The environmental estrogenic compound bisphenol A exerts estrogenic effects on mouse hippocampal (HT-22) cells: neuroprotection against glutamate and amyloid beta protein toxicity

We have examined using immortalized clonal mouse hippocampal cell line (HT-22) whether the environmental estrogenic compound bisphenol A (BPA), like estrogen, has any neuroprotective effect against glutamate and amyloid beta protein-induced neurotoxicity. BPA protects HT-cells against both 5 mM glutamate and 2 microM amyloid beta protein-induced cell death in a dose dependent manner. Optimum protection was attained at 1 microM and 500 nM BPA against 5 mM glutamate and 2 microM amyloid beta protein-induced HT-22 cell death, respectively. Using confocal immunoflourescence microscopy technique, we observed that 20 h of treatment with 5 mM glutamate resulted in intense nuclear localization of the glucocorticoid receptors (GR) in HT-22 cells as compared to control untreated cells. Interestingly, 1 microM BPA treatment for 24 h, followed by 20-h treatment with 5 mM glutamate, resulted in dramatic reduction in GR nuclear localization. We conclude that: (i) BPA mimics estrogen and exerts neuroprotective effects against both neurotoxins used; (ii) BPA inhibits enhanced nuclear localization of GR induced by glutamate; and (iii) HT-22 cells provide a good in vitro model system for screening the potencies of various environmental compounds for their estrogenic activity.     

Pregnenolone Protects Mouse Hippocampal (HT-22) Cells against Glutamate and Amyloid Beta Protein Toxicity

In the present work we have examined whether the neurosteroid pregnenolone has any neuroprotective effects against glutamate and amyloid beta protein neurotoxicity using immortalized clonal mouse hippocampal cell line (HT-22). The neurosteroid pregnenolone protects HT-22 cells against both 5 mM glutamate and 2 M amyloid beta protein induced cell death in a concentration dependent manner. Optimum protection was attained at 500 nM pregnenolone, against both 5 mM glutamate as well as 2 M amyloid beta protein induced HT-22 cell death. Furthermore, using confocal immunoflourescence microscopy we observed that 20 hours of treatment with 5 mM glutamate resulted in intense nuclear localization of the glucocorticoid receptor (GR) in HT-22 cells as compared to control untreated cells. Interestingly, 500 nM pregnenolone treatment for 24 hours, followed by 20 hours treatment with 5 mM glutamate resulted in dramatic reduction in GR nuclear localization. These results show that (i) pregnenolone has neuroprotective effects against both glutamate and amyloid beta protein neuropathology and (ii) prevention of glucocorticoid receptor (GR) localization to the nucleus may be involved in the observed neuroprotective effects of pregnenolone against glutamate neurotoxicity.     

Dehydroepiandrosterone protects hippocampal neurons against neurotoxin-induced cell death: mechanism of action

Dehydroepiandrosterone (DHEA), an adrenal cortex hormone secreted in large quantities in humans, protects cells of the clonal mouse hippocampal cell line HT-22 against the excitatory amino acid glutamate (5 mM), and amyloid beta-protein (2 microM) toxicity in a dose-dependent manner with optimum protection obtained at 5 microM concentration of DHEA. The protective effects of DHEA appear to be specific in that other related steroids and metabolites of DHEA, such as 5-androstene-3beta,17beta-diol, etiocholan-3alpha-ol-17-one, etiocholan-3beta-ol-17-one, testosterone, and 5alpha-androstane-3, 17-dione, offered no protection even at 50 microM concentrations. In addition, using immunocytochemical techniques, we observed that 20 hr of treatment with 5 mM glutamate remarkably increased glucocorticoid receptor (GR) nuclear localization in neuronal cells. Interestingly, 5 microM DHEA treatment for 24 hr, followed by 5 mM glutamate treatment for 20 hr almost completely reversed the copious nuclear localization of GR observed by glutamate treatment alone. Results obtained suggest that DHEA protects hippocampal neurons, at least in part, by its antiglucocorticoid action via decreasing hippocampal cells nuclear GR levels.     

