Trace LC/MS/MS quantitation of 17β‐estradiol as a biomarker for selective estrogen receptor modulator activity in the rat brain

A sensitive LC/MS/MS method has been developed by derivatization of 17β‐estradiol (E2) with dansyl chloride to quantitate 17β‐E2 in female rat serum. The use of E2‐d₅ minimized interferences from endogenous 17β‐E2 in order to achieve a limit of quantitation (LOQ) of 2.5 pg/ml using 150 µl of female rat serum. The recovery of the dansyl derivative was 95% or greater in quality control samples. The intra and interday assay precision was better than 8.2 and 6.2%, respectively, with accuracies ranging from 97 to 101% in the quality control samples. The assay was used for the quantitation of serum E2 as a biomarker for the estrogen receptor (ER) antagonist activity of small molecule SERMs (selective estrogen receptor modulators) in the female rat brain. The study revealed that a statistically significant upregulation of serum 17β‐E2 occurred for rats dosed with SERMs that are known to penetrate the brain and disrupt the hypothalamic‐pituitary‐ovarian (HPO) axis. Variations in 17β‐E2 in ascending dose studies also correlated with the corresponding trends in CYP17a1 levels, an mRNA biomarker for ovarian hyperstimulation. This biomarker assay has provided a useful screen for medicinal chemistry optimization to produce SERMs that do not interfere with negative feedback of estrogens on the brain and for biological hypothesis testing. Copyright © 2009 John Wiley & Sons, Ltd.     

INDOMETHACIN INHIBITS hCG AND GnRH AGONIST-INDUCED SECRETION OF PLASMINOGEN ACTIVATOR BY GRANULOSA AND THECA INTERSTITIAL CELLS OF HYPOPHYSECTOMIZED RATS

Considerable data have shown that plasminogen activator (PA) may play an important role in the mechanism of ovulation. A recent report argued that PA is not a primary proteoIytic enzyme for follicular rupture because a dose of indomethacin that blocked ovulation did not inhibit ovarian PA content. To further clarify the specific role of PA in the ovulation, we have carefully examined the effect of indomethacin on the secretion of PA in granulosa and theca-interstitial (TI) cells following hCG or GnRH agonist administration in PMSG-primed hypophysectomized immature rats. We have also studies in vitro action of this compound on PA secretion in granulosa cells. The results indicate that indomethacin can only suppress hCG- and GnRH agonist-induced PA secretion, but not suppress the ovarian content of these enzymes.     

B-RafV600E inhibits sodium iodide symporter expression via regulation of DNA methyltransferase 1

B-RafV600E mutant is found in 40–70% of papillary thyroid carcinoma (PTC) and has an important role in the pathogenesis of PTC. The sodium iodide symporter (NIS) is an integral plasma membrane glycoprotein that mediates active iodide transport into the thyroid follicular cells, and B-RafV600E has been known to be associated with the loss of NIS expression. In this study, we found that B-RafV600E inhibited NIS expression by the upregulation of its promoter methylation, and that specific regions of CpG islands of NIS promoter in B-RafV600E harboring PTC were highly methylated compared with surrounding normal tissue. Although DNA methyltransferase 3a and 3b (DNMT3a,3b) were not increased by B-RafV600E, DNMT1 expression was markedly upregulated in PTC and B-RafV600E expressing thyrocytes. Furthermore, DNMT1 expression was upregulated by B-RafV600E induced NF-κB activation. These results led us to conclude that NIS promoter methylation, which was induced by B-RafV600E, is one of the possible mechanisms involved in NIS downregulation in PTC.     

