysteine residues of human 11ß-HSD2.

The effect of cold exposure on the levels of glucocorticoids, 11-hydroxysteroid dehydrogenase 2, and placental vascularization in a rat model.

OBJECTIVE: Cold exposure (CE) before birth is one of the initial stressors that may impact mammalian pregnancy, changing placental and fetal development and affecting the health of the offspring. While glucocorticoids (GCs) participate in the body's response to the stress of CE, the specific mechanisms of their action are unclear. This study aims to determine the effect of CE stress on the placenta and to test whether stress, caused by cold exposure in pregnancy impairs fetal development by changing placental angiogenesis via excessive GC expression. MATERIALS AND METHODS: CE rat model was created by exposing 30 SD rats to cold preconception, or during the first, second, and third weeks of pregnancy. Serum cortisol and soluble fms-like tyrosine kinase-1 (sFlt-1) expression levels, physiological index changes (food intake, body weight change and blood pressure), and pregnancy outcomes (fetal rat weight, number of live fetal rats, and placental weight) were collected at baseline and at different time points after the conception. Protein expression levels of 11 ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2), glucocorticoid receptor, vascular endothelial growth factor A (VEGF-A), placental growth factor (PIGF), and sFlt-1 in placental tissues were measured by western blotting. Cytokeratin (CK) and laminin (LN) in trophoblasts, and α-actin in vascular smooth muscle of the spiral arteries of pregnant rats after the systemic cold treatment were assessed by immunofluorescence and visualized by fluorescent microscopy. To test the effect of 11ß-HSD2 levels on the placental recasting, human first-trimester extravillous trophoblast cells (HTR8/SVneo) underwent knockdown using specific 11ß-HSD2 siRNA constructs.  Expression levels of 11ß-HSD2 were analyzed by quantitative real-time PCR (qPCR) and into HTR8 cells, and the expression levels of the 11ß-HSD2 gene in each group were measured using qPCR. Cell migration and invasion was assessed by Transwell migration assay, and sFlt-1 levels in HTR8 cells were measured by ELISA. RESULTS: CE pre-conception led to consistently increasing serum corticosterone and sFlt-1 levels throughout pregnancy, and persistently increased diastolic blood pressure (DBP) in rat CE model compared to control animals. CE during the second week of gestation (Gp.3) was associated with significantly lower placental weight (p=0.0003). Cold exposure in the third week (Gp.4) was associated with significantly (p=0.001) lower fetal weight. CE pre-conception was associated with significantly decreased placental levels of 11ß-HSD2, glucocorticoid receptor, VEGF-A, PIGF, and sFlt-1 proteins and α-actin compared to the control group. Silencing 11ß-HSD2 by siRNA led to reduced cell migrations and invasion, and markedly increased expression levels of sFlt-1 in HTR8/SVneo cells (p<0.05). CONCLUSIONS: Pre-conception cold exposure and during early pregnancy leads to increased GCs levels and impaired placental 11ß-HSD2 activity. We suggest that the subsequent 11ß-HSD2-induced increase in the sFlt-1expression during early pregnancy may affect placental vascular remodeling and change placental morphological structure and function.

Transcriptome characterization of BPG axis and expression profiles of ovarian steroidogenesis-related genes in the Japanese sardine.

