Neuroprotective effect of 3,3'-Diindolylmethane against perinatal asphyxia involves inhibition of the AhR and NMDA signaling and hypermethylation of specific genes.

Each year, 1 million children die due to perinatal asphyxia; however, there are no effective drugs to protect the neonatal brain against hypoxic/ischemic damage. In this study, we demonstrated for the first time the neuroprotective capacity of 3,3'-diindolylmethane (DIM) in an in vivo model of rat perinatal asphyxia, which has translational value and corresponds to hypoxic/ischemic episodes in human newborns. Posttreatment with DIM restored the weight of the ipsilateral hemisphere and normalized cell number in the brain structures of rats exposed to perinatal asphyxia. DIM also downregulated the mRNA expression of HIF1A-regulated Bnip3 and Hif1a which is a hypoxic marker, and the expression of miR-181b which is an indicator of perinatal asphyxia. In addition, DIM inhibited apoptosis and oxidative stress accompanying perinatal asphyxia through: downregulation of FAS, CASP-3, CAPN1, GPx3 and SOD-1, attenuation of caspase-9 activity, and upregulation of anti-apoptotic Bcl2 mRNA. The protective effects of DIM were accompanied by the inhibition of the AhR and NMDA signaling pathways, as indicated by the reduced expression levels of AhR, ARNT, CYP1A1, GluN1 and GluN2B, which was correlated with enhanced global DNA methylation and the methylation of the Ahr and Grin2b genes. Because our study provided evidence that in rat brain undergoing perinatal asphyxia, DIM predominantly targets AhR and NMDA, we postulate that compounds that possess the ability to inhibit their signaling are promising therapeutic tools to prevent stroke.

The neuroprotective action of 3,3'-diindolylmethane against ischemia involves an inhibition of apoptosis and autophagy that depends on HDAC and AhR/CYP1A1 but not ERα/CYP19A1 signaling.

There are no studies examining the effects of 3,3'-diindolylmethane (DIM) in neuronal cells subjected to ischemia. Little is also known about the roles of apoptosis and autophagy as well as AhR and ERα signaling and HDACs in DIM action. We demonstrated for the first time the strong neuroprotective capacity of DIM in mouse primary hippocampal cell cultures exposed to ischemia at early and later stages of neuronal development. The protective effects of DIM were mediated via inhibition of ischemia-induced apoptosis and autophagy that was accompanied by a decrease in AhR/CYP1A1 signaling and an increase in HDAC activity. DIM decreased the levels of pro-apoptotic factors, i.e., Fas, Caspase-3, and p38 mitogen-activated protein kinase (MAPK). DIM also reduced the protein levels of autophagy-related Beclin-1 (BECN1) and microtubule-associated proteins 1A/1B light chain (LC3), partially reversed the ischemia-induced decrease in Nucleoporin 62 (NUP62) and inhibited autophagosome formation. In addition, DIM completely reversed the ischemia-induced decrease in histone deacetylase (HDAC) activity in hippocampal neurons. Although DIM inhibited AhR/CYP1A1 signaling, it did not influence the protein expression levels of ERα and ERα-regulated CYP19A1 which are known to be controlled by AhR. This study demonstrated for the first time, that the neuroprotective action of 3,3'-diindolylmethane against ischemia involves an inhibition of apoptosis and autophagy and depends on AhR/CYP1A1 signaling and HDAC activity, thus creating the possibility of developing new therapeutic strategies that target neuronal degeneration at specific molecular levels.

Posttreatment Strategy Against Hypoxia and Ischemia Based on Selective Targeting of Nonnuclear Estrogen Receptors with PaPE-1.

Newly synthesized Pathway Preferential Estrogen-1 (PaPE-1) selectively activates membrane estrogen receptors (mERs), namely, mERα and mERß, and has been shown to evoke neuroprotection; however, its effectiveness in protecting brain tissue against hypoxia and ischemia has not been verified in a posttreatment paradigm. This is the first study showing that a 6-h delayed posttreatment with PaPE-1 inhibited hypoxia/ischemia-induced neuronal death, as indicated by neutral red uptake in mouse primary cell cultures in vitro. The effect was accompanied by substantial decreases in neurotoxicity and neurodegeneration in terms of LDH release and Fluoro-Jade C staining of damaged cells, respectively. The mechanisms of the neuroprotective action of PaPE-1 also involved apoptosis inhibition demonstrated by normalization of both mitochondrial membrane potential and expression levels of apoptosis-related genes and proteins such as Fas, Fasl, Bcl2, FAS, FASL, BCL2, BAX, and GSK3ß. Furthermore, PaPE-1-evoked neuroprotection was mediated through a reduction in ROS formation and restoration of cellular metabolic activity that had become dysregulated due to hypoxia and ischemia. These data provide evidence that targeting membrane non-GPER estrogen receptors with PaPE-1 is an effective therapy that protects brain neurons from hypoxic/ischemic damage, even when applied with a 6-h delay from injury onset.