Regulation of steroidogenesis of infant and adult rhesus monkey granulosa cells in vitro

The present studies have demonstrated that infant monkey granulosa cells, like the adult ones, have the potential of responding markedly in vitro to human FSH, cyclic-AMP and forskolin, resulting in the increase of progesterone and estrogen production. Exogenous hCG was also capable of increasing FSH-stimulated progesterone biosynthesis in both infant and adult granulosa cells, but did not stimulate the infant granulosa cells to secrete estrogen. Addition of a synthetic estrogen, diethylstilbestrol, to the culture of monkey granulosa cells enhanced the FSH-stimulated progesterone and estrogen production. The steroidogenesis of monkey granulosa cells was also dramatically stimulated by a gonadotropin-releasing hormone agonist. Monkey granulosa cells, unlike the other animal cells, secrete measurable amount of estrogen in the absence of androgen substrate. The findings reported here are significant in regard to understanding of the mechanism of hormonal regulation of primate ovarian function.     

REGULATION OF STEROIDOGENESIS OF INFANT AND ADULT RHESUS MONKEY GRANULOSA CELLS in vitro

The present studies have demonstrated that infant monkey granulosa cells, like the adult ones, have the potential of responding markedly in vitro to human FSH, cyclic-AMP and forskolin, resulting in the increase of progesterone and estrogen production. Exogenous hCG was also capable of increasing FSH-stimulated progesterone biosynthesis in both infant and adult granulosa cells, but did not stimultate the infant granulosa cells to secrete estrogen. Addition of a synthetic estrogen, diethylstilestrol, to the culture of monkey granulosa cells enhanced the FSH-stimulated progesterone and estrogen production. The esteroidogenesis of monkey granulosa cells was also dramatically stimulated by a gonadotropin-releasing hormone agonist. Monkey granulosa cells, unlike the other animal cells, secrete measurable amount of estrogen in the absence of androgen substrate. The findings reported here are significant in regard to understanding of the mechanism of hormonal regulation of primate ovarian function.     

The Involvement of PDE4 in the Protective Effects of Melatonin on Cigarette-Smoke-Induced Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a significant disease threatening human health. Currently, roflumilast, a phosphodiesterase (PDE)4 inhibitor, is recommended as a therapeutic agent for COPD. In this study, we investigated the therapeutic effects of melatonin against COPD, focusing on determining whether it is a PDE4 inhibitor via in vivo and in vitro experiment using cigarette smoke (CS) and cigarette smoke condensate (CSC), respectively. In the in vivo experiments, melatonin treatment reduced inflammatory responses, including inflammatory cell counts. Melatonin treatment also suppressed the CS-exposure-induced upregulation of cytokine and matrix metalloproteinase (MMP)-9, reduced the PDE4B expression, and elevated cAMP levels. In addition, these effects were synergistic, as melatonin and roflumilast cotreatment eventually ameliorated the CS-exposure-induced worsening of lung function. In the CSC-stimulated NCI-H292 cells, melatonin inhibited elevation in the levels of inflammatory cytokines, MMP-9, and PDE4, and elevated cAMP levels. Furthermore, melatonin and roflumilast cotreatment was more effective on inflammatory responses than only melatonin or roflumilast treatment. Our results indicate that melatonin relieves inflammatory response and loss of lung function in COPD, which is associated with decreased PDE4 expression. Therefore, we suggest that melatonin is a putative candidate for the treatment of COPD.     

Laccase-Mediated Transformations of Endocrine Disrupting Chemicals Abolish Binding Affinities to Estrogen Receptors and Their Estrogenic Activity in Zebrafish

Endocrine disrupting chemicals (EDCs) are known to mainly affect aquatic organisms, producing negative effects in aquaculture. Transformation of the estrogenic compounds 17??-estradiol (E2), bisphenol-A (BPA), nonylphenol (NP), and triclosan (TCS) by laccase of Coriolopsis gallica was studied. Laccase is able to efficiently transform them into polymers. The estrogenic activity of the EDCs and their laccase transformation products was evaluated in vitro as their affinity for the human estrogen receptor alpha (hER??) and for the ligand binding domain of zebrafish (Danio rerio) estrogen receptor alpha (zfER??LBD). E2, BPA, NP, and TCS showed higher affinity for the zfER??LBD than for hER??. After laccase treatment, no affinity was found, except a marginal affinity of E2 products for the zfER??LBD. Endocrine disruption studies in vivo on zebrafish were performed using the induction of vitellogenin 1 as a biomarker (VTG1 mRNA levels). The use of enzymatic bioreactors, containing immobilized laccase, efficiently eliminates the endocrine activity of BPA and TCS, and significantly reduces the effects of E2. The potential use of enzymatic reactors to eliminate the endocrine activity of EDCs in supply water for aquaculture is discussed.     

