RESUMO
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.
Assuntos
Isquemia Encefálica , Congêneres do Estradiol , Hipóxia Encefálica , Indanos , Receptores de Estrogênio , Animais , Camundongos , Isquemia Encefálica/tratamento farmacológico , Caspase 3/metabolismo , Células Cultivadas , Congêneres do Estradiol/uso terapêutico , Hipóxia Encefálica/tratamento farmacológico , Indanos/farmacologia , Indanos/uso terapêutico , L-Lactato Desidrogenase/metabolismo , Neurônios/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Estrogênio/efeitos dos fármacosRESUMO
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.
Assuntos
Apoptose/genética , Asfixia/tratamento farmacológico , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Receptores de Hidrocarboneto Arílico/genética , Receptores de N-Metil-D-Aspartato/genética , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Asfixia/genética , Asfixia/patologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Metilação de DNA/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Indóis/farmacologia , Recém-Nascido , Masculino , Proteínas de Membrana/genética , MicroRNAs/genética , Proteínas Mitocondriais/genética , N-Metilaspartato/farmacologia , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , RatosRESUMO
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.
Assuntos
Anti-Infecciosos Locais/toxicidade , Carbanilidas/toxicidade , Receptores de Estrogênio/metabolismo , Animais , Metilação de DNA , Camundongos , NeurôniosRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Histona Desacetilases/metabolismo , Indóis/farmacologia , Fármacos Neuroprotetores/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Hipocampo/patologia , Isquemia/patologia , Camundongos , Neocórtex/patologia , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/metabolismo , Neurônios/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Carbanilidas/toxicidade , Epigênese Genética/efeitos dos fármacos , Neurônios/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Caspase 3/metabolismo , Células Cultivadas , Receptor Constitutivo de Androstano , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Hipocampo/patologia , Histona Desacetilases/metabolismo , L-Lactato Desidrogenase/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Neurônios/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Sirtuínas/metabolismo , Coloração e Rotulagem , Sumoilação/efeitos dos fármacos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
INTRODUCTION: Primary graft dysfunction (PGD) is a multifactorial syndrome related to the most adverse outcomes after liver transplantation. Ischemia-reperfusion injury is recognized as the predominant cause of this complication. PGD may be subdivided into early allograft dysfunction, diagnosed by the presence of a serum bilirubin level ≥10 mg/dL (171 µmol/L), International Normalized Ratio ≥1.6, or alanine and aspartate transaminase levels ≥2000 IU/L on the seventh postoperative day; and primary nonfunction, defined as either a need for retransplantation or patient death within the first 7 days. We aimed to determine the preoperative and intraoperative risk factors for PGD. MATERIALS AND METHODS: We enrolled 109 patients who underwent orthotopic liver transplantation between 2012 and 2016. Analysis included inter alia: biochemical parameters, morphology, blood transfusions, as well as intraoperative fluctuations of blood pressure. RESULTS: Fourteen percent of patients were diagnosed with PGD. Using logistic regression and multivariate and receiver operating characteristic and area under the curve analysis, a preoperative neutrophils level above 4030/µL (OR = 4.03, P = .012) and decrease of the mean arterial pressure after reperfusion were recognized as the major independent PGD risk factors. CONCLUSIONS: A high preoperative neutrophils level may be a novel recipient-related risk factor for PGD. A decrease of the arterial blood pressure after graft reperfusion may influence the development of PGD.
Assuntos
Complicações Intraoperatórias/etiologia , Transplante de Fígado/efeitos adversos , Disfunção Primária do Enxerto/etiologia , Reoperação/estatística & dados numéricos , Adulto , Feminino , Humanos , Transplante de Fígado/métodos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório , Período Pré-Operatório , Curva ROC , Estudos Retrospectivos , Fatores de Risco , Resultado do TratamentoRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Benzofenonas/toxicidade , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , PPAR gama/metabolismo , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Protetores Solares/toxicidade , Animais , Apoptose/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/agonistas , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , PPAR gama/antagonistas & inibidores , Receptores Acoplados a Proteínas G/agonistas , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
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.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Indóis/farmacologia , Neocórtex/patologia , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , PPAR gama/metabolismo , Cloridrato de Raloxifeno/farmacologia , Animais , Aromatase/genética , Aromatase/metabolismo , Caspase 3/metabolismo , Hipóxia Celular/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/genética , Hipocampo/patologia , L-Lactato Desidrogenase/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , PPAR gama/agonistas , PPAR gama/antagonistas & inibidores , PPAR gama/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
The connection between maternal hematological status and pregnancy outcome has been shown by many independent researchers. Attention was initially focused on the adverse effects of moderate and severe anemia. Interestingly, some studies revealed that mild anemia was associated with optimal fetal development and was not affecting pregnancy outcome. The explanation for this phenomenon became a target for scientists. Hemodilution, physiologic anemia and relative decrease in hemoglobin concentration are the changes observed during pregnancy but they do not explain the reasons for the positive influence of mild anemia on a fetomaternal unit. It is hypothesized that hemodilution facilitates placental perfusion because blood viscosity is reduced. Subsequently, it may lead to a decline in hemoglobin concentration. Anemia from its definition implies decreased oxygen carrying capacity of the blood and can result in hypoxemia and even hypoxia, which is a common factor inducing new blood vessels formation. Therefore, we raised the hypothesis that the lowered hemoglobin concentration during pregnancy may upregulate vascular growth factor receptors expression such as VEGFR-1 (Flt-1) and VEGFR-2 (FLK-1/KDR). Consecutively, increased fetoplacental vasculogenesis and angiogenesis provide further expansion of vascular network development, better placental perfusion and hence neither fetus nor the mother are affected.