Triclocarban impairs autophagy in neuronal cells and disrupts estrogen receptor signaling via hypermethylation of specific genes.

Although an increasing body of evidence suggests that triclocarban, a phenyl ether classified as a contaminant of emerging concern, presents a risk to development, there is limited data available on the potential interplay of triclocarban with the developing mammalian nervous system. This study was aimed to investigate the impact of environmentally pervasive chemical triclocarban on autophagy and estrogen receptor-mediated signaling pathways in mouse neurons. The study showed that triclocarban impaired autophagy and disrupted estrogen receptor signaling in mouse embryonic neurons in primary culture. Triclocarban used at environmentally relevant concentrations inhibited the mRNA and protein expression of ESR1 and GPER1 but not ESR2. The triclocarban-induced decrease in the expression of estrogen receptors was supported by the colocalization of the receptors in mouse neurons and corresponded to hypermethylation of the Esr1 and Gper1 genes. Selective antagonists increased the effects of triclocarban, which suggests that the neurotoxic effects of triclocarban, in addition to decreasing estrogen receptor expression, are mediated via inhibition of the neuroprotective capacity of the receptors. Furthermore, Becn1 and Atg7 siRNAs potentiated the caspase-3-dependent effect of triclocarban, which points to triclocarban-induced impairment of autophagy. Indeed, triclocarban dysregulated the expression of autophagy-related genes, and caused a time-dependent inhibition of the mRNA expression of Becn1, Map1lc3a, Map1lc3b, Nup62, and Atg7, which was correlated with a decrease in the protein levels of MAP1LC3B, BECN1 and autophagosomes, but not NUP62 protein level which was increased. Intriguingly, the Esr1 and Gper1 siRNAs did not affect the level of autophagosomes, suggesting that the triclocarban-induced impairment of autophagy is independent of the triclocarban-induced disruption of estrogen receptor signaling in mammalian neurons. Because our data provided evidence that triclocarban has the capacity to impair autophagy and disrupt estrogen receptor signaling in brain neurons at an early developmental stage, we postulate to categorize the compound as a neurodevelopmental risk factor.

Aryl hydrocarbon receptor-mediated apoptosis of neuronal cells: a possible interaction with estrogen receptor signaling.

Activation of aryl hydrocarbon receptors (AhRs) induces neuronal damage, but the mechanism by which this occurs is largely unknown. This study evaluated the effects of an AhR agonist, beta-naphthoflavone, on apoptotic pathways in mouse primary neuronal cell cultures. beta-Naphthoflavone (0.1-100 micronhanced caspase-3 activity and lactate dehydrogenase (LDH) release in neocortical and hippocampal cells. These data were supported at the cellular level with Hoechst 33342 and calcein AM staining. alpha-Naphthoflavone inhibited the action of beta-naphthoflavone, thus confirming specific activation of AhRs. A high-affinity estrogen receptor (ER) antagonist, ICI 182,780, and a selective estrogen receptor modulator (SERM), tamoxifen, enhanced beta-naphthoflavone-mediated apoptosis. Another SERM, raloxifene, and an ERalpha antagonist, methyl-piperidino-pyrazole, did not affect beta-naphthoflavone-induced caspase-3 activity. However, they inhibited beta-naphthoflavone-induced LDH release at a late hour of treatment, thus suggesting delayed control of AhR-mediated neuronal cell death. The apoptotic effects of beta-naphthoflavone were accompanied by increased levels of AhRs, and these receptors colocalized with ERbeta as demonstrated by confocal microscopy. These data strongly support apoptotic effects of AhR activation in neocortical and hippocampal tissues. Moreover, this study provides evidence for direct interaction of the AhR-mediated apoptotic pathway with estrogen receptor signaling, which provides insight into new strategies to treat or prevent AhR-mediated neurotoxicity.