A priming role of local estrogen on exogenous estrogen-mediated synaptic plasticity and neuroprotection

The localization of estrogen (E2) has been clearly shown in hippocampus, called local hippocampal E2. It enhanced neuronal synaptic plasticity and protected neuron form cerebral ischemia, similar to those effects of exogenous E2. However, the interactive function of hippocampal and exogenous E2 on synaptic plasticity activation and neuroprotection is still elusive. By using hippocampal H19-7 cells, we demonstrated the local hippocampal E2 that totally suppressed by aromatase inhibitor anastrozole. Anastrozole also suppressed estrogen receptor (ER)β, but not ERα, expression. Specific agonist of ERα (PPT) and ERβ (DPN) restored ERβ expression in anastrozole-treated cells. In combinatorial treatment with anastrozole and phosphoinositide kinase-3 (PI-3K) signaling inhibitor wortmannin, PPT could not improve hippocampal ERβ expression. On the other hand, DPN induced basal ERβ translocalization into nucleus of anastrozole-treated cells. Exogenous E2 increased synaptic plasticity markers expression in H19-7 cells. However, exogenous E2 could not enhance synaptic plasticity in anastrozoletreated group. Exogenous E2 also increased cell viability and B-cell lymphoma 2 (Bcl2) expression in H(2)O(2)-treated cells. In combined treatment of anastrozole and H(2)O(2), exogenous E2 failed to enhance cell viability and Bcl2 expression in hippocampal H19-7 cells. Our results provided the evidence of the priming role of local hippocampal E2 on exogenous E2-enhanced synaptic plasticity and viability of hippocampal neurons.     

A radioimmunoassay for serum estradiol

A radioimmunoassay procedure to measure estradiol levels in human serum was developed using¹²⁵I labeled estradiol and antiestradiol antibody raised in-house using the estradiol-6-CMO-BSA as the immunogen. Controlled process serum was used to maintain the serum matrix and sample-standard identity. The assay was validated by analysing several (n=40) samples and comparing with established commercial assays. The assay had a sensitivity of 35 pg/mL and a range of 50 pg/mL to 5 ng/mL. The interassay C.V. were between 2 to 10% while the intraassay C.V. were between 2 and 8%. This assay is sensitive enough for monitoring ovulation and hypersecretions in ovarian tumours and was primarily developed to be used as an in-house assay in the local hospitals.     

AGR2, a mucinous ovarian cancer marker, promotes cell proliferation and migration

Ovarian cancer is a leading cause of death in women. Early detection of ovarian cancer is essential to decrease mortality. However, the early diagnosis of ovarian cancer is difficult due to a lack of clinical symptoms and suitable molecular diagnostic markers. Thus, identification of meaningful tumor biomarkers with potential clinical application is clearly needed. To search for a biomarker for the early detection of ovarian cancer, we identified human anterior gradient 2 (AGR2) from our systematic analysis of paired normal and ovarian tumor tissue cDNA microarray. We noted a marked overexpression of AGR2 mRNA and protein in early stage mucinous ovarian tumors compared to normal ovarian tissues and serous type ovarian tumors by Western blot analysis and immunohistochemistry. To further elucidate the role of AGR2 in ovarian tumorigenesis, stable 2774 human ovarian cancer cell lines overexpressing AGR2 were established. Forced expression of AGR2 in 2774 cells enhanced the growth and migration of ovarian cancer cells. AGR2 protein was detected in the serum of mucinous ovarian cancer patients by Western blot and ELISA analysis. Thus, AGR2 is a potential biomarker for the diagnosis of mucinous ovarian cancer and an ELISA assay may facilitate the early detection of mucinous ovarian cancer using patient serum.     