Assuntos
Anemia/fisiopatologia , Neovascularização Patológica/fisiopatologia , Doenças Placentárias/fisiopatologia , Placenta/irrigação sanguínea , Placenta/fisiopatologia , Feminino , Humanos , Modelos Cardiovasculares , GravidezRESUMO
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.
Assuntos
Diclorodifenil Dicloroetileno/farmacologia , Neurônios/efeitos dos fármacos , Neurotoxinas/farmacologia , Receptores X de Retinoides/metabolismo , Animais , Apoptose , Benzoatos/farmacologia , Compostos de Bifenilo/farmacologia , Encéfalo/citologia , Caspase 3/metabolismo , Células Cultivadas , Metilação de DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Fluoresceínas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Camundongos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores X de Retinoides/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurotoxinas/toxicidade , Fenóis/toxicidade , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Receptores X de Retinoides/metabolismo , Animais , Células Cultivadas , Receptor Constitutivo de Androstano , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Receptor de Pregnano X , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genética , Receptores X de Retinoides/genética , Transdução de Sinais , Regulação para Cima/efeitos dos fármacosRESUMO
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.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Indóis/farmacologia , Neurônios/citologia , Neurônios/metabolismo , Neuroproteção/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Caspase 3/metabolismo , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocromo P-450 CYP1A1/metabolismo , Ativação Enzimática/efeitos dos fármacos , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/metabolismo , Inativação Gênica/efeitos dos fármacos , Hipocampo/metabolismo , L-Lactato Desidrogenase/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Coloração e RotulagemRESUMO
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".
Assuntos
Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Hipocampo/efeitos dos fármacos , Hipóxia/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Cloridrato de Raloxifeno/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Caspase 3/metabolismo , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Hipóxia/genética , Hipóxia/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , L-Lactato Desidrogenase/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Receptores de Estrogênio , Receptores Acoplados a Proteínas G/genética , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
4-para-Nonylphenol (NP) is a non-ionic surfactant that has widespread and uncontrolled distribution in the environment. Little is known, however, about its actions on neuronal cells during critical developmental periods. This study aimed to investigate the mechanisms underlying the apoptotic and toxic actions of NP on mouse embryonic neuronal cells and the possible interactions of NP with estrogen receptor (ER)- and retinoid X receptor (RXR)-mediated intracellular signaling. Treatment of mouse hippocampal neuronal cell cultures with NP (5 and 10µM) induced apoptotic and neurotoxic effects. The 2 and 7 day-old mouse hippocampal cultures were vulnerable to 5 and 10µM NP, whereas 12 day-old cultures responded only to the highest concentration of NP, thus suggesting an age-dependent action of the chemical on neuronal cells. The use of specific inhibitors did not support the involvement of calpains in NP-induced apoptosis, but indicated caspase-8- and caspase-9-dependent effects of NP. Specific ER antagonists MPP and PHTPP potentiated the NP-induced loss of mitochondrial membrane potential and increase in lactate dehydrogenase (LDH) release whereas, ER agonists PPT and DPN inhibited these effects. RXR antagonist HX531 diminished the NP-evoked loss of mitochondrial membrane potential, the activity of caspase-3 and LDH release. In addition, exposure to NP inhibited ERα- and ERß-specific immunofluorescence but stimulated RXR-specific immunolabeling in mouse hippocampal cells. In conclusion, our study demonstrated that the apoptotic and toxic actions of NP on neuronal cells in early development is accompanied by an impairment of ER- and stimulation of RXR-mediated signaling pathways. Taking into account NP-induced alterations in mRNA expression levels of particular types of RXRs, we suggest that NP affected mainly RXRα and RXRß, but not RXRγ signaling.
