RESUMO
The Health and Environmental Sciences Institute Developmental and Reproductive Toxicology (HESI-DART) group held a hybrid in-person and virtual workshop in Washington, DC, in 2022. The workshop was entitled, "Interpretation of DART in Regulatory Contexts and Frameworks." There were 154 participants (37 in person and 117 virtual) across 9 countries. The purpose of the workshop was to capture key consensus approaches used to assess DART risks associated with chemical product exposure when a nonclinical finding is identified. The decision-making process for determining whether a DART endpoint is considered adverse is critical because the outcome may have downstream implications (e.g., increased animal usage, modifications to reproductive classification and pregnancy labeling, impact on enrollment in clinical trials and value chains). The workshop included a series of webinar modules to train and engage in discussions with federal and international regulators, clinicians, academic investigators, nongovernmental organizations, contract research organization scientists, and private sector scientists on the best practices and principles of interpreting DART and new approach methodologies in the context of regulatory requirements and processes. Despite the differences in regulatory frameworks between the chemical and pharmaceutical sectors, the same foundational principles for data interpretation should be applied. The discussions led to the categorization of principles, which offer guidance for the systematic interpretation of data. Step 1 entails identifying any hazard by closely analyzing the data at the study endpoint level, while Step 2 involves assessing risk using weight of evidence. These guiding principles were derived from the collective outcomes of the workshop deliberations.
Assuntos
Reprodução , Animais , Gravidez , Feminino , Humanos , Medição de Risco/métodosRESUMO
Types I and II pyrethroid insecticides cause temporally distinct decreases in voltage-gated sodium channel (VGSC) inactivation rates that are proposed to underlie their characteristic differences in toxicity signs. How alterations in VGSC channel function give rise to the characteristic differences in signs of pyrethroid intoxication is not completely understood, particularly those changes that occur in functional networks of interconnected neurons. To characterize better pyrethroid actions at the network level, effects of the Type I pyrethroid permethrin (PM) and the Type II pyrethroid deltamethrin (DM) on spontaneous glutamate network-dependent spikes and bursts were investigated in primary cultures of frontal cortex or spinal cord neurons grown on microelectrode arrays (MEAs). Fast GABAergic transmission was blocked by BIC, and concentration-dependent effects of DM (1nM to 5microM) and PM (10nM to 50microM) were examined. Both compounds caused concentration-dependent reductions in the network spike and burst rates. DM was more potent than PM, with IC(50) values of approximately 0.13 and approximately 4microM for inhibition of spike rate in cortical and spinal cord neurons, respectively. Both compounds decreased the percentage of spikes that occurred within a burst and increased the interspike interval within bursts. Onset of effects was rapid, but recovery from total activity loss was not readily achievable. Individual neurons responded heterogeneously; activity of most declined monophasically, but activity in others exhibited biphasic responses with increases followed by decreases in activity. In spinal cord, DM caused a greater number of biphasic responses (29%) than PM (10%). These results demonstrate that both DM and PM inhibit activity of glutamatergic networks, but with different potencies.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Inseticidas/toxicidade , Rede Nervosa/efeitos dos fármacos , Inibição Neural , Neurônios/efeitos dos fármacos , Nitrilas/toxicidade , Permetrina/toxicidade , Piretrinas/toxicidade , Nervos Espinhais/efeitos dos fármacos , Animais , Células Cultivadas , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Relação Dose-Resposta a Droga , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Análise em Microsséries , Microeletrodos , Rede Nervosa/embriologia , Rede Nervosa/metabolismo , Neurônios/metabolismo , Nervos Espinhais/embriologia , Nervos Espinhais/metabolismo , Fatores de TempoRESUMO
Cyclic AMP (cAMP)-responsive element-binding protein (CREB) is a transcription factor important in developing nervous system cells and is activated by a variety of signaling molecules. Aroclor 1254 (A1254), a polychlorinated biphenyl mixture, perturbs Ca(2+) homeostasis and increases CREB phosphorylation in rat neonatal cortical cell cultures in a time- and concentration-dependent manner. The present experiments determined that the cell type responding to A1254 with Ca(2+) increases and phosphorylated CREB (phospho-CREB) was predominantly of neuronal morphology and microtubule-associated protein (MAP2)-positive phenotype. Similarly, glutamate (100 microM) increased phospho-CREB immunoreactivity selectively in MAP2-immunopositive cells. Using Western blotting and immunocytochemical techniques, we identified key signal transduction pathways operative in phosphorylating CREB in cortical cell cultures and examined their participation in 3 ppm A1254-induced CREB activation. Cortical cultures treated with glutamate, forskolin or the phorbol ester phorbol 12-myristate 13-acetate exhibited robust increases in phospho-CREB. Tetrodotoxin (1 microM) completely inhibited CREB phosphorylation by A1254, suggesting that synaptic activity is involved in A1254-induced CREB activation. Buffering [Ca(2+)](i) with bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester in the absence of extracellular Ca(2+) partially inhibited A1254-induced CREB phosphorylation. Inhibition of mitogen-activated protein kinase (10 microM U0126) or protein kinase C (PKC; bisindoylmaleimide, 5 microM) activation did not inhibit A1254-induced CREB phosphorylation. By contrast, inhibition of protein kinase A (PKA) with 100 microM PKA inhibitor peptide, PKI, blocked A1254-induced CREB phosphorylation. Thus, we examined whether A1254 activates PKA by increasing cAMP; 10 microM forskolin, but not A1254, elevated intracellular cAMP levels. These results indicate that in neocortical cells in culture, CREB phosphorylation occurs via Ca(2+)-, PKA-, and PKC-dependent pathways. Furthermore, A1254-induced CREB phosphorylation occurs predominantly in neurons, is dependent on synaptic activity and mediated by Ca(2+)- and PKA-dependent pathways.