Triclocarban Disrupts the Epigenetic Status of Neuronal Cells and Induces AHR/CAR-Mediated Apoptosis.

Triclocarban is a phenyl ether that has recently been classified as a contaminant of emerging concern. Evidence shows that triclocarban is present in human tissues, but little is known about the impact of triclocarban on the nervous system, particularly at early developmental stages. This study demonstrated that triclocarban that was used at environmentally relevant concentrations induced apoptosis in mouse embryonic neurons, inhibited sumoylation, and changed the epigenetic status, as evidenced by impaired activities of HDAC, sirtuins, and DNMT, global DNA hypomethylation, and alterations of methylation levels of bax, bcl2, Ahr, and Car genes. The use of selective antagonists and specific siRNAs, which was followed by the co-localization of aryl hydrocarbon receptor (AHR) and constitutive androstane receptor (CAR) in mouse neurons, points to the involvement of AHR and CAR in triclocarban-induced neurotoxicity. A 24-h treatment with triclocarban enhanced protein levels of the receptors which was paralleled by Car hypomethylation and Ahr hypermethylation. Car hypomethylation is in line with global DNA hypomethylation and explains the increased mRNA and protein levels of CAR in response to triclocarban. Ahr hypermethylation could reflect reduced Ahr mRNA expression and corresponds to lowered protein levels after 3- and 6-h exposures to triclocarban that is likely related to proteasomal degradation of activated AHR. We hypothesize that the triclocarban-induced apoptosis in mouse neurons and the disruption of epigenetic status involve both AHR- and CAR-mediated effects, which may substantiate a fetal basis of the adult onset of neurological diseases; however, the expression of the receptors is regulated in different ways.

Apoptosis Induced by the UV Filter Benzophenone-3 in Mouse Neuronal Cells Is Mediated via Attenuation of Erα/Pparγ and Stimulation of Erß/Gpr30 Signaling.

Although benzophenone-3 (BP-3) has frequently been reported to play a role in endocrine disruption, there is insufficient data regarding the impact of BP-3 on the nervous system, including its possible adverse effects on the developing brain. Our study demonstrated that BP-3 caused neurotoxicity and activated apoptosis via an intrinsic pathway involving the loss of mitochondrial membrane potential and the activation of caspases-9 and -3 and kinases p38/MAPK and Gsk3ß. These biochemical alterations were accompanied by ROS production, increased apoptotic body formation and impaired cell survival, and by an upregulation of the genes involved in apoptosis. The BP-3-induced effects were tissue-specific and age-dependent with the most pronounced effects observed in neocortical cells at 7 days in vitro. BP-3 changed the messenger RNA (mRNA) expression levels of Erα, Erß, Gpr30, and Pparγ in a time-dependent manner. At 3 h of exposure, BP-3 downregulated estrogen receptor mRNAs but upregulated Pparγ mRNA. After prolonged exposures, BP-3 downregulated the receptor mRNAs except for Erß mRNA that was upregulated. The BP-3-induced patterns of mRNA expression measured at 6 and 24 h of exposure reflected alterations in the protein levels of the receptors and paralleled their immunofluorescent labeling. Erα and Pparγ agonists diminished, but Erß and Gpr30 agonists stimulated the BP-3-induced apoptotic and neurotoxic effects. Receptor antagonists caused the opposite effects, except for ICI 182,780. This is in line with a substantial reduction in the effects of BP-3 in cells with siRNA-silenced Erß/Gpr30 and the maintenance of BP-3 effects in Erα- and Pparγ siRNA-transfected cells. We showed for the first time that BP-3-affected mRNA and protein expression levels of Erα, Erß, Gpr30, and Pparγ, paralleled BP-3-induced apoptosis and neurotoxicity. Therefore, we suggest that BP-3-evoked apoptosis of neuronal cells is mediated via attenuation of Erα/Pparγ and stimulation of Erß/Gpr30 signaling.

Bazedoxifene and raloxifene protect neocortical neurons undergoing hypoxia via targeting ERα and PPAR-γ.