BACKGROUND: The clupeoid fishes are ecologically and commercially important fish species worldwide that exhibit a high level of population fluctuation, accompanied by alteration of reproductive traits. However, knowledge about their reproductive physiology in order to understand mechanisms underlying such population dynamics is limited. The endocrine system along with the brain-pituitary-gonadal (BPG) axis is critical for regulating reproduction. The aims of this study were to provide transcript data and genes related to the BPG axis, and to characterize the expression profiles of ovarian steroidogenesis-related genes in the Japanese sardine (Sardinops melanostictus, Clupeidae). RESULTS: RNA sequencing was performed using the sardine brain, pituitary, and gonad in both sexes. A total of 290,119 contigs were obtained and 115,173 non-redundant ORFs were annotated. The genes differentially expressed between ovary and testis were strongly associated with GO terms related to gamete production. The tissue-specific profile of the abundance of transcripts was characterized for the major regulators in the BPG axis, such as gonadotropin-releasing hormone, gonadotropin, and steroidogenic enzyme. By comparing between ovary and testis, out of eight different 17ß-hydroxysteroid dehydrogenase (Hsd17b) genes identified, higher hsd17b7 expression was found in testis, whereas higher expression of hsd17b8, hsd17b10, hsd17b12a, and hsd17b12b was found in ovary. The cDNAs encoding key endocrine factors in the ovarian steroidogenic pathway were cloned, sequenced, and quantitatively assayed. In the pituitary, follicle-stimulating hormone beta peaked during vitellogenesis, while luteinizing hormone beta peaked at the completion of vitellogenesis. In the ovary, follicle-stimulating hormone receptor and luteinizing hormone receptor were upregulated from mid- to late phase of vitellogenesis. Furthermore, three steroidogenic enzyme genes (cyp11a1, cyp17a1, and cyp19a1a) gradually increased their expression during ovarian development, accompanying a rise in serum estradiol-17ß, while 3ß-hydroxysteroid dehydrogenase and steroidogenic acute regulatory protein did not change significantly. CONCLUSIONS: This is the first report of deep RNA sequencing analysis of Japanese sardine, in which many key genes involved in the BPG axis were identified. Expression profiles of ovarian steroidogenesis-related genes provide a molecular basis of the physiological processes underlying ovarian development in the sardine. Our study will be a valuable resource for clarifying the molecular biology of clupeoid fishes.

11ß-Hydroxysteroid dehydrogenases control access of 7ß,27-dihydroxycholesterol to retinoid-related orphan receptor γ.

Oxysterols previously were considered intermediates of bile acid and steroid hormone biosynthetic pathways. However, recent research has emphasized the roles of oxysterols in essential physiologic processes and in various diseases. Despite these discoveries, the metabolic pathways leading to the different oxysterols are still largely unknown and the biosynthetic origin of several oxysterols remains unidentified. Earlier studies demonstrated that the glucocorticoid metabolizing enzymes, 11ß-hydroxysteroid dehydrogenase (11ß-HSD) types 1 and 2, interconvert 7-ketocholesterol (7kC) and 7ß-hydroxycholesterol (7ßOHC). We examined the role of 11ß-HSDs in the enzymatic control of the intracellular availability of 7ß,27-dihydroxycholesterol (7ß27OHC), a retinoid-related orphan receptor γ (RORγ) ligand. We used microsomal preparations of cells expressing recombinant 11ß-HSD1 and 11ß-HSD2 to assess whether 7ß27OHC and 7-keto,27-hydroxycholesterol (7k27OHC) are substrates of these enzymes. Binding of 7ß27OHC and 7k27OHC to 11ß-HSDs was studied by molecular modeling. To our knowledge, the stereospecific oxoreduction of 7k27OHC to 7ß27OHC by human 11ß-HSD1 and the reverse oxidation reaction of 7ß27OHC to 7k27OHC by human 11ß-HSD2 were demonstrated for the first time. Apparent enzyme affinities of 11ß-HSDs for these novel substrates were equal to or higher than those of the glucocorticoids. This is supported by the fact that 7k27OHC and 7ß27OHC are potent inhibitors of the 11ß-HSD1-dependent oxoreduction of cortisone and the 11ß-HSD2-dependent oxidation of cortisol, respectively. Furthermore, molecular docking calculations explained stereospecific enzyme activities. Finally, using an inducible RORγ reporter system, we showed that 11ß-HSD1 and 11ß-HSD2 controlled RORγ activity. These findings revealed a novel glucocorticoid-independent prereceptor regulation mechanism by 11ß-HSDs that warrants further investigation.

Glucocorticoid-activation system mediated glucocorticoid-insulin-like growth factor 1 (GC-IGF1) axis programming alteration of adrenal dysfunction induced by prenatal caffeine exposure.

