Effects of Sulpiride on Prolactin and mRNA Levels of Steroid 5α-reductase Isozymes in Adult Rat Brain

Prolactin (PRL) promotes maternal behavior (MB), a complex pattern of behavior aimed at maximizing offspring survival. 3α,5α-reduced neurosteroids may also regulate MB. Indeed, PRL, 3α,5α-reduced neurosteroids, and 5α-reductase (5α-R), the key enzyme in the biosynthesis of these neuroactive steroids, are all increased in stress situations These facts led us to hypothesize a possible interrelation between PRL levels and 5α-R. In the present study we quantified mRNA levels of both 5α-R isozymes in prefrontal cortex of male and female rats after administration of sulpiride, an inductor of PRL secretion. Our results demonstrated that mRNA levels of both 5α-R isozymes were significantly increased in male and female rats by sulpiride, directly or via sulpiride-induced hyperprolactinemia. Since 3α,5α-reduced neurosteroids and PRL exert anxiolytic effects in response to stress, these molecules and 5α-R may possibly participate in a common pathway of significant adaptation to stress situations.     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

15β-hydroxysteroids (Part IV). Steroids of the human perinatal period: The synthesis of 3α,15β,17α-trihydroxy-5α-pregnan-20-one and its A/B-ring configurational isomers

In recent years several 15β-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3α,15β,17α-trihydroxy-5β-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3ξ,5ξ-isomers, namely 3α,15β,17α-trihydroxy-5α-pregnan-20-one (3), 3β,15β,17α-trihydroxy-5α-pregnan-20-one (7) and 3β,15β,17α-trihydroxy-5β-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3β,15β-Diacetoxy-17α-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15β,17α-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15β-acetoxy-3β,17α-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15β-acetoxy-3β,17α-dihydroxy-5α-pregnan-20-one (13) a common intermediate for the synthesis of the 3β(and α),5α-isomers. Hydrolysis of the 15β-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15β-acetoxy-17α-hydroxy-5α-pregnan-3,20-dione (14) which on reduction with -Selectride and hydrolysis of the 15β-acetate gave 3. Finally, hydrogenation of 4 gave 15β,17α-dihydroxy-5β-pregnan-3,20-dione (10) which on reduction with -Selectride gave 8.     

Stable isotope dilution analysis of human urinary metabolites of 17α-methyltestosterone

A method based on gas chromatography–mass spectrometry–selected-ion monitoring was developed to measure the main metabolites of 17α-methyltestosterone, 17α-methyl-5α-androstan-3α,17β-diol and 17α-methyl-5β-androstan-3α,17β-diol, in human urine. 17α-Methyl-[²H₃]-5α-androstan-3α,17β-diol and 17α-methyl-[²H₃]-5β-androstan-3α,17β-diol were used as internal standards. The methods involved purification using a Sep-Pak C₁₈ cartridge, hydrolysis by β-glucuronidase from Ampullaria and derivatization with N-methyl-N-trimethylsilyl-trifluoroacetamide/dithioerythriol/ammonium iodide. Quantitation was achieved by selected-ion monitoring of the characteristic fragment ions ([(M+H)−2×TMSOH]⁺) of the di-TMS derivatives on the chemical ionization mode. The method provides a specific, sensitive and reliable technique to determine the urine levels of 17α-methyl-5α-androstan-3α,17β-diol and 17α-methyl-5β-androstan-3α,17β-diol, and can be applied to pharmacokinetic studies of 17α-methyltestosterone.     

Parathyroid hormone stimulation of the renal 25-hydroxyvitamin D-1α-hydroxylase—Effect of age and free radicals