Selective estrogen receptor modulators (SERMs) such as bazedoxifene and raloxifene are recognized to mainly act via estrogen receptors (ERs), but there is no study examining the involvement of PPAR-γ in their actions, especially in neurons undergoing hypoxia. Little is also known about age-dependent actions of the SERMs on neuronal tissue challenged with hypoxia. In this study, bazedoxifene and raloxifene protected neocortical cells against hypoxia at early and later developmental stages. Both SERMs evoked caspase-3-independent neuroprotection and increased protein levels of ERα (66 and 46 kDa isoforms) and PPAR-γ. In addition, bazedoxifene enhanced expression of ERα-regulated Cyp19a1 mRNA. Using double siRNA silencing, for the first time we demonstrated a key role of ERα and PPAR-γ in the neuroprotective action of the SERMs in neocortical neurons undergoing hypoxia. This study provides prospects for the development of a new therapeutic strategies against hypoxic brain injury that selectively target ERα and/or PPAR-γ.

Genistein inhibits glutamate-induced apoptotic processes in primary neuronal cell cultures: an involvement of aryl hydrocarbon receptor and estrogen receptor/glycogen synthase kinase-3beta intracellular signaling pathway.

Phytoestrogens prevent neuronal damage, however, mechanism of their neuroprotective action has not been fully elucidated. This study aimed to evaluate the effects of genistein on glutamate-induced apoptosis in mouse primary neuronal cell cultures. Glutamate (1 mM) enhanced caspase-3 activity and lactate dehydrogenase (LDH) release in the hippocampal, neocortical and cerebellar neurons in time-dependent manner, and these data were confirmed at the cellular level with Hoechst 33342 and calcein AM staining. Genistein (10-10,000 nM) significantly inhibited glutamate-induced apoptosis, and the effect of this isoflavone was most prominent in the hippocampal cells. Next, we studied an involvement of estrogen and aryl hydrocarbon receptors in anti-apoptotic effects of genistein. A high-affinity estrogen receptor antagonist, ICI 182, 780 (1 microM), reversed, whereas less specific antagonist/partial agonist, tamoxifen (1 microM), either intensified or partially inhibited genistein effects. Aryl hydrocarbon receptor antagonist, alpha-naphthoflavone (1 microM), exhibited a biphasic action: it enhanced genistein action toward a short-term exposure (3 h) to glutamate, but antagonized genistein action toward prolonged exposure (24 h) to that insult. SB 216763 (1 microM), which preferentially inhibits glycogen synthase kinase-3beta (GSK-3beta), potentiated genistein effects. These data point to strong effects of genistein at low micromolar concentrations in various brain tissues against glutamate-evoked apoptosis. Moreover, this study provided evidence for involvement of aryl hydrocarbon receptor and estrogen receptor/GSK-3beta intracellular signaling pathway in anti-apoptotic action of genistein.

RXRα, PXR and CAR xenobiotic receptors mediate the apoptotic and neurotoxic actions of nonylphenol in mouse hippocampal cells.

In the present study, we investigated the role of the retinoid X receptor (RXR), the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), in the apoptotic and toxic effects of nonylphenol in mouse primary neuronal cell cultures. Our study demonstrated that nonylphenol activated caspase-3 and induced lactate dehydrogenase (LDH) release in hippocampal cells, which was accompanied by an increase in the mRNA expression and protein levels of RXRα, PXR and CAR. Nonylphenol stimulated Rxra, Pxr, and Car mRNA expression. These effects were followed by increase in the protein levels of particular receptors. Immunofluorescence labeling revealed the cellular distribution of RXRα, PXR and CAR in hippocampal neurons in response to nonylphenol, shortening of neurites and cytoplasmic shrinking, as indicated by MAP2 staining. It also showed NP-induced translocation of receptor-specific immunofluorescence from cytoplasm to the nucleus. The use of specific siRNAs demonstrated that Rxra-, Pxr-, and Car-siRNA-transfected cells were less vulnerable to nonylphenol-induced activation of caspase-3 and LDH, thus confirming the key involvement of RXRα/PXR/CAR signaling pathways in the apoptotic and neurotoxic actions of nonylphenol. These new data give prospects for the targeting xenobiotic nuclear receptors to protect the developing nervous system against endocrine disrupting chemicals.

Selective Aryl Hydrocarbon Receptor Modulator 3,3'-Diindolylmethane Impairs AhR and ARNT Signaling and Protects Mouse Neuronal Cells Against Hypoxia.