Assuntos
Hipocampo/citologia , Fenóis/toxicidade , Receptores de Estrogênio/metabolismo , Receptores X de Retinoides/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Benzoatos/farmacologia , Compostos de Bifenilo/farmacologia , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Antagonistas de Estrogênios/farmacologia , Estrogênios/metabolismo , L-Lactato Desidrogenase/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , RNA Mensageiro/metabolismo , Receptores de Estrogênio/genética , Receptores X de Retinoides/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Extended residual persistence of the pesticide dichlorodiphenyltrichloroethane (DDT) raises concerns about its long-term neurotoxic effects. Little is known, however, about DDT toxicity during the early stages of neural development. This study demonstrated that DDT-induced apoptosis of mouse embryonic neuronal cells is a caspase-9-, caspase-3-, and GSK-3ß-dependent process, which involves p,p'-DDT-specific impairment of classical ERs. It also provided evidence for DDT-isomer-nonspecific alterations of AhR- and GPR30-mediated intracellular signaling, including changes in the levels of the receptor and receptor-regulated mRNAs, and also changes in the protein levels of the receptors. DDT-induced stimulation of AhR-signaling and reduction of GPR30-signaling were verified using selective ligands and specific siRNAs. Co-localization of the receptors was demonstrated with confocal microscopy, and the presence of functional GPR30 was detected by electrophysiology. This study demonstrates that stimulation of AhR-signaling and impairment of GPR30-signaling play important roles in the propagation of DDT-induced apoptosis during the early stages of neural development.
Assuntos
Apoptose/efeitos dos fármacos , DDT/química , DDT/farmacologia , Neurônios/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Benzodioxóis/farmacologia , Benzoflavonas/farmacologia , Caspase 3/metabolismo , Inibidores de Caspase/farmacologia , Células Cultivadas , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Isomerismo , L-Lactato Desidrogenase/metabolismo , Camundongos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Quinolinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/genética , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Fatores de Tempo , beta-Naftoflavona/farmacologiaRESUMO
An intracellular arginine--specific aminopeptidase synthesized by Bacillus mycoides was purified and characterized. The purification procedure for studied aminopeptidase consisted of ammonium sulphate precipitation and three chromatographic steps: anion exchange chromatography and gel permeation chromatography. A molecular weight of -50 kDa was estimated for the aminopeptidase by gel permeation chromatography and SDS-PAGE. The optimal activity of the enzyme on arginyl-beta-naphthylamide as a substrate was at 37 degrees C and pH 9.0. The enzyme showed maximum specificity for basic amino acids: such as Arg and Lys but was also able to hydrolyze aromatic amino acids: Trp, Tyr, and Phe. Co2+ ions activated the enzyme, while Zn2+, Cu2+, Hg2+ and Mn2+ inhibited it. The enzyme is a metalloaminopeptidase whose activity is inhibited by typical metalloaminopeptidase inhibitors: EDTA and 1,10-phenanthroline. Analysis of fragments of the amino acid sequence of the purified enzyme demonstrated high similarity to AmpS of Bacillus cereus and AP II of B. thuringensis.
Assuntos
Aminopeptidases/metabolismo , Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Cobalto/metabolismo , Naftalenos/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Aminopeptidases/química , Proteínas de Bactérias/química , Cátions Bivalentes , Cromatografia em Gel , Cromatografia por Troca Iônica , Ácido Edético/química , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peso Molecular , Fenantrolinas/química , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade por Substrato , TemperaturaRESUMO
OBJECTIVE: Prospective pharmacogenetic screening for the human leucocyte antigen (HLA) B*5701 allele can significantly reduce the number of cases of abacavir-related hypersensitivity among HIV-infected patients treated with this drug. The aim of this study was to establish the frequency of the HLA B*5701 variant in HIV-infected Poles. METHODS: The sequence-specific primer (SSP) test was used to assess the feasibility of the introduction of such testing in clinical practice. For this purpose, 234 randomly selected HIV-positive patients were screened using a low-resolution SSP assay, with HLA B*5701-positive results confirmed using a high-resolution test. RESULTS AND CONCLUSIONS: The HLA B*5701 variant was found in 11 of 234 subjects (4.7%). Testing with the selected method proved quick and reliable.