Glucocorticoids play a major factor in fetal maturation and fate decision after birth. We have previously demonstrated that prenatal caffeine exposure (PCE) resulted in adrenal dysplasia. However, its molecular mechanism has not been clarified. In the present study, a rat model of intrauterine growth retardation (IUGR) was established by PCE, and offspring were sacrificed. Moreover, NCI-H295 A cells were used to confirm glucocorticoid-related molecular mechanism. Results showed that PCE fetal weight decreased, and the IUGR rate increased, while serum corticosterone levels increased but insulin-like growth factor 1 (IGF1) levels decreased. Fetal adrenals exhibited an activated glucocorticoid-activation system, and the downregulated expression of IGF1 signal pathway and steroidal synthetases. For adult rats, there was no significant change in the glucocorticoid-activation system in the PCE group, the IGF1 signal pathway showed increased trend, and the expression levels of adrenal steroidal synthetases were close to normal. The data in vitro showed that the cortisol of 1200 nM can inhibit the expression of adrenocortical cell steroidal synthetases and IGF1 signal pathway when compared with the control. Meanwhile, the glucocorticoid-activation system was activated while GR inhibitor mifepristone can reverse the effect of cortisol. Furthermore, cortisol can also promote GR into the nucleus after its activation. Based on these findings, we speculated that high concentrations of glucocorticoid in utero led to GR in the nucleus through its activation and then inhibited the IGF1 signaling pathway by activating the glucocorticoid-activation system, which could further downregulate steroid synthesis.

Glucocorticoid activation by 11ß-hydroxysteroid dehydrogenase enzymes in relation to inflammation and glycaemic control in chronic kidney disease: A cross-sectional study.

OBJECTIVE: Patients with chronic kidney disease (CKD) have dysregulated cortisol metabolism secondary to changes in 11ß-hydroxysteroid dehydrogenase (11ß-HSD) enzymes. The determinants of this and its clinical implications are poorly defined. METHODS: We performed a cross-sectional study to characterize shifts in cortisol metabolism in relation to renal function, inflammation and glycaemic control. Systemic activation of cortisol by 11ß-HSD was measured as the metabolite ratio (tetrahydrocortisol [THF]+5α-tetrahydrocortisol [5αTHF])/tetrahydrocortisone (THE) in urine. RESULTS: The cohort included 342 participants with a median age of 63 years, median estimated glomerular filtration rate (eGFR) of 28 mL/min/1.73 m2 and median urine albumin-creatinine ratio of 35.5 mg/mmol. (THF+5αTHF)/THE correlated negatively with eGFR (Spearman's ρ = -0.116, P = 0.032) and positively with C-reactive protein (ρ = 0.208, P < 0.001). In multivariable analysis, C-reactive protein remained a significant independent predictor of (THF+5αTHF)/THE, but eGFR did not. Elevated (THF+5αTHF)/THE was associated with HbA1c (ρ = 0.144, P = 0.008) and diabetes mellitus (odds ratio for high vs low tertile of (THF+5αTHF)/THE 2.57, 95% confidence interval 1.47-4.47). Associations with diabetes mellitus and with HbA1c among the diabetic subgroup were independent of eGFR, C-reactive protein, age, sex and ethnicity. CONCLUSIONS: In summary, glucocorticoid activation by 11ß-HSD in our cohort comprising a spectrum of renal function was associated with inflammation and impaired glucose control.

Toxicological Effects of Glycyrrhiza glabra (Licorice): A Review.