The neuroprotective potential of 3,3'-diindolylmethane (DIM), which is a selective aryl hydrocarbon receptor modulator, has recently been shown in cellular and animal models of Parkinson's disease and lipopolysaccharide-induced inflammation. However, there are no data concerning the protective capacity and mechanisms of DIM action in neuronal cells exposed to hypoxia. The aim of the present study was to investigate the neuroprotective potential of DIM against the hypoxia-induced damage in mouse hippocampal cells in primary cultures, with a particular focus on DIM interactions with the aryl hydrocarbon receptor (AhR), its nuclear translocator ARNT, and estrogen receptor ß (ERß). In the present study, 18 h of hypoxia induced apoptotic processes, in terms of the mitochondrial membrane potential, activation of caspase-3, and fragmentation of cell nuclei. These effects were accompanied by substantial lactate dehydrogenase release and neuronal cell death. The results of the present study demonstrated strong neuroprotective and anti-apoptotic actions of DIM in hippocampal cells exposed to hypoxia. In addition, DIM decreased the Ahr and Arnt mRNA expression and stimulated Erß mRNA expression level. DIM-induced mRNA alterations were mirrored by changes in protein levels, except for ERß, as detected by ELISA, Western blotting, and immunofluorescence labeling. We also demonstrated that DIM decreased the expression of AhR-regulated CYP1A1. Using specific siRNAs, we provided evidence that impairment of AhR and ARNT, but not ERß plays a key role in the neuroprotective action of DIM against hypoxia-induced cell damage. This study may have implication for identifying new agents that could protect neurons against hypoxia by targeting AhR/ARNT signaling.

The Crucial Involvement of Retinoid X Receptors in DDE Neurotoxicity.

Dichlorodiphenyldichloroethylene (DDE) is a primary environmental and metabolic degradation product of the pesticide dichlorodiphenyltrichloroethane (DDT). It is one of the most toxic compounds belonging to organochlorines. DDE has never been commercially produced; however, the parent pesticide DDT is still used in some developing countries for disease-vector control of malaria. DDT and DDE remain in the environment because these chemicals are resistant to degradation and bioaccumulate in the food chain. Little is known, however, about DDE toxicity during the early stages of neural development. The results of the present study demonstrate that DDE induced a caspase-3-dependent apoptosis and caused the global DNA hypomethylation in mouse embryonic neuronal cells. This study also provided evidence for DDE-isomer-non-specific alterations of retinoid X receptor α (RXRα)- and retinoid X receptor ß (RXRß)-mediated intracellular signaling, including changes in the levels of the receptor mRNAs and changes in the protein levels of the receptors. DDE-induced stimulation of RXRα and RXRß was verified using selective antagonist and specific siRNAs. Co-localization of RXRα and RXRß was demonstrated using confocal microscopy. The apoptotic action of DDE was supported at the cellular level through Hoechst 33342 and calcein AM staining experiments. In conclusion, the results of the present study demonstrated that the stimulation of RXRα- and RXRß-mediated intracellular signaling plays an important role in the propagation of DDE-induced apoptosis during early stages of neural development.

Effects of estrone on quisqualate-induced toxicity in primary cultures of rat cortical neurons.

Estrogens exert protective effects against neurotoxic changes induced by over-activation of ionotrophic glutamate receptors, whereas little is known about their interaction with changes mediated by metabotropic glutamate receptors. We evaluated effects of estrone on quisqualate (QA)-induced toxicity in neuronal cell cultures on 7 and 12 day in vitro (DIV). Twenty four hour exposure to QA (150 microM and 300 microM) significantly decreased cell survival in 7 day old cultures, but the 12 day old cultures were more resistant to its toxicity. DNQX (10 microM), an AMPA/kainate receptor antagonist, partly attenuated the toxic effects of QA, whereas LY 367 385 (100 microM), a selective mGluR1a antagonist, completely reversed the above effect. QA did not activate, but suppressed spontaneous caspase-3-like activity. Estrone (100 nM and 500 nM) attenuated QA-mediated neurotoxic effects independently of estrogen receptors, as indicated with ICI 182, 780 and without affecting the caspase-3-like activity. At early stage of development in vitro (7 DIV) toxic effects of QA were more profound and mediated mainly by metabotropic glutamate receptors of group I, whereas later (12 DIV) they were mediated mostly by ionotropic AMPA/kainate receptors. The toxic effects of QA were partly accompanied by anti-apoptotic action against spontaneous caspase-3-like activity, possibly due to modulation of neuronal plasticity.