Assuntos
Antitireóideos/farmacologia , Cálcio/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Animais , Carcinógenos/farmacologia , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos , Ratos Long-Evans , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Possible mechanisms of AlCl3-induced inhibition of agonist-stimulated inositol phosphate (IP) accumulation were investigated using rat brain cortex slices, synaptosomes or homogenates. Under conditions in which AlCl3 inhibits carbachol (CARB)-stimulated IP accumulation (Gp-mediated), AlCl3 did not affect CARB (100 microM)-induced decreases (Gi-mediated) in 30 microM forskolin-stimulated cAMP accumulation, suggesting that AlCl3 may be specific for Gp-mediated signal transduction. To determine whether AlCl3 interfered with Gp function and/or phosphatidylinositol-specific phospholipase C (PiPLC) activity, effects of AlCl3 on CARB- and Ca(2+)-stimulated IP accumulation were examined in cortical synaptosomes. AlCl3 (500 microM) decreased CARB (1 mM)- and Ca2+ (20 microM ionomycin)-stimulated IP accumulation to 77 and 75% of control, respectively, suggesting that AlCl3 may not directly affect Gp activity, but does inhibit PiPLC activity. In cortical homogenates, AlCl3 (10-500 microM) inhibited hydrolysis of [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) by PiPLC in a concentration-dependent manner with an estimated IC50 of 100 microM. The effects of AlCl3 on modulation of IP accumulation by extracellular Ca2+ and PKC were also examined as potential mechanisms. Decreasing the extracellular Ca2+ concentration ([Ca2+]e) from 1.0 to 0.1 mM decreased CARB-stimulated IP accumulation in slices. AlCl3 (500 microM) decreased significantly 1 mM CARB-stimulated IP accumulation in 1.0 and 0.1 mM Ca2+ solutions; however, the effect of AlCl3 on IP accumulation did not depend on [Ca2+]e. In cortical slices, inhibition of 1 mM CARB-stimulated IP accumulation by 500 microM AlCl3 was not altered by the PKC activator phorbol 12,13-dibutyrate (PdBu, 1 microM), or the PKC inhibitor H-7 (10 microM), suggesting that AlCl3 does not interfere with IP accumulation by activation of PKC. Other studies found that AlCl3 (10-100 microM) inhibited PKC activity in a concentration-dependent manner in both cytosolic and membrane fractions of cortical homogenates with an estimated IC50 of 60 microM. These results support the hypothesis that AlCl3 inhibition of agonist-stimulated IP accumulation may be mediated by inhibition of PiPLC activity, rather than disruption of G-protein function or modulation of the IP signalling system by Ca2+ or PKC.
Assuntos
Compostos de Alumínio/farmacologia , Cálcio/farmacologia , Cloretos/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Fosfatos de Inositol/biossíntese , Proteína Quinase C/metabolismo , Fosfolipases Tipo C/antagonistas & inibidores , Cloreto de Alumínio , Animais , Química Encefálica , Cálcio/metabolismo , Carbacol/antagonistas & inibidores , Técnicas In Vitro , Masculino , Fosfatos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
The developing nervous system has been identified as a potential target of pesticide exposure. Heptachlor is a cyclodiene pesticide that was widely used for many years, and for which inadvertent exposure to children and fetuses took place in the early 1980s; yet little is known regarding the developmental neurotoxicity of it and other cyclodienes. The aim of this study was to determine whether perinatal heptachlor exposure results in persistent alterations in nervous system function. Pregnant Sprague-Dawley dams were dosed from gestational day (GD) 12 to postnatal day (PND) 7, whereupon the rat pups were dosed directly until PND 21 (group A) or PND 42 (group B). Dose levels were 0, 0.03, 0.3, or 3 mg/kg/day, po. There were no dose-related effects on maternal weight, litter size, or pup growth. GABA(A) receptor binding (using [(35)S] tert-butylbicyclophosphorothionate; TBPS) and GABA-stimulated Cl- flux were evaluated in control and high-dose brain tissues taken on PND 7, 21, and 43. The B(max) values for [(35)S]-TBPS binding in brainstem, but not cortex, were decreased in female rats across all ages tested. There were no such changes in male rats, nor were K(D) values altered in either tissue or gender. GABA-stimulated Cl- flux was decreased in female cortex synaptoneurosomes only on PND 21. The ontogeny of the righting response (PND 2-5) was delayed in the high-dose females. All subsequent testing took place a week to months after dosing ceased. The functional observational battery (FOB) showed treatment-related, but not necessarily dose-related, changes in different aspects of the rat's reactivity and activity levels. Group-A rats also showed altered within-session habituation of motor activity. There were no heptachlor-related differences in motor activity following challenge with a range of chlordiazepoxide doses. Cognitive assessments were conducted in both groups of rats. There were no statistically significant differences among treatment groups in a one-trial passive avoidance test, although there was a trend toward less learning. In group B, rats (both sexes), heptachlor altered spatial learning in the Morris water maze during two weeks of daily training (2 trials/day). On probe trials, heptachlor-treated rats did not show significant preference for the correct quadrant (all dose groups in males, high dose in females). These rats did not show alterations on subsequent working-memory training (where the platform position was relearned each day). Thus, perinatal exposure to heptachlor produced neurochemical and persistent neurobehavioral changes, including alterations in spatial learning and memory.