Effects of 17-beta estradiol and estriol on NMDA-induced toxicity and apoptosis in primary cultures of rat cortical neurons.

Estrogens possess neuroprotective and antiapoptotic properties, however, the issue of involvement of estrogen receptors (ER)-dependent genomic pathway in these effects still remains controversial. Moreover, the majority of data on antiapoptotic effects of estrogens concern non-neuronal cells. In the present study we compared effects of the potent ER agonist, estradiol-17beta (E2), and its metabolite with a weak affinity for ER, estriol, on the neurotoxicity induced by high (1 and 5 mM) NMDA concentrations and on the apoptosis induced by low (0.1 mM) concentration of NMDA in rat primary cortical neurons. The obtained data showed that 24-hour exposure of cortical neurons to NMDA (0.1-5 mM) resulted in a dose-dependent increase in LDH level. Twenty four-hour pretreatment with estriol (100 nM and 500 nM) reduced the NMDA (1 and 5 mM)-induced toxicity by 16-26%, while estradiol-17beta (500 nM) reduced NMDA (5 mM)- induced toxicity by 14%. Twenty four hour exposure of cortical neurons to NMDA (0.1 mM) resulted in decrease of the level of antiapoptotic protein - Bcl-2 by 60% and increased the number of apoptotic cells by 50% compared to the control. Twenty four hour pretreatment with estradiol-17beta or estriol (100 and 1000 nM) prevented the NMDA-induced apoptotic changes. The specific estrogen receptor antagonist ICI 182,780 (100 nM) had no effect alone and did not antagonize the effects of estrogens on NMDA-induced toxicity as well as on changes in Bcl-2 level. The higher efficacy of estriol, together with the fact that the specific ER receptor antagonist, ICI 182,780, did not inhibit the above-described effects support the hypothesis about a nongenomic mechanism of the anti-NMDA action of estrogens.     

Soybean-derived phosphatidylinositol inhibits in vivo low concentrations of amyloid beta protein-induced degeneration of hippocampal neurons in V337M human tau-expressing mice

In our previous reports using primary cultured rat hippocampal neurons, pathophysiological concentrations (< or =10 nM) of amyloid beta proteins (Abetas) showed neurotoxicity via a phosphatidylinositol metabolism disorder, and soybean-derived phosphatidylinositol protected the neurons against the Abeta's neurotoxicity. In the present study, such a neurotoxic effect of Abeta and a neuroprotective effect of phosphatidylinositol were examined in vivo using transgenic mice expressing V337 M human tau. Intrahippocampal CA1 injection of 1.5 mul of 100 nM or 1 microM Abeta25-35 increased the number of degenerating neurons with an apoptotic feature in bilateral hippocampal CA1, CA2, CA3 and dentate gyrus regions in 1 month, demonstrating an in vivo neurotoxic effect of Abeta at lower concentrations after diffusion. Intrahippocampal co-injection or intracerebroventricular administration of 1.5 microl of 500 nM phosphatidylinositol prevented the Abeta25-35-induced neuronal degeneration in all the hippocampal regions, while co-injection of another acidic phospholipid, phosphatidylserine (1.5 microl, 500 nM) with Abeta25-35 showed no protective effects. Thus, exogenously applied phosphatidylinositol appeared to minimize the toxic effects of Abeta in vivo. These results suggest that soybean-derived phosphatidylinositol may be effective in the treatment of Alzheimer's disease.     