The capacity of parathyroid hormone (PTH) to increase serum 1,25(OH)₂D levels declines with age in both rats and humans. In young rats, PTH stimulates renal 1,25(OH)₂D production and increases mRNA levels for the terminal mitochondrial P450 of the 1α-hydroxylase complex (CYP27B1 or CYP1α). However, in older rats PTH increases mRNA levels but not 1,25(OH)₂D production. This suggests that in old animals there is either decreased CYP1α protein levels in response to PTH or that the protein produced lacks functionality. The CYP1α protein is located on the inner mitochondrial membrane, the site of increased free radical production with age. To study these possibilities, we examined the effect of PTH and free radicals on CYP1α expression in a model system—AOK-B50 renal tubular cells. PTH increased CYP1α mRNA and protein in a similar time-dependent manner, suggesting that CYP1α protein levels were largely regulated by mRNA levels. The effect of free radicals was determined by preincubation with hydrogen peroxide (H₂O₂), a standard model for studying free radical damage. H₂O₂ inhibited PTH-stimulated CYP1α protein levels and 1,25(OH)₂D production in a dose dependent manner. However, 1,25(OH)₂D production was more sensitive to H₂O₂ than was CYP1α protein levels. This suggests that the catalytic activity of the CYP1α protein may be reduced by free radical damage in these cells. Future studies will focus on detecting oxidative damage in this model system and in vivo.     

Bisphenol A Exposure during Adulthood Alters Expression of Aromatase and 5α-Reductase Isozymes in Rat Prostate

The high incidence of prostate cancer (PCa) and benign prostatic hypertrophy (BPH) in elderly men is a cause of increasing public health concern. In recent years, various environmental endocrine disruptors, such as bisphenol A (BPA), have been shown to disrupt sexual organs, including the prostate gland. However, the mechanisms underlying these effects remain unclear. Because androgens and estrogens are important factors in prostate physiopathology, our objective was to examine in rat ventral prostate the effects of adult exposure to BPA on 5α-Reductase isozymes (5α-R types 1, 2, and 3) and aromatase, key enzymes in the biosynthesis of dihydrotestosterone and estradiol, respectively. Adult rats were subcutaneously injected for four days with BPA (25, 50, 300, or 600 µg/Kg/d) dissolved in vehicle. Quantitative RT-PCR, western blot and immunohistochemical analyses showed lower mRNA and protein levels of 5α-R1 and 5α-R2 in BPA-treated groups versus controls but higher mRNA levels of 5α-R3, recently proposed as a biomarker of malignancy. However, BPA treatment augmented mRNA and protein levels of aromatase, whose increase has been described in prostate diseases. BPA-treated rats also evidenced a higher plasma estradiol/testosterone ratio, which is associated with prostate disease. Our results may offer new insights into the role of BPA in the development of prostate disease and may be of great value for studying the prostate disease risk associated with exposure to BPA in adulthood.     

Determination of a novel selective inhibitor of type 1 5α-reductase in human plasma by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

A sensitive and specific assay of human plasma for the determination of (5α,7β,16β)-16[(4-chlorophenyl)oxy]-4,7-dimethyl-4-aza-andronstan-3-one (I), a selective inhibitor of human type 1 5α-reductase, has been developed. The method is based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS–MS) detection. The analyte (I) and internal standard, Proscar (II), were isolated from the basified biological matrix using a liquid–liquid extraction with methyl-tert.-butyl ether (MTBE). The organic extract was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The MS–MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer using a heated nebulizer interface. Multiple reaction monitoring using the precursor→product ion combinations of m/z 430→114 and 373→305 was used to quantify I and internal standard (II), respectively. The assay was validated in the concentration range of 0.5 to 500 ng/ml in human plasma. The precision of the assay, expressed as coefficient of variation (C.V.), was less than 7% over the entire concentration range, with adequate assay specificity and accuracy. The HPLC–MS–MS method provided sufficient sensitivity to completely map the 24 h pharmacokinetic time-course following a single 0.5 mg dose of I.     

Angiotensin II type-1 receptor antagonist attenuates LPS-induced acute lung injury

Angiotensin II is able to trigger inflammatory responses through an angiotensin II type 1 (AT1) receptor. The role of AT1 receptor in acute lung injury (ALI) is poorly understood. Mice were randomly divided into three groups (n = 40 each groups): NS group; LPS group (2 mg/kg LPS intratracheally); and LPS + ZD 7155 group, 10 mg/kg ZD 7155 (an AT1 receptor antagonist) intraperitoneally 30 min prior to LPS exposure. Samples from the lung were isolated and assayed for histopathology analyses or proinflammatory gene expressions, angiotensin II receptors expressions and nuclear factors activities. LPS exposure resulted in severe ALI, elevated levels of TNF-α and IL-1β mRNA expressions, and increased activities of NF-κB and activated protein (AP)-1. Upregulation of AT1 receptor and down-regulation of AT2 receptor were also observed after LPS challenge. Pretreatment with ZD 7155 significantly inhibited the increase of AT1 receptor expression and upregulated AT2 receptor expression. ZD 7155 also reduced the mRNA expression of TNF-α and IL-1β, inhibited the activation of NF-κB and AP-1, and improved lung histopathology. These findings suggest that antagonism of AT1 receptor inhibits the activation of NF-κB and AP-1 in the lung, which may mediate the release of TNF-α and IL-1β and contribute to LPS-induced ALI.     