Assuntos
Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Desenvolvimento Embrionário e Fetal/efeitos dos fármacos , Heptacloro/toxicidade , Inseticidas/toxicidade , Efeitos Tardios da Exposição Pré-Natal , Animais , Animais Recém-Nascidos , Peso Corporal/efeitos dos fármacos , Encéfalo/embriologia , Encéfalo/fisiopatologia , Química Encefálica , Cloretos/metabolismo , Feminino , Heptacloro/farmacocinética , Inseticidas/farmacocinética , Tamanho da Ninhada de Vivíparos/efeitos dos fármacos , Masculino , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores de GABA/metabolismo , Distribuição Tecidual , Testes de Toxicidade , Ácido gama-Aminobutírico/farmacologiaRESUMO
The ability of acute application of the neurotoxicant methylmercury (MeHg) to disrupt the function of presynaptic Ca2+ and Na+ channels at intact neuromuscular junctions was examined using mouse triangularis sterni motor nerves. In Ba(2+)-containing solutions, potential changes arising from Na+ and Ca2+ channel function could be recorded from the perineurial sheath surrounding motor neurons when K+ channels were blocked by tetraethylammonium chloride and 3,4-diaminopyridine. MeHg (100 microM) reduced both Na(+)- and Ba(2+)-dependent components to block within 3-5 min at apparently equivalent rates. Time to block was approximately 7 min after exposure to 50 microM MeHg. In 2 of 5 preparations exposed to 50 microM MeHg, the Ca2+ channel-mediated component was blocked prior to the Na+ channel-mediated component. In the remaining three preparations, Na(+)- and Ba(2+)-dependent potentials were blocked at similar times. Following block by MeHg, neither perfusing the preparation in MeHg-free solutions nor increasing the intensity and/or duration of stimulus to the intercostal nerves resulted in recovery of Na+ or Ca2+ potentials. In the presence of K+ channel blockers, repetitive firing of nerves in response to a single stimulus was observed in 20-30% of the triangularis preparations; in the two preparations treated with MeHg in which repetitive firing was observed, it decreased prior to block of the stimulus-induced Na+/Ba2+ potentials. These results corroborate the results obtained in isolated synaptosomes and pheochromocytoma cells, and suggest that MeHg decreases motor nerve excitability by disrupting Na+ channel function and may block neurotransmitter release by disrupting Na+ and Ca2+ channel function.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Compostos de Metilmercúrio/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Canais de Sódio/efeitos dos fármacos , 4-Aminopiridina/análogos & derivados , 4-Aminopiridina/farmacologia , Amifampridina , Animais , Bário/fisiologia , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Placa Motora/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Compostos de Tetraetilamônio/farmacologiaRESUMO
Effects of aluminum chloride (AlCl3) (0.1 to 1000 microM) on inositol phosphate (IP) accumulation stimulated by carbachol (CARB), norepinephrine (NE) or quisqualate (QUIS) were examined in rat hippocampal and cortical slices. In the absence of agonist, only 1000 microM AlCl3 significantly reduced basal accumulation of IPs. For CARB-stimulated IP accumulation, 100 microM and greater AlCl3 significantly inhibited IP accumulation. In cortical slices, 1000 microM AlCl3 reduced CARB-stimulated IP accumulation by 55% and in hippocampal slices 1000 microM AlCl3 inhibited IP accumulation by 40%. Similar effects of AlCl3 were observed for NE-stimulated IP accumulation. In cortical slices, the concentration-response for AlCl3 effects on agonist-stimulated IP accumulation was significantly different from that in hippocampal slices. For QUIS-stimulated accumulation of IPs, 1000 microM AlCl3 significantly inhibited IP accumulation in hippocampal slices. However, in cortical slices a biphasic effect of AlCl3 was observed. 500 and 1000 microM AlCl3 significantly inhibited IP accumulation, whereas 10 and 50 microM AlCl3 significantly enhanced QUIS-stimulated IP accumulation. In both hippocampal and cortical slices, 500 microM AlCl3 significantly inhibited CARB-, NE- or QUIS-stimulated IP accumulation at all agonist concentrations (0.1 to 10000 microM) tested, indicating a post-receptor effect on agonist-mediated IP accumulation. Stimulation of G-proteins with NaF (5-30 mM) resulted in accumulation of IPs in hippocampal and cortical slices in the absence of added agonists. NaF (5-30 mM) plus 1 mM CARB produced increased accumulation of IPs over CARB or NaF alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Compostos de Alumínio/farmacologia , Carbacol/farmacologia , Córtex Cerebral/metabolismo , Cloretos/farmacologia , Hipocampo/metabolismo , Fosfatos de Inositol/metabolismo , Norepinefrina/farmacologia , Fosfatidilinositóis/metabolismo , Ácido Quisquálico/farmacologia , Cloreto de Alumínio , Animais , Córtex Cerebral/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hidrólise , Técnicas In Vitro , Cinética , Masculino , Ratos , Receptores Adrenérgicos/efeitos dos fármacos , Receptores Adrenérgicos/fisiologia , Receptores de Glutamato/efeitos dos fármacos , Receptores de Glutamato/fisiologia , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/fisiologia , Fluoreto de Sódio/farmacologiaRESUMO