Licorice (Glycyrrhiza glabra) has been considered as an herbal drug since ancient time. Nowadays, it is a well-known spice that possesses worth pharmacological effects. However, some relevant articles have revealed negative impacts of licorice in health. By considering the great wishes in using herbal medicine, it is important to show adverse effects of herbal medicine in health. At present, there are misunderstandings toward the safety of herbal medicines. Herein, we gathered scientific research projects on the toxicity effects of licorice and glycyrrhizin to highlight their safety. In this regards, we categorized our findings about the toxicity effects of licorice and glycyrrhizin in acute, sub-acute, sub-chronic, and chronic states. Besides, we discussed on the cytotoxicity, genotoxicity, mutagenicity, and carcinogenicity of licorice and glycyrrhizin as well as their developmental toxicity. This review disclosed that G. glabra and glycyrrhizin salts are moderately toxic. They need to be used with caution during pregnancy. G. glabra and glycyrrhizin possess selective cytotoxic effects on cancerous cells. The most important side effects of licorice and glycyrrhizin are hypertension and hypokalemic-induced secondary disorders. Licorice side effects are increased by hypokalemia, prolonged gastrointestinal transient time, decreased type 2 11-beta-hydroxysteroid dehydrogenase activities, hypertension, anorexia nervosa, old age, and female sex. Copyright © 2017 John Wiley & Sons, Ltd.

Stimulatory Effect of Intermittent Hypoxia on the Production of Corticosterone by Zona Fasciculata-Reticularis Cells in Rats.

Hypoxia or intermittent hypoxia (IH) have known to alter both synthesis and secretion of hormones. However, the effect of IH on the production of adrenal cortical steroid hormones is still unclear. The aim of present study was to explore the mechanism involved in the effect of IH on the production of corticosterone by rat ZFR cells. Male rats were exposed at 12% O2 and 88% N2 (8 hours per day) for 1, 2, or 4 days. The ZFR cells were incubated at 37 °C for 1 hour with or without ACTH, 8-Br-cAMP, calcium ion channel blockers, or steroidogenic precursors. The concentration of plasma corticosterone was increased time-dependently by administration of IH hypoxia. The basal levels of corticosterone production in cells were higher in the IH groups than in normoxic group. IH resulted in a time-dependent increase of corticosterone production in response to ACTH, 8-Br-cAMP, progesterone and deoxycorticosterone. The production of pregnenolone in response to 25-OH-C and that of progesterone in response to pregnenolone in ZFR cells were enhanced by 4-day IH. These results suggest that IH in rats increases the secretion of corticosterone via a mechanism at least in part associated with the activation of cAMP pathway and steroidogenic enzymes.

11ß-hydroxysteroid dehydrogenases-2 decreases the apoptosis of MC3T3/MLO-Y4 cells induced by glucocorticoids.

The aim of the present study was to confirm the role of 11ß-hydroxysteroid dehydrogenases type 2(11ß-HSD-2) in steroid induced osteonecrosis of the femoral head(SANFH). We cultured mouse bone-like cells (MLO-Y4) and mouse osteoblast-like cells (MC3T3-E1). After overexpressed 11ß-HSD-2 successfully, we induced cell apoptosis by dexamethasone (DXM). The level of cell apoptosis, the expression of Bcl-2 in MLO-Y4 cells and the expression of Fas and caspase8 in MC3T3-E1 cells were detected. Then, we constructed 11ß-HSD-2 siRNA plasmid and represented it on MLO-Y4/MC3T3-E1 Cells, to down-regulate the 11ß-HSD-2 expression. After that, we used dexamethasone to induce cell apoptosis. The level of cell apoptosis, the expression of Bcl-2 in MLO-Y4 cells and the expression of Fas and caspase8 in MC3T3-E1 cells were detected again. In the overexpression model of cells, we found that the amount of cell apoptosis, the expression of Fas and caspase8 in MC3T3-E1 cells are lower than that of control groups. The amount of cell apoptosis, the expression of Fas and caspase8 in MC3T3-E1 cells were more than before when we reduced the expression of 11ß-HSD-2. In our study, we concluded that 11ß-HSD-2 plays an important role in the development of bone or osteoblast cell apoptosis, and the decreased expression of 11ß-HSD-2 may aggravate steroid induced bone/osteoblast cell apoptosis.

Aldosterone and Mineralocorticoid Receptors-Physiology and Pathophysiology.

