RESUMO
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor activated by dioxin and polyaromatic hydrocarbons. Recent studies have revealed that AhR activity in central neurons depends on the NMDA receptor. In this study, we investigated how the neuronal activity influence AhR-mediated dioxin-responsive gene expression and neurotoxicity. Our results show that activation of AhR by the selective agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin induced dioxin-responsive gene expression and calcium entry, which were attenuated by AhR small interfering RNA, the NMDA receptor channel blocker MK801, and the action potential blocker tetrodotoxin (TTX). In addition, AhR-mediated gene expression was enhanced in neurons during synaptogenesis (10 days in vitro) compared with younger neurons (4 days in vitro), as was sensitivity to TTX and MK801. Furthermore, TTX and MK801 differentially affected the association of AhR and its transcriptional co-activator cAMP-responsive-element binding protein with the cytochrome P450 1A1 (cyp1A1) gene enhancer. Calcium/calmodulin-dependent protein kinase IV, the cAMP-responsive-element binding protein activating enzyme, was also activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin in an activity-dependent manner. Finally, we found that neuronal susceptibility to dioxin insult was also maturation and activity-dependent. Together, the results suggest that neuronal activity may facilitate AhR-mediated calcium signaling, which in turn enhances AhR-mediated gene regulation and mediated maturation-dependent dioxin neurotoxicity.
Assuntos
Córtex Cerebral/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/biossíntese , Animais , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/fisiologia , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores de Hidrocarboneto Arílico/genéticaRESUMO
Recent epidemiological studies have shown that dioxin, a persistent organic pollutant, is related to cognitive and behavioral abnormalities in the offspring of exposed cohort. In order to investigate the possible impact of dioxin in survival gene expression during brain development, we established an animal model of gestational and lactational dioxin-exposed rat offspring. The expressions of dioxin-responsive gene cytochrome P450 1A1 (CYP1A1), apoptotic gene Bax, and anti-apoptotic genes Bcl-2 and Bcl-xL were examined in rat liver and brains using Western blot analysis and RT-PCR. The results showed that treatment of pregnant rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (2 microg/kg body weight through oral delivery) at gestation day 15 resulted in an increase of Bcl-xL in offspring male liver and cerebral cortex, but a decrease in female offspring. In contrast, the expression of Bcl-xL in the cerebellum was decreased in male, but increased in female. Bcl-2, another anti-apoptotic gene, was also downregulated in P0 female liver, cerebral cortex, but was not observed in male. In the 4-month-old offspring, however, the Bcl-2 protein levels in the liver and cerebellum of both male and female pups were higher in the TCDD group as compared with the control group. However, the Bcl-2 level in the cerebral cortex of TCDD-treated groups was higher than the control group only in female but not male offspring at 4 months old. The expression of Bax showed no significant changes upon TCDD exposure at P0 stage, but was significantly reduced in the 4-month-old male cortex. These results indicate that early exposure of dioxin could affect the development of certain brain regions with gender difference, in terms of its differential effect on expressions of Bcl-xL, Bcl-2, and Bax.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Dioxinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Lactação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/classificação , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Cruzamento , Citocromo P-450 CYP1A1/genética , Feminino , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
Neuronal injury in the central nervous system following ischemic insult is believed to result from glutamate toxicity and glucose deprivation. In this study, polyphenols isolated from Scutellaria baicalensis Georgi, including baicalin, baicalein, and wogonin, were investigated for their neuroprotective effects against glutamate/NMDA (Glu/NMDA) stimulation and glucose deprivation in primary cultured rat brain neurons. Cell death was accessed by lactate dehydrogenase (LDH) release assay for necrosis, and mitochondrial activity was accessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction activity assay. It was found that both baicalin and baicalein decreased LDH release of the cultured neurons after 24 h treatment, whereas wogonin profoundly increased LDH release after 2 h treatment and resulted in neuronal death after 24 h. Glu/NMDA treatment profoundly increased LDH release and moderately decreased MTT reduction activity in an NMDA receptor-dependent manner. Both baicalin and baicalein significantly reduced Glu/NMDA-increased LDH release, in which baicalein is much more potent than baicalin. Glu/NMDA-increased intracellular calcium was also significantly attenuated by baicalin and baicalein. Baicalin and baicalein did not affect glutamate receptor binding activity, but baicalein did moderately decrease Glu/NMDA-induced nitric oxide (NO) production. In the glucose deprivation (GD) study, baicalein but not baicalin showed significant protective effects on the GD-increased LDH release, without affecting the GD-induced NO production, in cultured rat brain neurons. These results suggest that baicalein is the most effective compound among three polyphenols tested in preventing neurotoxicity induced by both glutamate and GD, whereas baicalin was only effective in preventing glutamate toxicity. Wogonin might have a neurotoxic effect on the brain.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Flavonoides/farmacologia , Degeneração Neural/tratamento farmacológico , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fenóis/farmacologia , Animais , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiopatologia , Medicamentos de Ervas Chinesas/uso terapêutico , Agonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Flavanonas/farmacologia , Flavanonas/uso terapêutico , Flavonoides/uso terapêutico , Glucose/deficiência , Ácido Glutâmico/toxicidade , L-Lactato Desidrogenase/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Neurônios/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Óxido Nítrico/metabolismo , Fenóis/uso terapêutico , Polifenóis , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato/efeitos dos fármacos , Receptores de Glutamato/metabolismo , Scutellaria baicalensis/químicaRESUMO
We cultured a P19 mouse teratocarcinoma cell line and induced its neuronal differentiation to study the function of ionotropic glutamate receptors (GluRs) in early neuronal development. Immunocytochemical studies showed 85% neuronal population at 5 days in vitro (DIV) with microtubule-associated protein 2-positive staining. Thirty percent and 50% of the cells expressed the alpha-amino-3-hydroxy-5-methyl-4-isopropinonate (AMPA) receptor subunit, GluR2/3, and the kainate (kainic acid; KA) receptor subunit, GluR5/6/7, respectively. In Western blot analysis, the temporal expression of GluR2/3 began to appear at 3 DIV, whereas GluR5/6/7 was already expressed in the undifferentiated cells. P19-derived neurons began to respond to glutamate, AMPA and KA, but not to the metabotropic GluR agonist trans-1-aminocyclopentane-1,3-decarboxylic acid, by 5 DIV in terms of increases in intracellular calcium and phospholipase C-mediated poly-phosphoinositide turnover. Furthermore, KA reduced cell death of P19-derived neurons in both atmospheric and hypobaric conditions in a phospholipase C-dependent manner. The common AMPA/KA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, but not the AMPA receptor antagonist, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium, profoundly increased hypobaric insult-induced neurotoxicity. In a flow cytometry study, the nerve growth factor-mediated antiapoptotic effect was facilitated by AMPA, with an induction of TrkA, but not p75(NTR) expression. Therefore, AMPA and KA receptors might mediate neurotrophic functions to facilitate neurotrophic factor signaling to protect neurons against hypoxic insult in early neuronal development.