Previous reports from our laboratory have suggested that the neuroactivity of some polychlorinated biphenyl (PCB) congeners is associated with perturbations in cellular Ca(2+)-homeostasis. We have characterized further the neurochemical effects of PCBs on signal transduction in primary cultures of cerebellar granule cells. The present experiments found that neither 2,2'-dichlorobiphenyl (DCBP), an ortho-substituted congener, nor 3,3',4,4',5-pentachlorobiphenyl (PCBP), a non-ortho-substituted congener, affected basal phosphoinositide (PI) hydrolysis in cerebellar granule cells. However, at concentrations up to 50 microM, DCBP potentiated carbachol-stimulated PI hydrolysis, while decreasing it at 100 microM. PCBP, on the other hand, had no effect on carbachol-stimulated PI hydrolysis in concentrations up to 100 microM. [3H]Phorbol ester ([3H]PDBu) binding was used to determine protein kinase C (PKC) translocation. DCBP increased [3H]PDBu binding in a concentration-dependent manner and a twofold increase was observed at 100 microM in cerebellar granule cells. PCBP had no effect on [3H]PDBu binding at concentrations up to 100 microM. The effect of DCBP on [3H]PDBu binding was time-dependent and was also dependent on the presence of external Ca2+ in the medium. To test the hypothesis that DCBP increases [3H]PDBu binding by acting on receptor-activated calcium channels, the effects of DCBP were compared to those of L-glutamate. The effects of DCBP (50 microM) and glutamate (20 microM) were additive.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Cerebelo/metabolismo , Fosfatidilinositóis/metabolismo , Bifenilos Policlorados/farmacologia , Proteína Quinase C/metabolismo , Animais , Cálcio/metabolismo , Carbacol/metabolismo , Células Cultivadas , Cerebelo/efeitos dos fármacos , Cerebelo/enzimologia , Antagonistas de Aminoácidos Excitatórios , Ácido Glutâmico/farmacologia , Hidrólise , Ésteres de Forbol/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Protein kinase C (PKC)-mediated phosphorylation has been implicated in neuronal growth and differentiation [R.S. Turner, R.L. Mazzei, G.J. Raynor, P.R. Girard, J.F. Kuo, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 3143-3147.]. We examined effects of gestational exposure to the neurotoxicant, methylmercury (CH3Hg), on the developmental profile of immunoreactivity (IR) for alpha, beta, gamma and epsilon PKC isoforms and cytosolic PKC activity. Long-Evans dams were dosed on gestational days (GD)6-15 (p.o.) with 0, 1, or 2 mg kg-1 day-1 CH3Hg dissolved in saline. Pups were sacrificed and perfused with buffered paraformaldehyde on post-natal days (PND) 1, 4, 10, 21, 45 and 85. The brains were sectioned sagittally, stained immunohistochemically, and examined throughout the medial to lateral extent. IR in neuronal cell bodies for PKC isoforms alpha, beta, gamma, and epsilon was densest in the olfactory bulb, hippocampus, shell of the inferior colliculus, pons, cerebral, piriform, and cerebellar cortex, whereas axonal staining was prominent in the brainstem, internal capsule, corpus callosum, anterior commissure, fornix and olfactory tract. In controls, the PKC alpha and epsilon IR was highest on PND1-4, decreased dramatically by PND10, and decreased further by PND21. In the neonate, the regional and cellular distributions of alpha and epsilon IR were similar. The PKC gamma IR was greater at post-weaning ages (PND21-85) with the greatest regional density apparent in the hippocampus, cortex, and cerebellum. Only the highest dose of CH3Hg (2 mg kg-1 day-1; GD6-15) produced a persistent decrease in regional alpha and epsilon, but not beta or gamma IR during the post-natal period. These regional and time-dependent changes in PKC isoforms were complemented by the examination of PKC activity in cortex, olfactory bulb, cerebellum and brainstem. Cytosolic PKC activity increased from PND1 to 10 in cortex, olfactory bulb, and cerebellum. On PND21, PKC activity decreased in the cortex and olfactory bulb, but remained high in the cerebellum. By contrast, PKC activity in the brainstem was highest on PND1 and 4 and decreased dramatically by PND21. CH3Hg (2 mg kg-1 day-1) significantly decreased PKC activity on PND1 and 4 in the cortex. The present results characterize the cellular and regional ontogeny of PKC isoenzymes alpha, beta, gamma and epsilon, and indicate that developmental exposure to CH3Hg can alter the ontogeny of specific isoforms and regional PKC activity.
