Melatonin reduces LH, 17 beta-estradiol and induces differential regulation of sex steroid receptors in reproductive tissues during rat ovulation

Abstract Background Melatonin is associated with direct or indirect actions upon female reproductive function. However, its effects on sex hormones and steroid receptors during ovulation are not clearly defined. This study aimed to verify whether exposure to long-term melatonin is able to cause reproductive hormonal disturbances as well as their role on sex steroid receptors in the rat ovary, oviduct and uterus during ovulation. Methods Twenty-four adult Wistar rats, 60 days old (+/- 250 g) were randomly divided into two groups. Control group (Co): received 0.9% NaCl 0.3 mL + 95% ethanol 0.04 mL as vehicle; Melatonin-treated group (MEL): received vehicle + melatonin [100 μg/100 g BW/day] both intraperitoneally during 60 days. All animals were euthanized by decapitation during the morning estrus at 4 a.m. Results Melatonin significantly reduced the plasma levels of LH and 17 beta-estradiol, while urinary 6-sulfatoximelatonin (STM) was increased at the morning estrus. In addition, melatonin promoted differential regulation of the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) and melatonin receptor (MTR) along the reproductive tissues. In ovary, melatonin induced a down-regulation of ER-alpha and PRB levels. Conversely, it was observed that PRA and MT1R were up-regulated. In oviduct, AR and ER-alpha levels were down-regulated, in contrast to high expression of both PRA and PRB. Finally, the ER-beta and PRB levels were down-regulated in uterus tissue and only MT1R was up-regulated. Conclusions We suggest that melatonin partially suppress the hypothalamus-pituitary-ovarian axis, in addition, it induces differential regulation of sex steroid receptors in the ovary, oviduct and uterus during ovulation.

Lung function monitoring in patients with duchenne muscular dystrophy on steroid therapy

Background Duchenne muscular dystrophy (DMD) is a sex-linked inherited muscle disease characterized by a progressive loss in muscle strength and respiratory muscle involvement. After 12 years of age, lung function declines at a rate of 6 % to 10.7 % per year in patients with DMD. Steroid therapy has been proposed to delay the loss of motor function and also the respiratory involvement. Method In 21 patients with DMD aged between seven and 16 years, the forced vital capacity (FVC) and the forced expiratory volume in one second (FEV1) were evaluated at three different times during a period of two years. Results We observed in this period of evaluation the maintenance of the FVC and the FEV1 in this group of patients independently of chronological age, age at onset of steroid therapy, and walking capacity. Conclusion The steroid therapy has the potential to stabilize or delay the loss of lung function in DMD patients even if they are non-ambulant or older than 10 years, and in those in whom the medication was started after 7 years of age.

Neuropeptide Y modulates steroid production of human adrenal H295R cells through Y1 receptors

Neuropeptide Y (NPY) is abundantly expressed in the nervous system and acts on target cells through NPY receptors. The human adrenal cortex and adrenal tumors express NPY receptor subtype Y1, but its function is unknown. We studied Y1-mediated signaling, steroidogenesis and cell proliferation in human adrenal NCI-H295R cells. Radioactive ligand binding studies showed that H295R cells express Y1 receptor specifically. NPY treatment of H295R cells stimulated the MEK/ERK1/2 pathway, confirming that H295R cells express functional Y1 receptors. Studies of the effect of NPY and related peptide PYY on adrenal steroidogenesis revealed a decrease in 11-deoxycortisol production. RIA measurements of cortisol from cell culture medium confirmed this finding. Co-treatment with the Y1 antagonist BIBP2336 reversed the inhibitory effect of NPY on cortisol production proving specificity of this effect. At mRNA level, NPY decreased HSD3B2 and CYP21A2 expression. However NPY revealed no effect on cell proliferation. Our data show that NPY can directly regulate human adrenal cortisol production.

Control of Steroid 21-oic Acid Synthesis by Peroxisome Proliferator-activated Receptor and Role of the Hypothalamic-Pituitary-Adrenal Axis