Neuroprotective action of raloxifene against hypoxia-induced damage in mouse hippocampal cells depends on ERα but not ERß or GPR30 signalling.

Raloxifene is the selective estrogen receptor modulator (SERM) currently used in clinical practice to activate estrogen receptors (ERs) in bone tissue and to antagonise ERs in breast and uterine cancers. Little is known, however, about mechanisms of action of raloxifene on hypoxia-induced neuronal cell damage. The aim of the present study was to investigate the neuroprotective potential of raloxifene against hypoxia-induced damage of mouse hippocampal cells in primary cultures, with a particular focus on raloxifene interactions with the classical nuclear ERs (ERα, ERß) and the recently identified membrane ER G-protein-coupled receptor 30 (GPR30). In this study, 18 h of hypoxia increased hypoxia inducible factor 1 alpha (Hif1α) mRNA expression and induced apoptotic processes, such as loss of the mitochondrial membrane potential, activation of caspase-3 and fragmentation of cell nuclei based on Hoechst 33342 staining. These effects were accompanied by reduced ATPase and intracellular esterase activities as well as substantial lactate dehydrogenase (LDH) release from cells exposed to hypoxia. Our study demonstrated strong neuroprotective and anti-apoptotic caspase-3-independent actions of raloxifene in hippocampal cells exposed to hypoxia. Raloxifene also inhibited the hypoxia-induced decrease in Erα mRNA expression and attenuated the hypoxia-induced rise in Erß and Gpr30 mRNA expression levels. Impact of raloxifene on hypoxia-affected Erα mRNA was mirrored by fluctuations in the protein level of the receptor as demonstrated by Western blot and immunofluorescent labelling. Raloxifene-induced changes in Erß mRNA expression level were in parallel with ERß immunofluorescent labeling. However, changes in Gpr30 mRNA level were not reflected by changes in the protein levels measured either by ELISA, Western blot or immunofluorescent staining at 24h post-treatment. Using specific siRNAs, we provided evidence for a key involvement of ERα, but not ERß or GPR30 in neuroprotective action of raloxifene against hypoxia-induced cell damage. This study may have implications for the treatment or prevention of hypoxic brain injury and the administration of current or new generations of SERMs specific to ERα. This article is part of a Special Issue entitled "Sex steroids and brain disorders".

Effects of estrone on N-methyl-D-aspartic acid- and staurosporine-induced changes in caspase-3-like protease activity and lactate dehydrogenase-release: time- and tissue-dependent effects in neuronal primary cultures.

A growing body of evidence indicates that estrogens affect apoptotic processes in neuronal cells. However, their effects seem to depend on type of neuronal tissue, stage of development and apoptosis inducing factors. In the present study we compared effects of estrone (100 and 500 nM) on N-methyl-D-aspartic acid (NMDA) (1 mM)- and staurosporine (1 microM)-induced caspase-3-like activity and lactate dehydrogenase (LDH)-release in primary cultures of rat hippocampal and neocortical neurons. Fluorometric and colorimetric determination of enzyme activity was performed 6 h, 14 h, and 24 h after exposure to apoptotic agents. In the hippocampal cell cultures on 7 days in vitro (DIV), a time-dependent NMDA-induced activation of caspase-3-like proteases was accompanied by increased LDH-release. In neocortical cell cultures on 7 DIV NMDA did not affect caspase activity and decreased LDH-release. In neocortical cell cultures on 12 DIV NMDA inhibited spontaneous caspase activity, but was toxic to neurons after 24 h exposure suggesting that these cells underwent necrotic rather than apoptotic death. Estrone has attenuated both pro- and anti-apoptotic NMDA-induced changes in rat primary neuronal cultures acting independently of estrogen receptors, as detected with ICI 182, 780. In hippocampal neurons estrone antagonized not only the NMDA-induced caspase-3-like activity, but also NMDA-mediated LDH-release. However, in neocortical neurons estrone either attenuated NMDA-induced inhibition of caspase-3-like activity (12 DIV) or partly blocked NMDA-mediated decrease in LDH-release (7 DIV). In contrast to NMDA, staurosporine elevated caspase-3-like activity and LDH-release in a time-dependent manner in all used culture systems. Estrone inhibited pro-apoptotic effects of staurosporine in neocortical neurons, but only at later stage of development in vitro, which points to the protective role of estrogens during the brain tissue maturation. Since estrone triggered its effects via non-genomic mechanisms, it suggests that the other estradiol metabolites exhibiting low affinity to hormone receptors may be potent neuroprotective agents, which could retain the favorable and minimize the adverse side effects of estrogens.