Aldosterone is a uniquely terrestrial hormone, first appearing in lungfish, which have both gills and lungs. Mineralocorticoid receptors (MRs), on the other hand, evolved much earlier, and are found in cartilaginous and bony fish, presumptive ligand cortisol. MRs have equivalent high affinity for aldosterone, progesterone, and cortisol; in epithelia, despite much higher cortisol circulating levels, aldosterone selectively activates MRs by co-expression of the enzyme 11ß-hydroxysteroid dehydrogenase, Type 11. In tissues in which the enzyme is not expressed, MRs are overwhelmingly occupied but not activated by cortisol, which normally thus acts as an MR antagonist; in tissue damage, however, cortisol mimics aldosterone and acts as an MR agonist. The risk profile for primary aldosteronism (PA) is much higher than that in age-, sex-, and blood pressure-matched essential hypertensives. High levels of aldosterone per se are not the problem: in chronic sodium deficiency, as seen in the monsoon season in the highlands of New Guinea, plasma aldosterone levels are extraordinarily high, but cause neither hypertension nor cardiovascular damage. Such damage occurs when aldosterone levels are out of the normal feedback control, and are inappropriately elevated for the salt status of the individual (or experimental animal). The question thus remains of how excess salt can synergize with elevated aldosterone levels to produce deleterious cardiovascular effects. One possible mechanism is through the agency of the elusive ouabain-like factors (OLFs). Such factors are secreted from the adrenal in response to ACTH (adrenalocortical tropic hormone), to angiotensin via AT2R, and-the polar opposite of aldosterone-to sodium loading. They act on blood vessels to cause vasoconstriction and thus elevate blood pressure to dump excess sodium through pressure natriuresis. Their levels are chronically elevated in PA in response to the continually elevated sodium status, and they thus act to constrict coronary and systemic arteries. In the context of the elevated blood volume and total body sodium in a PA patient, this raises blood pressure and acts as the proximate cause of cardiovascular damage. If this is the case, it would appear to offer new insights into therapy for PA. One would be the use of digibindin, or its more recent successors as antagonists of OLFs acting on Na/K ATPase at the vessel wall. A second would be to routinely combine a low dose MR antagonist, an ENaC inhibitor, and sodium restriction as first-line therapy for bilateral aldosterone overproduction. Finally, for unilateral cases post-surgery, there is good reason to include low-dose MRs in drug therapy if required, given the ability of cortisol in damaged blood vessels to mimic aldosterone vasoconstrictor action.

Maternal chronic hypoxia increases expression of genes regulating lung liquid movement and surfactant maturation in male fetuses in late gestation.

KEY POINTS: Chronic fetal hypoxaemia is a common pregnancy complication associated with intrauterine growth restriction that may influence respiratory outcome at birth. We investigated the effect of maternal chronic hypoxia for a month in late gestation on signalling pathways regulating fetal lung maturation and the transition to air-breathing at birth using isobaric hypoxic chambers without alterations to maternal food intake. Maternal chronic hypoxia in late gestation increases fetal lung expression of genes regulating hypoxia signalling, lung liquid reabsorption and surfactant maturation, which may be an adaptive response in preparation for the successful transition to air-breathing at birth. In contrast to other models of chronic fetal hypoxaemia, late gestation onset fetal hypoxaemia promotes molecular regulation of fetal lung maturation. This suggests a differential effect of timing and duration of fetal chronic hypoxaemia on fetal lung maturation, which supports the heterogeneity observed in respiratory outcomes in newborns following exposure to chronic hypoxaemia in utero. ABSTRACT: Chronic fetal hypoxaemia is a common pregnancy complication that may arise from maternal, placental and/or fetal factors. Respiratory outcome of the infant at birth likely depends on the duration, timing and severity of the hypoxaemic insult. We have isolated the effect of maternal chronic hypoxia (MCH) for a month in late gestation on fetal lung development. Pregnant ewes were exposed to normoxia (21% O2 ) or hypoxia (10% O2 ) from 105 to 138 days of gestation (term ∼145 days). At 138 days, gene expression in fetal lung tissue was determined by quantitative RT-PCR. Cortisol concentrations were determined in fetal plasma and lung tissue. Numerical density of surfactant protein positive cells was determined by immunohistochemistry. MCH reduced maternal PaO2 (106 ± 2.9 vs. 47 ± 2.8 mmHg) and fetal body weight (4.0 ± 0.4 vs. 3.2 ± 0.9 kg). MCH increased fetal lung expression of the anti-oxidant marker CAT and decreased expression of the pro-oxidant marker NOX-4. MCH increased expression of genes regulating hypoxia signalling and feedback (HIF-3α, KDM3A, SLC2A1, EGLN-3). There was no effect of MCH on fetal plasma/lung tissue cortisol concentrations, nor genes regulating glucocorticoid signalling (HSD11B-1, HSD11B-2, NR3C1, NR3C2). MCH increased expression of genes regulating sodium (SCNN1-B, ATP1-A1, ATP1-B1) and water (AQP-4) movement in the fetal lung. MCH promoted surfactant maturation (SFTP-B, SFTP-D, ABCA3) at the molecular level, but did not alter the numerical density of surfactant positive cells in lung tissue. MCH in late gestation promotes molecular maturation of the fetal lung, which may be an adaptive response in preparation for the successful transition to air-breathing at birth.