A previous study identified the peroxisome proliferator-activated receptor alpha (PPARalpha) activation biomarkers 21-steroid carboxylic acids 11beta-hydroxy-3,20-dioxopregn-4-en-21-oic acid (HDOPA) and 11beta,20-dihydroxy-3-oxo-pregn-4-en-21-oic acid (DHOPA). In the present study, the molecular mechanism and the metabolic pathway of their production were determined. The PPARalpha-specific time-dependent increases in HDOPA and 20alpha-DHOPA paralleled the development of adrenal cortex hyperplasia, hypercortisolism, and spleen atrophy, which was attenuated in adrenalectomized mice. Wy-14,643 activation of PPARalpha induced hepatic FGF21, which caused increased neuropeptide Y and agouti-related protein mRNAs in the hypothalamus, stimulation of the agouti-related protein/neuropeptide Y neurons, and activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased adrenal cortex hyperplasia and corticosterone production, revealing a link between PPARalpha and the HPA axis in controlling energy homeostasis and immune regulation. Corticosterone was demonstrated as the precursor of 21-carboxylic acids both in vivo and in vitro. Under PPARalpha activation, the classic reductive metabolic pathway of corticosterone was suppressed, whereas an alternative oxidative pathway was uncovered that leads to the sequential oxidation on carbon 21 resulting in HDOPA. The latter was then reduced to the end product 20alpha-DHOPA. Hepatic cytochromes P450, aldehyde dehydrogenase (ALDH3A2), and 21-hydroxysteroid dehydrogenase (AKR1C18) were found to be involved in this pathway. Activation of PPARalpha resulted in the induction of Aldh3a2 and Akr1c18, both of which were confirmed as target genes through introduction of promoter luciferase reporter constructs into mouse livers in vivo. This study underscores the power of mass spectrometry-based metabolomics combined with genomic and physiologic analyses in identifying downstream metabolic biomarkers and the corresponding upstream molecular mechanisms.

Reduction in hepatic drug metabolizing CYP3A4 activities caused by P450 oxidoreductase mutations identified in patients with disordered steroid metabolism

Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizes approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.

Assessment of letrozole and tamoxifen alone and in sequence for postmenopausal women with steroid hormone receptor-positive breast cancer: the BIG 1-98 randomised clinical trial at 8·1 years median follow-up

Postmenopausal women with hormone receptor-positive early breast cancer have persistent, long-term risk of breast-cancer recurrence and death. Therefore, trials assessing endocrine therapies for this patient population need extended follow-up. We present an update of efficacy outcomes in the Breast International Group (BIG) 1-98 study at 8·1 years median follow-up.

Role of AMP-activated protein kinase on steroid hormone biosynthesis in adrenal NCI-H295R cells

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

Steroid-responsive encephalopathy associated with Hashimoto thyroiditis

An 11-year-old girl presented with sudden sensory disturbance and left-sided muscle weakness. MRI revealed ischaemic change in the right lateral thalamus and the right internal capsule. During sonographic work-up of the cervical arteries, inflammation of the thyroid gland was noted. The results of the thyroid function tests and antibody titers confirmed Hashimoto thyroidits. Under high-dose corticosteroids, the girl had a full neurological recovery.

Therapy of steroid-resistant inflammatory bowel disease

Although systemic corticosteroids are successfully administered for the induction of clinical response and remission in the majority of patients with inflammatory bowel disease (IBD) presenting with a flare, a proportion of these patients demonstrate a primary nonresponse to steroids or in the case of an initial response, they develop a resistance or a steroid dependence. Long-term therapy with corticosteroids for treatment of IBD should be avoided, given the high frequency of adverse treatment effects. Knowledge about treatment strategies in case of steroid nonresponse is therefore highly relevant.

Agonist specific L-type Ca(2+)-current stimulation in ventricular myocytes by a novel steroid-like compound

In cardiac muscle the amplitude of Ca(2+) transients can be increased by enhancing Ca(2+) influx. Among the processes leading to increased Ca(2+) influx, agonists of the L-type Ca(2+)-channel can play an important role. Known pharmacological Ca(2+)-channel agonists act on different binding sites on the channel protein, which may lead not only to enhanced peak currents, but also to distinct changes in other biophysical characteristics of the current. In this study, membrane currents were recorded with the patch-clamp technique in the whole-cell configuration in guinea pig isolated ventricular myocytes in combination with confocal fluorescence Ca(2+) imaging techniques and a variety of pharmacological tools. Testing a new positive inotropic steroid-like compound, we found that it increased the L-type Ca(2+)-current by 2.5-fold by shifting the voltage-dependence of activation by 20.2 mV towards negative potentials. The dose-response relationship revealed two vastly different affinities (EC(50(high-affinity))=4.5+/-1.7 nM, EC(50(low-affinity))=8.0+/-1.1 microM) exhibiting differential pharmacological interactions with three classes of Ca(2+)-current antagonists, suggesting more than one binding site on the channel protein. Therefore, we identified and characterized a novel positive inotropic compound (F90927) as a member of a new class of Ca(2+)-channel agonists exhibiting unique features, which set it apart from other presently known L-type Ca(2+)-channel agonists.