Antidepressant drugs inhibit glucocorticoid receptor-mediated gene transcription - a possible mechanism.

1. Antidepressant drugs are known to inhibit some changes evoked by glucocorticoids, as well as a hyperactivity of hypothalamic-pituitary-adrenal (HPA) axis, often observed in depression. 2. The aim of present study was to investigate effects of various antidepressant drugs on the glucocorticoid-mediated gene transcription in fibroblast cells, stably transfected with an MMTV promoter (LMCAT cells). 3. The present study have shown that antidepressants (imipramine, amitriptyline, desipramine, fluoxetine, tianeptine, mianserin and moclobemide), but not cocaine, inhibit the corticosterone-induced gene transcription in a concentration- and a time-dependent manner. 4. Drugs which are known to augment clinical effects of medication in depressed patients (lithium chloride, amantadine, memantine), do not affect the inhibitory effects of imipramine on the glucocorticoid receptor (GR)-mediated gene transcription. 5. Inhibitors of phospholipase C (PLC), protein kinase C (PKC), Ca(2+)/calmodulin-dependent protein kinase (CaMK) and antagonists of the L-type Ca(2+) channel also inhibit the corticosterone-induced gene transcription. 6. Inhibitors of protein kinase A (PKA) and protein kinase G (PKG) are without effect on the GR-induced gene transcription. 7. Phorbol ester (an activator of PKC) attenuates the inhibitory effect of imipramine on the GR-induced gene transcription. 8. Imipramine decreases binding of corticosterone-receptor complex to DNA. 9. It is concluded that antidepressant drugs inhibit the corticosterone-induced gene transcription, and that the inhibitory effect of imipramine depends partly on the PLC/PKC pathway.

Protective effects of TRH and its stable analogue, RGH-2202, on kainate-induced seizures and neurotoxicity in rodents.

Thyrotropin-releasing hormone (TRH) has been postulated to be involved in the regulation of seizures and neural degeneration. We examined the effects of TRH and its stable analogue, RGH-2202, on the kainate-induced seizures and excitotoxicity in mice - a model of a drug-resistant temporal lobe epilepsy. We found that TRH (2.0 and 5.0 mg/kg) and RGH-2202 (2.5 and 5 mg/kg) elevated the ED(50) for kainate-induced convulsions and tended to decrease mortality. A histological analysis showed that kainate caused a neuronal loss of CA(1) and CA(3) hippocampal fields. TRH (10, 20 and 50 mg/kg) and RGH-2202 (2.5, 7.5 and 10.0 mg/kg) markedly reduced the excitotoxic effect of kainate. Further studies showed that TRH (1-100 microM) and RGH-2202 (100 microM) significantly attenuated the kainate (150 microM)-induced lactate dehydrogenase release in a primary cortical cell culture from rat embryos. In conclusion, the present study showed that TRH and RGH-2202 attenuated the kainate-induced seizures and inhibited the kainate-evoked neurotoxicity in vivo and in vitro. These results support the hypothesis of a potential utility of TRH and its analogues in the treatment of seizures and some neurodegenerative diseases.

Polichlorinated biphenyls (PCB126 and PCB 153) action on proliferation and progesterone secretion by cultured in vitro porcine luteal cells.