17α-Ethyl-5β-estrane-3α,17β-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle

The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17α-ethyl-5β-estrane-3α,17β-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.     

Laminin 5 Promotes Activation and Apoptosis of the T Cells Expressing α3β1 Integrin

By introducing an α3 gene-containing plasmid into a human T cell line Jurkat, we prepared the T cells, which express a high level of the α3β1 integrin, to assess the role of laminin 5 in the skin immune system. The α3β1-expressing T cells adhered to laminin 5 and exhibited spreading. These adhered T cells showed a significant tyrosine phosphorylation of intracellular proteins including p59^fynupon T-cell receptor (TCR) stimulation. Six hours after cross-linking TCR, these cells on laminin 5 secreted a three times higher level of IL-2 than those on a BSA-coated plate. Twenty hours after the stimulation, 48% of the α3β1-expressing T cells on laminin 5 caused apoptosis. The protein level of cyclin D3 and E decreased, while that of p53 increased in these T cells. These data suggest that laminin 5 may play at least two regulatory roles for T cell functions: augmentation of IL-2 production by antigen-stimulated T cells and induction of apoptosis in these T cells.     

Increased A‐ring Reduction of Glucocorticoids in Obese Zucker Rats: Effects of Insulin Sensitization

Objective: Obesity is associated with altered glucocorticoid metabolism, which may impact on hypothalamic‐pituitary‐adrenal axis activity. Here we characterize hepatic 5α‐ and 5β‐reductase in obese rats and their responses to insulin sensitization. Research Methods and Procedures: Hepatic A‐ring reductase protein and mRNA were assessed in lean and obese Zucker rats after insulin sensitization with metformin or rosiglitazone (n = 7 to 8/group). Results: Hepatic 5α‐reductase 1 and 5β‐reductase mRNA and protein (p < 0.01) were increased in obese rats. Insulin sensitization ameliorated increased 5α‐reductase 1 mRNA in obese rats (p < 0.01) and partially reversed increased 5β‐reductase activity. Discussion: Hepatic clearance of glucocorticoids by 5α‐ and 5β‐reductase is increased in obese Zucker rats, and this increase in clearance is attenuated by insulin sensitization. This increased hepatic clearance may underpin compensatory activation of the hypothalamic‐pituitary‐adrenal axis in obesity.     

PPARγ activation induces autophagy in breast cancer cells

It has been previously shown that PPARγ ligands induce apoptotic cell death in a variety of cancer cells. Given the evidence that these ligands have a receptor-independent function, we further examined the specific role of PPARγ activation in this biological process. Surprisingly, we failed to demonstrate that MDA-MB-231 breast cancer cells undergo apoptosis when treated with sub-saturation doses of troglitazone and rosiglitazone, which are synthetic PPARγ ligands. Acridine orange (AO) staining showed acidic vesicular formation within ligand-treated cells, indicative of autophagic activity. This was confirmed by autophagosome formation as indicated by redistribution of LC3, an autophagy-specific protein, and the appearance of double-membrane autophagic vacuoles by electron microscopy following exposure to ligand. To determine the mechanism by which PPARγ induces autophagy, we transduced primary mammary epithelial cells with a constitutively active mutant of PPARγ and screened gene expression associated with PPARγ activation by genome-wide array analysis. HIF1α and BNIP3 were among 42 genes up-regulated by active PPARγ. Activation of PPARγ induced HIF1α and BNIP3 protein and mRNA abundance. HIF1α knockdown by shRNA abolished the autophagosome formation induced by PPARγ activation. In summary, our data shows a specific induction of autophagy by PPARγ activation in breast cancer cells providing an understanding of distinct roles of PPARγ in tumorigenesis.