Suppression of protein kinase Cepsilon mediates 17beta-estradiol-induced neuroprotection in an immortalized hippocampal cell line

Although estrogens are neuroprotective in a variety of neuroprotection models, the precise underlying mechanisms are currently not well understood. Here, we examined the role of protein kinase C (PKC) in mediating estrogen-induced neuroprotection in the HT-22 immortalized hippocampal cell line. The neuroprotection model utilized calcein fluorescence to quantitate cell viability following glutamate insults. 17beta-Estradiol (betaE2) protected HT-22 cells when treatment was initiated before or after the glutamate insult. The inhibition of PKC by bis-indolylmaleimide mimicked and enhanced betaE2-induced neuroprotection. In contrast, the inhibition of specific PKC isozymes (alpha and beta) by Go6976, inhibition of 1-phosphatidylinositol 3 kinase by wortmannin, or inhibition of protein kinase A by H-89, did not alter cell viability, suggesting a specific involvement of PKC in an isozyme-dependent manner. We further examined whether estrogen interacts with PKC in a PKC isozyme-specific manner. Protein levels and activity of PKC isozymes (alpha, delta, epsilon, and zeta) were assessed by western blot analysis and radiolabeled phosphorylation assays respectively. Among the isozymes tested, betaE2 altered only PKCepsilon; it reduced the activity and membrane translocation of PKCepsilon in a manner that correlated with its protection against glutamate toxicity. Furthermore, betaE2 reversed the increased activity of membrane PKCepsilon induced by glutamate. These data suggest that the neuroprotective effects of estrogens are mediated in part by inhibition of PKCepsilon activity and membrane translocation.     

The excitoprotective effect of N-methyl-D-aspartate receptors is mediated by a brain-derived neurotrophic factor autocrine loop in cultured hippocampal neurons

The neuroprotective effect and molecular mechanisms underlying preconditioning with N-methyl-D-aspartate (NMDA) in cultured hippocampal neurons have not been described. Pre-incubation with subtoxic concentrations of the endogenous neurotransmitter glutamate protects vulnerable neurons against NMDA receptor-mediated excitotoxicity. As a result of physiological preconditioning, NMDA significantly antagonizes the neurotoxicity resulting from subsequent exposure to an excitotoxic concentration of glutamate. The protective effect of glutamate or NMDA is time- and concentration-dependent, suggesting that sufficient agonist and time are required to establish an intracellular neuroprotective state. In these cells, the TrkB ligand, brain-derived neurotrophic factor (BDNF) attenuates glutamate toxicity. Therefore, we tested the hypothesis that NMDA protects neurons via a BDNF-dependent mechanism. Exposure of hippocampal cultures to a neuroprotective concentration of NMDA (50 microM) evoked the release of BDNF within 2 min without attendant changes in BDNF protein or gene expression. The accumulated increase of BDNF in the medium is followed by an increase in the phosphorylation (activation) of TrkB receptors and a later increase in exon 4-specific BDNF mRNA. The neuroprotective effect of NMDA was attenuated by pre-incubation with a BDNF-blocking antibody and TrkB-IgG, a fusion protein known to inhibit the activity of extracellular BDNF, suggesting that BDNF plays a major role in NMDA-mediated survival. These results demonstrate that low level stimulation of NMDA receptors protect neurons against glutamate excitotoxicity via a BDNF autocrine loop in hippocampal neurons and suggest that activation of neurotrophin signaling pathways plays a key role in the neuroprotection of NMDA.     

Roles of antiestrogen binding sites in human endometrial cancer cells

Estrogen-noncompatible antiestrogen binding sites (AEBS) as well as estrogen receptors (ER), and the growth-inhibitory effect of tamoxifen were investigated in two human endometrial cancer cell lines, IK-90 and HEC-IA cells. IK-90 cells contained specific AEBS, but no ER was found in these cells. Scatchard plot analysis of AEBS in 12,000 g supernatant from IK-90 cells showed a high affinity binding site for tamoxifen (Kd:5.6 +/- 1.0 nM) with the maximum binding site of 457 +/- 47 fmol/mg protein. However, no measurable ER or AEBS was found in HEC-IA cells. The effect of tamoxifen on the growth of cells was found to be identical in both cell lines; the addition of 10 microM tamoxifen to culture medium was cytocidal whereas tamoxifen at lower concentrations (1 nM-1 microM) did not significantly affect the growth of both IK-90 and HEC-IA cells. These results demonstrate for the first time the presence of AEBS in human endometrial cancer cells. The present results also suggest that AEBS does not play a fundamental role in mediating the growth-inhibitory effect of tamoxifen in endometrial cancer cells.     