Assuntos
Encéfalo/efeitos dos fármacos , Compostos de Metilmercúrio/toxicidade , Efeitos Tardios da Exposição Pré-Natal , Proteína Quinase C/biossíntese , Animais , Animais Recém-Nascidos , Encéfalo/enzimologia , Encéfalo/crescimento & desenvolvimento , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/crescimento & desenvolvimento , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Hipocampo/crescimento & desenvolvimento , Imuno-Histoquímica , Isoenzimas/biossíntese , Gravidez , Ratos , Ratos Long-EvansRESUMO
Recent studies from the laboratory indicate that polychlorinated biphenyl (PCB) congeners can alter signal transduction and calcium homeostasis in neuronal preparations. These effects were more pronounced for the ortho-substituted, non-coplanar congeners, although the mechanisms underlying these effects are not clear. In the present study the time-course and concentration-dependent effects of coplanar and non-coplanar PCBs on intracellular free calcium concentration ([Ca2+]i) in cerebellar granule cell cultures were compared using the fluorescent probe fura-2. The ortho-substituted congeners 2,2'-dichlorobiphenyl (DCB) and 2,2',4,6,6'-pentachlorobiphenyl (PeCB) caused a gradual increase of [Ca2+]i while the non-ortho-substituted congeners 4,4'-DCB and 3,3',4,4',5-PeCB had no effect. The increase of [Ca2+]i produced by 2,2'-DCB was time- and concentration-dependent. Further studies examined possible mechanisms for this rise in [Ca2+]i. In contrast to the muscarinic agonist carbachol, the effects of 2,2'-DCB on [Ca2+]i were not blocked by thapsigargin and required the presence of extracellular calcium. The effects of ortho-substituted PCBs may depend on their ability to inhibit calcium sequestration as 2,2'-DCB significantly inhibited 45Ca2+-uptake by microsomes and mitochondria while 3,3',4,4',5-PeCB had no effect. In addition, 2,2'-DCB significantly increased the binding of [3H]inositol 1,4,5-trisphosphate to receptors on cerebellar microsomes, suggesting another possible mechanism by which ortho-substituted PCBs can mobilize [Ca2+]i. These results show that PCBs increase [Ca2+]i in vitro via a mechanism that requires extracelluar calcium, and support previous structure-activity studies indicating that ortho-substituted PCBs are more potent than non-ortho-substituted PCBs.