Assuntos
Didesoxinucleosídeos/efeitos adversos , Hipersensibilidade a Drogas/genética , Testes Genéticos , Infecções por HIV/tratamento farmacológico , Antígenos HLA-B/genética , Inibidores da Transcriptase Reversa/efeitos adversos , Adulto , Alelos , Estudos de Viabilidade , Feminino , Frequência do Gene , Genótipo , Infecções por HIV/genética , Antígenos HLA-B/análise , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Polônia , Reação em Cadeia da Polimerase/métodos , Valor Preditivo dos Testes , Testes CutâneosRESUMO
INTRODUCTION: Lymphocele is a lymph collection that forms after surgery following injury to lymph nodes and vessels. The aim of the study was to perform a retrospective analysis of different treatment modalities of lymphocele in patients after kidney transplantation. MATERIAL AND METHODS: A lymphocele located in renal graft area was observed in 25 of 386 transplanted patients (6.5%). Mean patient age was 45 (95% confidence interval [CI], 40 to 50) years. Mean observation time was 35 (95% CI, 27 to 43) months. RESULTS: Mean time from transplantation to diagnosis of lymphocele was 29 days (range, 4 to 127). In 13 patients (54.2%), the lymphocele was symptomatic, requiring initial treatment by repeated needle aspirations or percutaneous drainage. Among 7 patients with persistence of the lesion treatment by sclerotherapy with doxycycline, povidone-iodine, and/or ethanol was successful in 4 cases who showed maximal lymphocele volume of 500 mL. Three other patients, namely, volumes of 120, 874, and 2298 mL were referred for surgery; in two cases, internal marsupialization was performed and in one case external drainage was necessary due to abscess formation. Mean time from the diagnosis to recovery in patients requiring surgical treatment was 15 (range, 8 to 24) weeks. Eleven patients with asymptomatic lymphoceles (mean volume 45 mL; range, 8 to 140) were monitored to resolution after a mean of 4 (range, 1 to 11) weeks. CONCLUSION: All lymphoceles with the maximal volume exceeding 140 mL were clinically symptomatic. Initial percutaneous drainage with or without sclerotherapy was an effective method of treatment. Punctures, drainage, and sclerotherapy were not effective in patients with lymphoceles (>500 mL).
Assuntos
Transplante de Rim/efeitos adversos , Linfocele/etiologia , Linfocele/patologia , Complicações Pós-Operatórias/patologia , Adulto , Biópsia por Agulha , Cadáver , Doxiciclina/uso terapêutico , Etanol/uso terapêutico , Seguimentos , Humanos , Doadores Vivos , Linfocele/tratamento farmacológico , Linfocele/epidemiologia , Pessoa de Meia-Idade , Complicações Pós-Operatórias/tratamento farmacológico , Complicações Pós-Operatórias/epidemiologia , Povidona-Iodo/uso terapêutico , Estudos Retrospectivos , Escleroterapia , Fatores de Tempo , Doadores de TecidosRESUMO
BACKGROUND: The aim of this study was to investigate the hepatic disorders in a cohort of HIV-infected patients including clinical, biochemical, serological and histopathological data. MATERIAL AND METHODS: 125 patients, 23 (18.4%) females and 102 (81.6%) males with an average age of 30.2+/-8.4 yrs, were retrospectively analysed. 61 patients (49.2%) were i. v. drug users (IVDU) and 64 patients (51.8%) were infected HIV by sexual contact. AIDS was diagnosed in 52 (41.6%) patients. RESULTS: Hepatomegaly was detected in 62 (49.6%) patients. An increased serum aspartate aminotransferase (AST) level was found in 110 (88%) patients. Testing showed 61 (48.8%) patients with evidence of a history of hepatitis B infection; 71 (56.8%) were anti-HCV positive and 40 (32%) had serological markers of both infections. 36 (28.8%) patients underwent liver biopsy. Abnormalities were detected in 32 (88%) patients. The biopsies of 13 (37.1%) patients revealed features of chronic viral hepatitis. CONCLUSIONS: HCV and HBV co-infection is frequently encountered in this cohort of HIV-infected patients. In the majority of cases the liver diseases were asymptomatic or mildly symptomatic. The hepatic disorders found in HIV-infected patients are due to different etiopathogenic factors.
Assuntos
Infecções por HIV/complicações , Hepatopatias/complicações , Adulto , Aspartato Aminotransferases/sangue , Biópsia , Estudos de Coortes , Feminino , Infecções por HIV/patologia , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Hepatite B/complicações , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite C/complicações , Hepatomegalia , Humanos , Fígado/patologia , Hepatopatias/patologia , Masculino , RNA Viral/sangue , Carga ViralRESUMO
Flow cytometry analysis has been used to measure the S phase in paraffin embedded osteosarcoma tissues of 22 children and adolescents who were surgically treated. The aim of this study was the assessment of S phase cell cycle in relation to survival, localization of tumour and histopathological grading. Our findings demonstrate the usefulness of flow cytometry determination of S phase cell cycle as a prognostic factor. Higher percentage of S phase cells predict worse prognosis.