Role of nonfeminizing estrogen analogues in neuroprotection of rat retinal ganglion cells against glutamate-induced cytotoxicity

Glaucoma is a family of eye disorders whose ultimate cause of vision loss is apoptosis of retinal ganglion cells. Although several etiologies of glaucoma exist, oxidative stress is thought to be a key mechanism by which ganglion cells die. From this perspective, the work presented here was designed to examine the efficacy of 17beta-estradiol and three synthetic estrogen analogues (ZYC-1, ZYC-3, ZYC-10) as retinal ganglion cell neuroprotectants. Compound ZYC-1 and its enantiomer ZYC-10, containing an additional double bond in the steroid C ring of 17beta-estradiol, had similar (ZYC-1) or modestly reduced (ZYC-10) affinity for estrogen receptors compared to the parent estrogen. In the case of ZYC-3, the addition of an adamantyl group to the C2 position of the A ring of estrone abolished its binding to the estrogen receptors. RGC-5 cells (an established clonal rat retinal ganglion cell line) and rat retinas were shown to predominantly express estrogen receptor alpha, with minimal detectable levels of estrogen receptor beta. The affinity of the synthetic compounds for estrogen receptors was as follows: ZYC-3 < ZYC-10 < ZYC-1. An in vitro model of glutamate-induced RGC-5 cell death was used. Glutamate treatment resulted in 50-60% RGC-5 cell death with respect to control untreated cells. 17beta-estradiol and the three estrogen analogues (0.5 to 1.0 microM) protected the RGC-5 cells against glutamate cytotoxicity. The efficacy of neuroprotection by the estrogen analogues was as follows: ZYC-3 > ZYC-1 > ZYC-10. EC(50) values for inhibition of TBARS levels were as follows: ZYC-3 > ZYC-10 > ZYC-1. Furthermore, these compounds worked independent of estrogen receptors, as inclusion of 100 nM ICI 182,780 did not reverse their neuroprotective properties against glutamate insult. These compounds seem to affect neuroprotection via pathways independent of the classical estrogen receptors. The data support the hypothesis that estrogen analogues may be useful in the treatment of neurodegenerative diseases, particularly in neuroprotection of retinal ganglion cells in ocular pathologies such as glaucoma.     

Catechin and epicatechin from Smilacis chinae rhizome protect cultured rat cortical neurons against amyloid beta protein (25-35)-induced neurotoxicity through inhibition of cytosolic calcium elevation

We previously reported that the Smilacis chinae rhizome inhibits amyloid beta protein (25-35) (Abeta (25-35))-induced neurotoxicity in cultured rat cortical neurons. Here, we isolated catechin and epicatechin from S. chinae rhizome and also studied their neuroprotective effects on Abeta (25-35)-induced neurotoxicity in cultured rat cortical neurons. Catechin and epicatechin inhibited 10 microM Abeta (25-35)-induced neuronal cell death at a concentration of 10 microM, which was measured by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. Catechin and epicatechin inhibited 10 microM Abeta (25-35)-induced elevation of cytosolic calcium concentration ([Ca2+]c), which was measured by a fluorescent dye, Fluo-4 AM. Catechin and epicatechin also inhibited glutamate release into medium induced by 10 microM Abeta (25-35), which was measured by HPLC, generation of reactive oxygen species (ROS) and activation of caspase-3. These results suggest that catechin and epicatechin prevent Abeta (25-35)-induced neuronal cell damage by interfering with the increase of [Ca2+]c, and then by inhibiting glutamate release, generation of ROS and caspase-3 activity. Furthermore, these effects of catechin and epicatechin may be associated with the neuroprotective effect of the S. chinae rhizome.     