A novel steroid-like compound F90927 exerting positive-inotropic effects in cardiac muscle

Here we report a novel steroid-like compound F90363, exhibiting positive inotropy in vivo and in vitro in various cardiac muscle preparations. F90363 is a racemic mixture composed of the stereoisomers (-)-F90926 and (+)-F90927. Only F90927 exerted positive inotropy, while F90926 induced a weak negative inotropy, but only at concentrations 10(3) times higher than F90927 and most likely resulting from an unspecific interaction. The rapid time course of the action of F90927 suggested a direct interaction with a cellular target rather than a genomic alteration. We could identify the L-type Ca2+ current I(Ca(L)) as a main target of F90927, while excluding other components of cardiac Ca2+ signalling as potential contributors. In addition, several other signaling pathways known to lead to positive inotropy (e.g. alpha- and beta-adrenergic stimulation, cAMP pathways) could be excluded as targets of F90927. However, vessel contraction and stiffening of the cardiac muscle at high doses (>30 microM, 0.36 mg kg(-1), respectively) prevent the use of F90927 as a candidate for drug development. Since the compound may still find valuable applications in research, the aim of the present study was to identify the cellular target and the mechanism of inotropy of F90927.

Steroid hormone related male biased parasitism in chamois, Rupicapra rupicapra rupicapra

Parasites are linked with their host in a trophic interaction with implications for both hosts and parasites. Interaction stretches from the host's immune response to the structuring of communities and the evolution of biodiversity. As in many species sex determines life history strategy, response to parasites may be sex-specific. Males of vertebrate species tend to exhibit higher rates of parasites than females. Sex-associated hormones may influence immunocompetence and are hypothesised to lead to this bias. In a field study, we tested the prediction of male biased parasitism (MBP) in free ranging chamois (Rupicapra rupicapra rupicapra), which are infested intensely by gastrointestinal and lung helminths. We further investigated sex differences in faecal androgen (testosterone and epiandrosterone), cortisol and oestrogen metabolites using enzyme immunoassays (EIA) to evaluate the impact of these hormones on sex dependent parasite susceptibility. Non-invasive methods were used and the study was conducted throughout a year to detect seasonal patterns. Hormone levels and parasite counts varied significantly throughout the year. Male chamois had a higher output of gastrointestinal eggs and lungworm larvae when compared to females. The hypothesis of MBP originating in sex related hormone levels was confirmed for the elevated output of lungworm larvae, but not for the gastrointestinal nematodes. The faecal output of lungworm larvae was significantly correlated with androgen and cortisol metabolite levels. Our study shows that sex differences in steroid levels play an important role to explain MBP, although they alone cannot fully explain the phenomenon.

Pharmacokinetics and clinical efficacy of cyclosporine treatment of dogs with steroid-refractory inflammatory bowel disease

The usual treatment of dogs with inflammatory bowel disease (IBD) consists of administration of immunosuppressive doses of steroids. However, some dogs are refractory to steroid treatment and pose a significant challenge to the veterinarian. Because cyclosporine A (cyA) has been shown to be effective in steroid-resistant IBD in humans, the purpose of this study was to investigate the pharmacokinetics and clinical efficacy of PO cyA treatment in dogs with steroid-refractory IBD (n = 14). All dogs were treated with cyA 5 mg/kg PO q24h for a period of 10 weeks. A clinical activity score was assigned to assess severity of clinical signs before and after treatment. The total number of infiltrating lymphocytes and T cells in duodenal biopsies were assessed before and after treatment in 9 dogs. In addition, serum concentration of cyA was measured in 8 dogs over a 24-hour period. Pharmacokinetic profiles in dogs with IBD were similar to those of healthy dogs. Improvement of clinical signs was observed in 12 of 14 dogs with IBD. Median clinical activity score after treatment with cyA was significantly reduced from a median score of 9 to a median score of 5 (P = 0.001). T cell numbers in duodenal biopsies were significantly decreased after treatment from a median +/- 95% range in the villous region of 28 (19-30) cells/10,000 microm2 before versus 7 (0-10)/10,000 microm2 after treatment, P = 0.01; and from a median +/- 95% range number in the crypt region of 15 (6-23) cells/10,000 microm2 before versus 4 (0-9)/10,000 microm2 after treatment, P = 0.02, implying T cell lysis as a possible mechanism of action. In conclusion, based on this small study, cyA appears to be an effective alternative drug in dogs with IBD that are refractory to immunosuppressive doses of steroids.