Blueberry (Vaccinium ashei Reade) extract ameliorates ovarian damage induced by subchronic cadmium exposure in mice: Potential δ-ALA-D involvement.

Females are born with a finite number of oocyte-containing follicles and ovary damage results in reduced fertility. Cadmium accumulates in the reproductive system, damaging it, and the cigarette smoke is a potential exposure route. Natural therapies are relevant to health benefits and disease prevention. This study verified the effect of cadmium exposure on the ovaries of mice and the blueberry extract as a potential therapy. Blueberry therapy was effective in restoring reactive species levels and δ-aminolevulinate dehydratase activity, and partially improved the viability of cadmium-disrupted follicles. This therapy was not able to restore the 17 ß-hydroxysteroid dehydrogenase activity. Extract HPLC evaluation indicated the presence of quercetin, quercitrin, isoquercetin, and ascorbic acid. Ascorbic acid was the major substance and its concentration was 620.24 µg/mL. Thus, cadmium accumulates in the ovaries of mice after subchronic exposure, inducing cellular damage, and the blueberry extract possesses antioxidant properties that could protect, at least in part, the ovarian tissue from cadmium toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 188-196, 2017.

Claudin tight junction proteins in rainbow trout (Oncorhynchus mykiss) skin: Spatial response to elevated cortisol levels.

This study examined regional distribution and corticosteroid-induced alterations of claudin (cldn) transcript abundance in teleost fish skin. Regional comparison of mRNA encoding 20 Cldns indicated that 12 exhibit differences in abundance along the dorsoventral axis of skin. However, relative abundance of cldns (i.e. most to least abundant) remained similar in different skin regions. Several cldns appear to be present in the epidermis and dermal vasculature whereas others are present only in the epidermis. Increased circulating cortisol levels significantly altered mRNA abundance of 10 cldns in a region specific manner, as well as corticosteroid receptors and 11ß-hydroxysteroid dehydrogenase (type 2). Epidermis and epidermal mucous cell morphometrics also altered in response to cortisol, exhibiting changes that appear to enhance skin barrier properties. Taken together, data provide a first look at spatial variation in the molecular physiology of the teleost fish integument TJ complex and region-specific sensitivity to an endocrine factor.

Carbenoxolone Disodium Treatment for Canine Pituitary-Dependent Hyperadrenocorticism.