Daphnetin, a Natural Coumarin Derivative, Provides the Neuroprotection Against Glutamate-Induced Toxicity in HT22 Cells and Ischemic Brain Injury

Daphnetin (DAP), a coumarin derivative, has been reported to have multiple pharmacological actions including analgesia, antimalarial, anti-arthritic, and anti-pyretic properties. It is unclear whether DAP has neuroprotective effects on ischemic brain injury. In this study, we found that DAP treatment (i.c.v.) reduced the infarct volume at 24 h after ischemia/reperfusion injury and improved neurological behaviors in a middle cerebral artery occlusion mouse model. Moreover, we provided evidences that DAP had protective effects on infarct volume in neonate rats even it was administrated at 4 h after cerebral hypoxia/ischemia injury. To explore its neuroprotective mechanisms of DAP, we examined the protection of DAP on glutamate toxicity-induced cell death in hippocampal HT-22 cells. Our results demonstrated that DAP protected against glutamate toxicity in HT-22 cells in a concentration-dependent manner. Further, we found that DAP maintained the cellular levels of glutathione and superoxide dismutase activity, suggesting the anti-oxidatant activity of DAP. Since DAP has been used for the treatment of coagulation disorder and rheumatoid arthritis for long time with a safety profile, DAP will be a promising agent for the treatment of stroke.     

Glutamate Receptor Requirement for Neuronal Death from Anoxia–Reoxygenation: An in Vitro Model for Assessment of the Neuroprotective Effects of Estrogens

1.Previous studies demonstrated that estrogens, specifically 17-estradiol, the potent, naturally occurring estrogen, are neuroprotective in a variety of models including glutamate toxicity. The aim of the present study is twofold: (1) to assess the requirement for glutamate receptors in neuronal cell death associated with anoxia–reoxygenation in three cell types, SK-N-SH and HT-22 neuronal cell lines and primary rat cortical neuronal cultures, and (2) to evaluate the neuroprotective activity of both 17-estradiol and its weaker isomer, 17-estradiol, in both anoxia-reoxygenation and glutamate toxicity.2.SK-N-SH and HT-22 cell lines, both of which lack NMDA receptors as assessed by MK-801 binding assays, were resistant to both anoxia–reoxygenation and glutamate-induced cell death. In contrast, primary rat cortical neurons, which exhibit both NMDA and AMPA receptors, were sensitive to brief periods of exposure to anoxia–reoxygenation or glutamate. As such, there appears to be an obligatory requirement for NMDA and/or AMPA receptors in neuronal cell death resulting from brief periods of anoxia followed by reoxygenation.3.Using primary rat cortical neuronal cultures, we evaluated the neuroprotective activity of 17-estradiol (1.3 or 133 nM) and 17-estradiol (133 nM) in both anoxia–reoxygenation and excitotoxicity models of cell death. We found that the 133 nM but not the 1.3 nM dose of the potent estrogen, 17-estradiol, protected 58.0, 57.5, and 85.3% of the primary rat cortical neurons from anoxia–reoxygenation, glutamate, or AMPA toxicity, respectively, and the 133 nM dose of the weak estrogen, 17-estradiol, protected 74.6, 81.7, and 85.8% of cells from anoxia–reoxygenation, glutamate, or AMPA toxicity, respectively. These data demonstrate that pretreatment with estrogens can attenuate glutamate excitotoxicity and that this protection is independent of the ability of the steroid to bind the estrogen receptor.     