Assuntos
Cálcio/metabolismo , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Poluentes Ambientais/toxicidade , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Bifenilos Policlorados/toxicidade , Animais , Animais Recém-Nascidos , Canais de Cálcio/metabolismo , Carbacol/farmacologia , Células Cultivadas , Cerebelo/citologia , Relação Dose-Resposta a Droga , Receptores de Inositol 1,4,5-Trifosfato , L-Lactato Desidrogenase/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Agonistas Muscarínicos/farmacologia , Ratos , Ratos Long-Evans , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-Atividade , Tapsigargina/farmacologiaRESUMO
Chlorotriazine herbicides disrupt luteinizing hormone (LH) release in female rats following in vivo exposure. Although the mechanism of action is unknown, significant evidence suggests that inhibition of LH release by chlorotriazines may be mediated by effects in the central nervous system. GABA(A) receptors are important for neuronal regulation of gonadotropin releasing hormone and LH release. The ability of chlorotriazine herbicides to interact with GABA(A) receptors was examined by measuring their effects on [3H]muscimol, [3H]Ro15-4513 and [35S]tert-butylbicyclophosphorothionate (TBPS) binding to rat cortical membranes. Cyanazine (1-400 microM) inhibited [3H]Ro15-4513 binding with an IC50 of approximately 105 microM (n=4). Atrazine (1-400 microM) also inhibited [3H]Ro15-4513 binding, but was less potent than cyanazine (IC50 = 305 microM). However, the chlorotriazine metabolites diaminochlorotriazine, 2-amino-4-chloro-6-ethylamino-s-triazine and 2-amino-4-chloro-6-isopropylamino-s-triazine were without significant effect on [3H]Ro15-4513 binding. Cyanazine and the other chlorotriazines were without effect on [3H]muscimol or [35S]TBPS binding. To examine whether cyanazine altered GABA(A) receptor function, GABA-stimulated 36Cl- flux into synaptoneurosomes was examined. Cyanazine (50-100 microM) alone did not significantly decrease GABA-stimulated 36Cl- flux. Diazepam (10 microM) and pentobarbital (100 microM) potentiated GABA-stimulated 36Cl- flux to 126 and 166% of control, respectively. At concentrations of 50 and 100 microM, cyanazine decreased potentiation by diazepam to 112 and 97% of control, respectively, and decreased potentiation by pentobarbital to 158 and 137% of control (n = 6). Interestingly, at lower concentrations (5 microM), cyanazine shifted the EC50 for GABA-stimulated 36Cl- flux into synaptoneurosomes from 28.9 to 19.4 microM, respectively (n = 5). These results suggest that cyanazine modulates benzodiazepine, but not the muscimol (GABA receptor site) or TBPS (Cl- channel), binding sites on GABA(A) receptors. Furthermore, at low concentrations, cyanazine may slightly enhance function of GABA(A) receptors, but at higher concentrations, cyanazine antagonizes GABA(A) receptor function and in particular antagonizes the positive modulatory effects of diazepam and pentobarbital.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Herbicidas/toxicidade , Receptores de GABA-A/metabolismo , Triazinas/toxicidade , Animais , Azidas/metabolismo , Azidas/farmacologia , Benzodiazepinas/metabolismo , Benzodiazepinas/farmacologia , Sítios de Ligação , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Córtex Cerebral/metabolismo , Canais de Cloreto/metabolismo , Cloro/metabolismo , Agonistas GABAérgicos/metabolismo , Agonistas GABAérgicos/farmacologia , Herbicidas/metabolismo , Masculino , Muscimol/metabolismo , Muscimol/farmacologia , Radioisótopos , Ratos , Ratos Long-Evans , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Triazinas/metabolismoRESUMO
The increased desire to use in vitro techniques in neurotoxicology has resulted in the search for clonal cell lines which may be useful for studying disruption by neurotoxicants of various aspects of neuronal physiology and biochemistry. One such cell line is the PC12 cell, a clonal cell line derived from a pheochromocytoma of the rat adrenal medulla. When cultured under normal conditions, PC12 cells resemble adrenal chromaffin cells in morphology, physiology and biochemistry. However, when cultured in the presence of nerve growth factor (NGF) or several other compounds, PC12 cells differentiate to resemble sympathetic neurons morphologically and functionally. Differentiation and the resultant physiological and biochemical changes are some of the most attractive and useful features of this cell line. PC12 cells release, depending on the conditions, dopamine, norepinephrine and acetylcholine and contain Na, K and Ca channels and other membrane receptors, including receptors coupled to G-proteins. Moreover, the relative proportion of various subtypes of Ca channels changes during differentiation. Thus, PC12 cells provide an excellent model for studying chemical disruption of processes associated with neuronal differentiation, synthesis, storage and release of neurotransmitters, function and regulation of ion channels and interactions of compounds with membrane bound receptors. The ability of PC12 cells to differentiate in response to NGF and other compounds allows for selective expression of certain channels and proteins and for comparisons of responses in undifferentiated and differentiated cells. The prominent neurotoxicant methylmercury causes potent reductions in uptake of 45Ca and binding of ligands associated with various subpopulations of Ca channels in the PC12 cells, as well as currents carried through putative Ca channels.
Assuntos
Canais Iônicos/fisiologia , Neurônios/metabolismo , Neurotransmissores/metabolismo , Células PC12/metabolismo , Receptores de Neurotransmissores/metabolismo , Animais , Diferenciação Celular , Células PC12/efeitos dos fármacosRESUMO
Recent evidence indicates that the neurotoxic metal aluminum interferes with the phosphoinositide second messenger system in adult rats both in vitro and in vivo. We have examined the age-related effects of aluminum chloride (AlCl3) on receptor-stimulated inositol phosphate (IP) accumulation in brain slices from neonatal and adult rats in vitro. Carbachol-stimulated (1 mM) IP accumulation was greatest in frontal cortex slices from 7 day old rats, decreased in 14 day old and 21 day old rats, and was lowest in adults (120 days old). AlCl3 (500 microM) inhibited both basal and carbachol-stimulated IP accumulation in neonatal and adult rats. The effects of AlCl3 were concentration-related and produced significant decreases (15-25%) in IP accumulation at 500 and 1000 microM. The concentration-response curve for AlCl3 was similar in 7 day old and adult rats. AlCl3 reduced carbachol-, norepinephrine- and quisqualate-stimulated IP accumulation in both 7 day old and adult rats. The effects of 500 microM AlCl3 were examined on carbachol-stimulated IP accumulation in slices prepared from frontal cortex, hippocampus, striatum, and cerebellum. Although IP accumulation was greater in slices from the 7 day old rats compared to adults in each tissue, AlCl3 (500 microM) decreased IP accumulation by approximately 20% in all regions at both ages. Aluminum produced concentration-dependent inhibition of phospholipase C in cortical homogenates which was similar in 7 day old and adult rats. These results show that in vitro exposure to aluminum decreases IP accumulation through a mechanism which is not age-dependent.