Pituitary-dependent hyperadrenocorticism (PDH) is mainly caused by pituitary corticotroph tumors in dogs. A characteristic feature of corticotroph tumors is their resistance to negative feedback by glucocorticoids. In some animal species, including dogs, the aberrant expression of 11ß-hydroxysteroid dehydrogenase (11HSD), a cortisol metabolic enzyme, is observed in corticotroph tumors. We previously reported that carbenoxolone (CBX), an inhibitor of 11HSD, suppressed ACTH secretion from the pituitary gland, and decreased cortisol concentrations in healthy dogs. Therefore, the aim of this study was to investigate the therapeutic effects of CBX on dogs with PDH. Six dogs with PDH were treated with 60 to 80 mg/kg/day of CBX for 6 weeks, followed by trilostane, which is a commonly used agent for canine PDH. CBX treatment led to a gradual decrease in both basal and in corticotropic releasing hormone (CRH)-stimulated plasma ACTH concentrations and CRH-stimulated serum cortisol concentrations, without side effects. However, basal and stimulated ACTH and cortisol concentrations remained higher than those of healthy dogs, and clinical symptoms such as polydipsia and polyuria were not ameliorated. After a 2-week wash-out interval, trilostane was administered for 2 weeks. Although basal plasma ACTH concentrations were higher after trilostane treatment than CBX treatment, polydipsia and polyuria resolved in all six dogs. The reason for the lack of improvement in polydipsia and polyuria with CBX treatment is unclear. Other mechanisms, in addition to a partial decrease in ACTH secretion, are likely to be involved. In conclusion, this is the first study to report the in vivo effects of CBX in dogs with PDH. The findings suggest that CBX inhibits ACTH secretion from canine pituitary tumors, resulting in lower cortisol concentrations.

Effects of Ziram on Rat and Human 11ß-Hydroxysteroid Dehydrogenase Isoforms.

Ziram is a widely used fungicide for crops. Its endocrine disrupting action is largely unknown. 11ß-Hydroxysteroid dehydrogenases, isoforms 1 (HSD11B1) and 2 (HSD11B2), have been demonstrated to be the regulators of the local levels of active glucocorticoids, which have broad physiological actions. In the present study, the potency of ziram was tested for its inhibition of rat and human HSD11B1 and HSD11B2. Ziram showed the inhibition of rat HSD11B1 reductase with IC50 of 87.07 µM but no inhibition of human enzyme at 100 µM. Ziram showed the inhibition of both rat and human HSD11B2 with IC50 of 90.26 and 34.93 µM, respectively. Ziram exerted competitive inhibition of rat HSD11B1 when 11-dehydrocorticosterone was used and mixed inhibition when NADPH was supplied. Ziram exerted a noncompetitive inhibition of both rat and human HSD11B2 when steroid substrates were used and an uncompetitive inhibition when NAD(+) was supplied. Increased DTT concentrations antagonized rat and human HSD11B2 activities, suggesting that the cysteine residues are associated with the inhibition of ziram. In conclusion, for humans, ziram is a selective inhibitor of HSD11B2, implying that this agent may cause excessive glucocorticoid action in local tissues such as the kidney, brain, and placenta.

Placental 11B-Hydroxysteroid Dehydrogenase Type 2 mRNA Levels in Intrauterine Growth Restriction versus Small-for-Gestational-Age Fetuses.

INTRODUCTION: The objective of this study was to evaluate placental 11B-hydroxysteroid dehydrogenase type 2 (11B-HSD-2) mRNA levels in intrauterine growth-restricted fetuses (IUGR) as compared with small-for-gestational-age (SGA) fetuses according to clinical criteria. MATERIAL AND METHODS: Placental levels of 11B-HSD-2 mRNA levels were measured in SGA (birth weight <10th centile) and gestational-age-matched, appropriate-for-gestational-age (AGA) births. SGA was classified as IUGR (birth weight <3rd centile or <10th percentile with abnormal uterine artery Doppler or cerebroplacental ratio) or non-IUGR SGA. After RNA extraction, mRNA levels were determined by reverse transcription and quantitative PCR. RESULTS: A total of 38 placentas were analyzed (20 AGA and 18 SGA). Among the SGA pregnancies, 13 qualified as IUGR. The activity of 11B-HSD-2 in IUGR pregnancies [0.105 (SD 0.328)] was significantly reduced compared to non-IUGR SGA [0.304 (SD 0.261); p = 0.018] and AGA [0.294 (SD 0.328); p = 0.001]. These differences remained significant after adjusting for potential confounders (such as smoking or maternal cortisol levels). Activity levels did not significantly differ between non-IUGR SGA and AGA. DISCUSSION: IUGR fetuses had reduced 11B-HSD-2 activity in comparison with SGA and normally grown fetuses. This finding provides opportunities to develop new placental biomarkers for the phenotypic characterization of fetal smallness.