SUMMARY: To characterise PCBs action on luteal cell steroidogenesis and cell viability two PCB congeners were selected as model substances. PCB 126 because of its dioxin-like configuration and high toxicity while 153 because it is one of the most commonly detected congeners in breast milk. Luteal cells collected from mature corpora lutea were cultured in M199 medium at 37 degrees C. Control cultures were maintained in that medium alone, while other cultures were supplemented with either PCB 126 (5, 10, 50 and 100 pg/ml) or PCB 153 (5, 10, 50 and 100 ng/ml). After 24 h, 48 h and 72 h of culture media were collected for P4 content analysis. Cell viability was measured using LDH cytotoxicity test. Exposure of luteal cells to all doses of PCB 126 for 24 h had no effect on progesterone secretion while longer, 48 h and 72 h exposure decreased progesterone secretion in a statistically significant manner. Concentration dependent decrease in progesterone secretion by luteal cells was seen after 24 h and 48 h exposure to PCB153 while concentration dependent increase in progesterone secretion was noted after 72 h exposition to this congener. The toxic effect of both congeners was observed only after 72 h exposition to 50 pg/ml and 100 pg/ml in the case of PCB 126 and 50 ng/ml and 100 ng/ml in the case of PCB 153. In conclusion, these results suggest that there are differences in PCB 153 and 126 action in luteal cells. Since information concerning mechanism of PCBs action on luteal cells is scarce, these preliminary experiments are of pioneering character.

Estrone, but not 17 beta-estradiol, attenuates kainate-induced seizures and toxicity in male mice.

Estrogens change the susceptibility to seizures in humans and experimental animals. In this study, the effect of estrone and 17 beta-estradiol on kainate-induced seizures and neurotoxicity was investigated in male mice. Pre-treatment with estrone (250-1000 micrograms/kg) at 24 and 2 hours before kainate (40 mg/kg) administration significantly decreased both the percentage of animals with clonic seizures and their mortality (the latter at a dose of 1000 micrograms/kg only). On the other hand, 17 beta-estradiol (10-500 micrograms/kg) had no effect on seizures, and its dose of 10 micrograms/kg increased mortality. When given alone at a dose of 1 mg/kg, tamoxifen, an antagonist at estrogene receptors, did not affect the kainate-induced seizures, but prevented the anticonvulsant effect of estrone. A histological analysis showed that 73% of mice injected with vehiculum and kainate incurred hippocampal damage. Estrone (2000 micrograms/kg) decreased the percentage of animals with hippocampal neuronal loss down to 43%, and that effect was not antagonized by tamoxifen. Pretreatment of mice with 17 beta-estradiol had no effect on the kainate-induced neuronal loss. Additionally, we found that kainate injected i.p. had a profound effect on the immune system of mice, as reflected by a decrease in the thymus weight and an increased metabolic activity of splenocytes. The anticonvulsive dose of estrone (1000 micrograms/kg) did not change the immunoreactivity of either control or kainate-treated mice. In conclusion, the obtained data indicate that estrone, but not 17 beta-estradiol, attenuates the kainate-induced seizures, mortality and excitotoxicity in male mice. Moreover, it is suggested that the suppressive effect of estrone on clonic seizures involves intracellular receptors, whereas its antineurotoxic activity seems to depend on a non-genomic mechanism.

Effect of PCB 126 and PCB 153 on incidence of apoptosis in cultured theca and granulosa cells collected from small, medium and large preovulatory follicles.

The aim of the presented study was to evaluate the effects of PCB 126 and PCB 153 on granulosa and theca cell apoptosis. Granulosa and theca cells were collected from small, medium, and large preovulatory porcine follicles and cultured as monolayers. Cells were initially cultured for 24 h to allow attachment to the plates. Media were changed and 100 pg/ml PCB 126 or 100 ng/ml PCB 153 were added. After 48 h, granulosa and theca cells were fixed for assessment of the number of apoptotic cells utilizing a Hoechst staining technique or frozen for measurement of caspase-3 activity. Media were collected for testosterone concentration analysis from theca cell cultures or estradiol from granulosa cell cultures. Neither PCB 153 nor PCB 126 had an effect on testosterone secretion by theca cells collected from small and medium size follicles, while both PCBs decreased testosterone secretion by large follicles. The decrease in testosterone secretion by large follicles under the influence of both PCBs was paralleled by a suppression of caspase-3 activity and a decreased incidence of apoptotic bodies. Neither of the PCBs had an effect on estradiol secretion by granulosa cells collected from small and medium size follicles, while both PCBs increased estradiol in granulosa cells collected from large follicles. PCB-associated increased estradiol secretion by granulosa cells collected from large follicles was accompanied by suppression of caspase-3 activity and a decreased incidence of apoptotic bodies. In conclusion, we have presented evidence that in preovulatory follicles PCBs inhibit both theca and granulosa cells apoptosis. Therefore, an exposure to PCBs may cause alterations in the pattern of terminal differentiation of follicles and attenuate spontaneous elimination of atretic follicles.