Assuntos
Compostos de Alumínio/toxicidade , Encéfalo/metabolismo , Cloretos/toxicidade , Fosfatos de Inositol/metabolismo , Fatores Etários , Cloreto de Alumínio , Compostos de Alumínio/farmacologia , Animais , Animais Recém-Nascidos , Encéfalo/efeitos dos fármacos , Carbacol/farmacologia , Cerebelo/efeitos dos fármacos , Cloretos/farmacologia , Relação Dose-Resposta a Droga , Feminino , Lobo Frontal/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Técnicas In Vitro , Masculino , RatosRESUMO
Effects of the neurotoxic heavy metals Cd2+, Pb2+ and CH3Hg+ on current carried by Ca2+ ions (I(Ca)) through high-voltage activated Ca2+ channels in nerve growth factor (NGF)-differentiated pheochromocytoma (PC12) cells were examined to characterize possible differences in the mechanism of action of these metals on Ca2+ channel function. Specifically, the potency and reversibility of effect on I(Ca) by each metal was examined, as well as the relationship between extracellular [Ca2+] and potency of block of I(Ca) by Cd2+ and Pb2+. In addition, the effect of each of these metals on Ca2+ channels when applied to the intracellular side of the membrane was also examined. When extracellular solution contained 20, 10 or 5 mM Ca2+, the estimated IC50 values (total metal concentration) for block of I(Ca) were 15, 10, and 6.5 microM for Cd2+ and 7.5, 2.0 and 1.1 microM for Pb2+, respectively. CH3Hg+ (1-10 microM) blocked I(Ca) (20 mM Ca2+) in a time- and concentration-dependent manner. When cells were washed with metal-free solutions, block of I(Ca) by Cd2+ was reversed rapidly, whereas block by Pb2+ was reversed only partially, and block of I(Ca) by CH3Hg+ was not reversed. When Pb2+ and CH3Hg+ treated cells were washed in metal-free solutions containing 50 microM D-penicillamine (DPEN), block of I(Ca) by 10 microM Pb2+ was rapidly and completely reversed, whereas, block of I(Ca) by 5 microM CH3Hg+ was not reversed. Higher concentrations (500 microM) of 2,3-dimercapto-1-propane sulfonic acid (DMPS) did reverse partially the block of I(Ca) by 5 and 10 microM CH3Hg+. When Cd2+, Pb2+ or CH3Hg+ was present in the intracellular solution, Ca2+ channel currents were significantly reduced. These results characterize effects of Cd2+ on Ca2+ channels and demonstrate that Cd2+, Pb2+ and CH3Hg+ differ in their actions on Ca2+ channels.
Assuntos
Cádmio/toxicidade , Canais de Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Chumbo/toxicidade , Compostos de Metilmercúrio/toxicidade , Animais , Cálcio/fisiologia , Canais de Cálcio/fisiologia , Quelantes/farmacologia , Espaço Extracelular/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Células PC12 , Técnicas de Patch-Clamp , Penicilamina/farmacologia , Ratos , Unitiol/farmacologiaRESUMO
Aluminum has been reported to inhibit long-term potentiation (LTP) following in vivo administration and decrease glutamate release following in vitro exposure. Because glutamate release is critical for synaptic transmission and the development and maintenance of LTP in the hippocampus, we examined the effects of aluminum chloride (AlCl3) on depolarization-induced glutamate release and LTP in rat hippocampal slices. The effects of AlCl3 on [14C]glutamate release were examined by incubation of slices in depolarizing (56 mM)K+ buffer solution in the absence or presence of 2 mM CaCl2. After 15 min depolarization, AlCl3 (100-1000 microM) did not significantly affect Ca(2+)-dependent [14C]glutamate release from slices, whereas a known Ca2+ channel blocker (100 microM CdCl2) decreased Ca(2+)-dependent [14C]glutamate release by approximately 50%. In contrast to a previous report, acute exposure to AlCl3 was without effect on depolarization-dependent glutamate release. LTP of the population spike (PS) in CA1 of hippocampus was induced by the delivery of stimulus trains to the stratum radiatum. LTP of the PS was observed in both control slices and slices bathed in solution containing 100 microM AlCl3. Neither the magnitude nor longevity (measured up to 1 h posttrain) of LTP distinguished control from aluminum-exposed slices. The lack of sensitivity in rat to the encephalopathic changes induced by aluminum, or methodological differences in exposure conditions may account for the lack of effect of aluminum on in vitro LTP in rat hippocampus.