Propofol hemisuccinate protects neuronal cells from oxidative injury

Oxidative stress contributes to the neuronal death observed in neurodegenerative disorders and neurotrauma. Some antioxidants for CNS injuries, however, have yet to show mitigating effects in clinical trials, possibly due to the impermeability of antioxidants across the blood-brain barrier (BBB). Propofol (2,6-diisopropylphenol), the active ingredient of a commonly used anesthetic, acts as an antioxidant, but it is insoluble in water. Therefore, we synthesized its water-soluble prodrug, propofol hemisuccinate sodium salt (PHS), and tested for its protective efficacy in neuronal death caused by non-receptor-mediated, oxidative glutamate toxicity. Glutamate induces apoptotic death in rat cortical neurons and the mouse hippocampal cell line HT-22 by blocking cystine uptake and causing the depletion of intracellular glutathione, resulting in the accumulation of reactive oxygen species (ROS). PHS has minimal toxicity and protects both cortical neurons and HT-22 cells from glutamate. The mechanism of protection is attributable to the antioxidative property of PHS because PHS decreases the ROS accumulation caused by glutamate. Furthermore, PHS protects HT-22 cells from oxidative injury induced by homocysteic acid, buthionine sulfoximine, and hydrogen peroxide. For comparison, we also tested alpha-tocopherol succinate (TS) and methylprednisolone succinate (MPS) in the glutamate assay. Although TS is protective against glutamate at lower concentrations than PHS, TS is toxic to HT-22 cells. In contrast, MPS is nontoxic but also nonprotective against glutamate. Taken together, PHS, a water-soluble prodrug of propofol, is a candidate drug to treat CNS injuries owing to its antioxidative properties, low toxicity, and permeability across the BBB.     

Coenzyme Q10 protects SHSY5Y neuronal cells from beta amyloid toxicity and oxygen-glucose deprivation by inhibiting the opening of the mitochondrial permeability transition pore

Coenzyme Q10 (CoQ10) is an essential biological cofactor which increases brain mitochondrial concentration and exerts neuroprotective effects. In the present study, we exposed SHSY5Y neuroblastoma cells to neurotoxic beta amyloid peptides (Abeta) and oxygen glucose deprivation (OGD) to investigate the neuroprotective effect of 10 microM CoQ10 by measuring (i) cell viability by the MTT assay, (ii) opening of the mitochondrial permeability transition pore via the fluorescence intensity of calcein-AM, and (iii) superoxide anion concentration by hydroethidine. Cell viability (mean +/- S.E.M.) was 55.5 +/- 0.8% in the group exposed to Abeta + OGD, a value lower than that in the Abeta or OGD group alone (P < 0.01). CoQ10 had no neuroprotective effect on cell death induced by either Abeta or OGD, but increased cell survival in the Abeta + OGD group to 57.3 +/- 1.7%, which was higher than in the group treated with vehicle (P < 0.05). The neuroprotective effect of CoQ10 was blocked by administration of 20 microM atractyloside. Pore opening and superoxide anion concentration were increased in the Abeta + OGD group relative to sham control (P < 0.01), and were attenuated to the sham level (P > 0.05) when CoQ10 was administered. Our results demonstrate that CoQ10 protects neuronal cells against Abeta neurotoxicity together with OGD by inhibiting the opening of the pore and reducing the concentration of superoxide anion.     

Estrogen induces a rapid secretion of amyloid beta precursor protein via the mitogen-activated protein kinase pathway.

The female sex hormone estrogen (17beta-estradiol; E2) may function as a neurohormone and has multiple neuromodulatory functions in the brain. Its potent neuroprotective activities can be dependent and independent of estrogen receptors (ERs). In addition, E2 influences the processing of the amyloid beta precursor protein (APP), one central step in the pathogenesis of Alzheimer's disease. Here, we show: (a) that physiological concentrations of E2 very rapidly cause an increased release of secreted nonamyloidogenic APP (sAPPalpha) in mouse hippocampal HT22 and human neuroblastoma SK-N-MC cells; and (b) that this effect is mediated through E2 via the phosphorylation of extracellular-regulated kinase 1 and 2 (ERK1/2), prominent members of the mitogen-activated protein kinase (MAPK) pathway. Furthermore, we show that the activation of MAPK-signaling pathway and the enhancement of the sAPP release is independent of ERs and could be induced by E2 to a similar extent in neuronal cells either lacking or overexpressing a functional ER.     