A comparative study on involvement of tPA activity in ovulation induced by hCG and GnRH agonist in hypophysectomized rats.

A GnRH agonist (5-50 micrograms) is capable of inducing ovulation in PMSG-primed hypophysectomized immature rats, as is the case in hCG-induced ovulation, but 2-4 h earlier than hCG. GnRH-induced ovulation is effectively blocked by the concomitant administration of the GnRH-antagonist which failed to interfere with hCG-induced ovulation, indicating that GnRH and its agonists do not share a receptor with LH/hCG. Like hCG, GnRH is also capable of inducing tissue type (tPA), but not urokinase type (uPA) PA. The plasminogen activator activity in ovarian homogenates and the granulosa and theca-interstitial cells increase in a time-dependent manner, reaching maximum levels just prior to ovulation. Similar to hCG, GnRH also increases tPA activity in cumulus-oocyte complexes in a time-dependent fashion.     

An enzyme-linked immunosorbent assay system for quantitative determination of calphobindin I, a new placental anticoagulant protein, and its application to various specimens

We developed a sandwich enzyme-linked immunosorbent assay (ELISA) system for calphobindin I (CPB-I), a new placental coagulation inhibitor, using two monoclonal antibodies. This ELISA system can detect CPB-I at concentrations of between 0.4 and 25 ng/ml in buffer and allow almost quantitative determination of it in human plasma. Using this ELISA system, CPB-I levels in many kinds of specimens were measured. Levels in the plasma and urine of women were as low as 10 ng/ml, and no significant differences were observed throughout the trimesters of pregnancy and during different stages of the menstrual cycle. Toxemic patients were slightly higher in CPB-I levels than normal pregnant women, and levels in body fluids such as the amniotic fluid, saliva, milk, ascites, and semen were higher than those in the plasma. The high levels of CPB-I were found, being in the order of micrograms/ml, in the ascites of carcinomatous peritonitis as well as seminal plasma. Measurements of the levels in ovarian follicular fluid samples at different stages of the menstrual cycle showed that those in the immature and atretic stages were higher than those in mature stages. CPB-I levels in many types of cultured human cells ranged from 0.023 to 10.30 micrograms/mg protein, and levels in cultured human lymphocytes were less than those in other types of cells measured. Little of this inhibitor was secreted into media from cultured human lymphocytes, and it was found in all measured tissues of Macacus irus at levels ranging from 0.232 to 1.557 micrograms/mg protein. From these results, it was suggested that CPB-I might be a ubiquitous protein in the body that has an important physiological role.     

Dysfunction of Shh signaling activates autophagy to inhibit trophoblast motility in recurrent miscarriage

In early pregnancy, the placenta anchors the conceptus and supports embryonic development and survival. This study aimed to investigate the underlying functions of Shh signaling in recurrent miscarriage (RM), a serious disorder of pregnancy. In the present study, Shh and Gli2 were mainly observed in cytotrophoblasts (CTBs), Ptch was mainly observed in syncytiotrophoblasts (STBs), and Smo and Gli3 were expressed in both CTBs and STBs. Shh signaling was significantly impaired in human placenta tissue from recurrent miscarriage patients compared to that of gestational age-matched normal controls. VEGF-A and CD31 protein levels were also significantly decreased in recurrent miscarriage patients. Furthermore, inhibition of Shh signaling impaired the motility of JAR cells by regulating the expression of Gli2 and Gli3. Intriguingly, inhibition of Shh signaling also triggered autophagy and autolysosome accumulation. Additionally, knockdown of BECN1 reversed Gant61-induced motility inhibition. In conclusion, our results showed that dysfunction of Shh signaling activated autophagy to inhibit trophoblast motility, which suggests the Shh pathway and autophagy as potential targets for RM therapy.Subject terms: Embryology, Endocrine reproductive disorders, Autophagosomes