Inhibition of 11ß-Hydroxysteroid Dehydrogenase Type II Suppresses Lung Carcinogenesis by Blocking Tumor COX-2 Expression as Well as the ERK and mTOR Signaling Pathways.

Lung cancer is by far the leading cause of cancer death. Early diagnosis and prevention remain the best approach to reduce the overall morbidity and mortality. Experimental and clinical evidence have shown that cyclooxygenase-2 (COX-2) derived prostaglandin E2 (PGE2) contributes to lung tumorigenesis. COX-2 inhibitors suppress the development and progression of lung cancer. However, increased cardiovascular risks of COX-2 inhibitors limit their use in chemoprevention of lung cancers. Glucocorticoids are endogenous and potent COX-2 inhibitors, and their local actions are down-regulated by 11ß-hydroxysteroid dehydrogenase type II (11ßHSD2)-mediated metabolism. We found that 11ßHSD2 expression was increased in human lung cancers and experimental lung tumors. Inhibition of 11ßHSD2 activity enhanced glucocorticoid-mediated COX-2 inhibition in human lung carcinoma cells. Furthermore, 11ßHSD2 inhibition suppressed lung tumor growth and invasion in association with increased tissue active glucocorticoid levels, decreased COX-2 expression, inhibition of ERK and mTOR signaling pathways, increased tumor endoplasmic reticulum stress as well as increased lifespan. Therefore, 11ßHSD2 inhibition represents a novel approach for lung cancer chemoprevention and therapy by increasing tumor glucocorticoid activity, which in turn selectively blocks local COX-2 activity and/or inhibits the ERK and mTOR signaling pathways.

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

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

11ß-Hydroxyandrostenedione: Downstream metabolism by 11ßHSD, 17ßHSD and SRD5A produces novel substrates in familiar pathways.

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

C21-steroids inactivation and glucocorticoid synthesis in the developing lung.

Glucocorticoids (GCs) are important regulators of lung development. The genes normally involved in GC synthesis in adrenals are co-expressed with 20α-hydroxysteroid dehydrogenase (20α-HSD) in the developing lung. In this study, C21-steroid metabolism was investigated in fetal and postnatal mouse lungs. Incubation of [(3)H]-progesterone with lung explant cultures of different perinatal developmental time points revealed two different (antenatal vs. postnatal) complex metabolization patterns. Progesterone inactivation was predominant. 20αOH-derivatives were more abundant after birth and some metabolites were 5α-reduced. Using [(3)H]-progesterone as substrate, corticosterone synthesis was only observed in a fraction of lung explants from gestation day (GD) 15.5. Neither aldosterone synthase nor P450c17 activity was observed. With epithelial-enriched primary cell cultures, deoxycorticosterone synthesis from [(3)H]-progesterone was observed. With lung explants incubated with [(3)H]-corticosterone as substrate, [(3)H]-4-pregnen-21-ol-3,11,20-trione (11-dehydrocorticosterone), the product of 11ß-HSD2, accumulated in higher proportion on GD 15.5 than at later developmental time points. The temporal correlation observed between levels of progesterone inactivation by 20α-HSD (higher after birth) and the sensitivity of lung development to GCs suggests a role for 20α-HSD in the modulation of GR occupancy through the control of 21-hydroxylase substrate and product levels. In conclusion, the developing lung is characterized by effective inactivation of c21-steroids by 20α-HSD. The formation of active GCs from the "adrenal"-like pathway was observed with some lung explants and primary epithelial cell cultures. Coexistence of this GC synthesis pathway with 20α-HSD activity strongly suggests local regulation of GC action and is compatible with intracrine/paracrine actions of GC.