Antioxidant activity and neuroprotective effects of zolpidem and several synthesis intermediates

Structural relationship between the antioxidant melatonin and the non-benzodiazepine hypnotic zolpidem (ZPD) suggests possible direct antioxidant and neuroprotective properties of this compound. In the present work, these effects were analyzed for zolpidem and four of its synthesis intermediates. In vitro assays include lipid peroxidation and protein oxidation studies in liver and brain homogenates. Intracellular antioxidant effects were analyzed by evaluation of free radical formation prevention in HT-22 hippocampal cells treated with glutamate 10mM and measured by flow cytometer DCF fluorescence. The neuroprotective effect of these compounds was evaluated as neuronal death prevention of HT-22 cells treated with the same concentration of glutamate. Zolpidem was found to prevent induced lipid peroxidation in rat liver and brain homogenates showing figures similar to melatonin, although it failed to prevent protein oxidation. ZPD-I was the most effective out of the several zolpidem intermediates studied as it prevented lipid peroxidation with an efficiency higher than melatonin or zolpidem and with an effectiveness similar to estradiol and trolox. ZPD-I prevents protein oxidation, which trolox is known to be unable to prevent. When cellular experiments were undertaken, ZPD-I prevented totally the increase of intracellular free radicals induced by glutamate 10mM in culture medium for 12h, while zolpidem and ZPD-III partially prevented this increase. Also the three compounds protected hippocampal neurons from glutamate-induced death in the same conditions, being their comparative efficacy, ZPD-III > ZPD-I = ZPD.     

Estrogenic activity of procymidone in rainbow trout (Oncorhynchus mykiss) hepatocytes: a possible mechanism of action

It is known that procymidone modifies sexual differentiation in vivo and in vitro, and that it induces vitellogenin (Vtg) synthesis in primary cultured rainbow trout hepatocytes. The aim of this study was to evaluate the mechanism underlying this latter in vitro estrogenic action. The cells were treated for 24 h with procymidone 150 microM (with 17beta-estradiol [E2] 20 microM as a positive control) combined with an estrogen receptor (ER) antagonist (tamoxifen 20 microM or ICI 182,780 1 microM) or, given the drug toxic action on the production of reactive oxygen species (ROS), a free radical scavenger (alpha-tocopherol 30 microM). The results from ELISA experiments provided evidence that procymidone Vtg-induction is inhibited by ER antagonists and by alpha-tocopherol suggesting that both ER and ROS are involved in this effect. The ROS detection revealed that the treatment with alpha-tocopherol and tamoxifen completely prevented ROS induction by procymidone, that was not inhibited by ICI 182,780. In exploring the mechanism mediating these events and its timing, we found that procymidone induced mitogen-activated protein kinase (MAPK) at 30 and 60 min, and that this effect was blocked by co-treatment with alpha-tocopherol. In summary, the results of the study clearly support the idea that the estrogenic activity of procymidone in primary cultured trout hepatocytes is mediated by ROS production, and that this activity is similar to that of the ligand-independent ER activation involving MAPK.     

Amyloid beta proteins inhibit Cl(-)-ATPase activity in cultured rat hippocampal neurons

Cl(-)-ATPase in the CNS is a candidate for an outwardly directed neuronal Cl(-) transporter requiring phosphatidylinositol-4-phosphate (PI4P) for its optimal activity. To test its pathophysiological changes in a phosphatidylinositol (PI) metabolism disorder, the effects of neurotoxic factors in Alzheimer's disease (AD), amyloid beta proteins (Abetas), on the Cl(-)-ATPase activity were examined using primary cultured rat hippocampal neurons. Amyloid beta proteins (1-40, 1-42 and 25-35) concentration-dependently (1-100 nM) and time-dependently (from 1 h to 6 day) decreased Cl(-)-ATPase activity and elevated intracellular Cl(-) concentrations ([Cl(-)]i), Abeta25-35 being the most potent. Addition of inositol or 8-Br-cyclic GMP completely reversed these Abeta-induced changes. The recoveries in enzyme activity were attenuated by an inhibitor of PI 4-kinase, 10 microM wortmannin or 20 microM quercetin, but not by a PI 3-kinase inhibitor, 50 nM wortmannin or 10 microM LY294002. The PI, PIP and PIP2 levels of the plasma membrane-rich fraction were lower in the Abeta-treated cells as compared with each control. In the Abeta-exposed culture, but not in control, stimulation by 10 microM glutamate for 10 min significantly increased fragmentation of DNA and decreased cell viability. Addition of inositol or 8-Br-cyclic GMP prevented the effect of Abeta-treatment on the neurotoxicity of glutamate. Thus, Abetas reduce neuronal Cl(-)-ATPase activity, resulting in an increase in [Cl(-)]i probably by lowering PI4P levels, and this may reflect a pre-apoptotic condition in early pathophysiological profiles of AD.     

Tributyltin disturbs bovine adrenal steroidogenesis by two modes of action

Tributyltin, an environmental pollutant, affected adrenal steroid hormone biosynthesis by two modes of action. Treatment of bovine adrenal cultured cells with 10-100 nM tributyltin for 48 h suppressed cortisol and androstenedione secretion, but induced the accumulation of 17alpha-hydroxyprogesterone and deoxycortisol, indicating that the P450(C21) and P450(11beta) activities were specifically suppressed. Direct inhibition of the enzymatic activities due to tributyltin was not observed in isolated organelles of untreated cells at concentrations less than 10 microM. Western blotting experiments using specific antibodies against steroidogenic enzymes showed that treatment with 1-100 nM tributyltin caused a decrease in cellular P450(C21) and P450(11beta) protein levels, and real-time PCR experiments showed that the decrease in protein content was attributable to decreases in mRNA of the enzymes. Tributyltin at concentrations higher than 100 nM suppressed all steroid biosynthesis in the adrenal cells. This suppression was closely correlated to the decrease in steroidogenic acute regulatory protein. Since nanomolar concentrations of tributyltin disturbed steroidogenesis in mammalian cells, there is the possibility that steroid hormone synthesis in polluted wild animals is affected by this compound.     

Synergistic induction of ER stress by homocysteine and beta-amyloid in SH-SY5Y cells

Clinical studies have raised the possibility that elevated plasma levels of homocysteine increase the risk of atherosclerosis, stroke and possibly neurodegenerative diseases such as Alzheimer's disease (AD); however, the direct impact of homocysteine on neuron cells and the mechanism by which it could induce neurodegeneration have yet to be clearly demonstrated. Here, we investigated the effect of homocysteine on endoplasmic reticulum (ER) stress, the suggested mechanism of neurotoxicity, in human neuroblastoma SH-SY5Y cells. The effect of homocysteine on amyloid-beta (Abeta)-induced neurotoxicity and the protective activity of folate were also investigated. Homocysteine led to increased expressions of the binding protein (BiP) and the spliced form of X-box-protein (XBP)-1 mRNAs, suggesting activation of the unfolded-protein response and an increase in apoptosis. When cells were cotreated with homocysteine and Abeta, caspase-3 activity was significantly increased, and expressions of BiP and the spliced form of XBP-1 mRNAs were significantly induced. The neurotoxicity of homocysteine was attenuated by the treatment of cells with folate, as determined by caspase-3 activity and apoptotic body staining. These findings indicate that homocysteine induces ER stress and, ultimately, apoptosis and sensitizes neurons to amyloid toxicity via the synergistic induction of ER stress. Furthermore, a neuroprotective effect of folate against homocysteine-induced toxicity was also observed. Therefore, the findings of our study suggest that ER stress-induced homocysteine toxicity may play an important physiological role in enhancing the pathogenesis of Abeta-induced neuronal degeneration.