Assuntos
Alumínio/toxicidade , Glutamatos/metabolismo , Hipocampo/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Animais , Cádmio/farmacologia , Cloreto de Cádmio , Cloretos/farmacologia , Relação Dose-Resposta a Droga , Eletrofisiologia/métodos , Hipocampo/metabolismo , Técnicas In Vitro , Masculino , RatosRESUMO
Neuronal assemblies within the nervous system produce electrical activity that can be recorded in terms of action potential patterns. Such patterns provide a sensitive endpoint to detect effects of a variety of chemical and physical perturbations. They are a function of synaptic changes and do not necessarily involve structural alterations. In vitro neuronal networks (NNs) grown on micro-electrode arrays (MEAs) respond to neuroactive substances as well as the in vivo brain. As such, they constitute a valuable tool for investigating changes in the electrophysiological activity of the neurons in response to chemical exposures. However, the reproducibility of NN responses to chemical exposure has not been systematically documented. To this purpose six independent laboratories (in Europe and in USA) evaluated the response to the same pharmacological compounds (Fluoxetine, Muscimol, and Verapamil) in primary neuronal cultures. Common standardization principles and acceptance criteria for the quality of the cultures have been established to compare the obtained results. These studies involved more than 100 experiments before the final conclusions have been drawn that MEA technology has a potential for standard in vitro neurotoxicity/neuropharmacology evaluation. The obtained results show good intra- and inter-laboratory reproducibility of the responses. The consistent inhibitory effects of the compounds were observed in all the laboratories with the 50% Inhibiting Concentrations (IC(50)s) ranging from: (mean ± SEM, in µM) 1.53 ± 0.17 to 5.4 ± 0.7 (n = 35) for Fluoxetine, 0.16 ± 0.03 to 0.38 ± 0.16 µM (n = 35) for Muscimol, and 2.68 ± 0.32 to 5.23 ± 1.7 (n = 32) for Verapamil. The outcome of this study indicates that the MEA approach is a robust tool leading to reproducible results. The future direction will be to extend the set of testing compounds and to propose the MEA approach as a standard screen for identification and prioritization of chemicals with neurotoxicity potential.
Assuntos
Benchmarking , Família/psicologia , Encaminhamento e Consulta/organização & administração , Obtenção de Tecidos e Órgãos/organização & administração , Cuidados Críticos/organização & administração , Cuidados Críticos/psicologia , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Consentimento Livre e Esclarecido , Resultado do Tratamento , Estados Unidos , Listas de EsperaAssuntos
Comportamento Animal/efeitos dos fármacos , Desenvolvimento Fetal/efeitos dos fármacos , Inseticidas/toxicidade , Anormalidades Musculoesqueléticas/induzido quimicamente , Nitrilas/toxicidade , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Piretrinas/toxicidade , Animais , Feminino , Masculino , GravidezRESUMO
In vivo exposure levels for neurotoxicants are often reported in parts per million (ppm) concentration in tissue, whereas exposure levels in experiments utilizing in vitro models are most commonly reported in micromolar (muM) concentration in the exposure solution. The present experiments sought to determine whether or not in vitro solution concentration was an appropriate dose-metric for comparison to in vivo tissue levels for lipophilic compounds. To do so, the accumulation of the polychlorinated biphenyl (PCB) mixture Aroclor 1254 (A1254) or methylmercury (MeHg) was examined in three commonly utilized in vitro neuronal tissue models: nerve growth factor differentiated pheochromocytoma (PC12) cells, primary cultures of rat neocortical cells, and adult rat hippocampal slices. Tissues were exposed to A1254 (0.65 ppm) or to MeHg (0.0033-0.33 ppm) in serum-free media for 1 or 24 h. Total PCB or mercury accumulation was measured by dual column gas chromatography with electron capture detection or by cold vapor atomic absorption, respectively. PC12 cells accumulated 66.7 and 103.8 ppm PCBs after 1 and 24 h exposure to A1254. Neocortical neurons also accumulated significant concentrations of PCBs, but less so than PC12 cells. After 1 h exposure to 0.65 ppm A1254, slices contained 3.46 and 0.81 ppm PCBs when exposed in a static and perfused system, respectively. After 1 h exposure to 0.0033, 0.033, and 0.33 ppm MeHg, PC12 cells contained 0.3, 2.2, and 17.7 ppm mercury, respectively; after 24 h, PC12 cells contained 0.4, 2.8, and 21.9 ppm. Hippocampal slices accumulated 1.7 and 4.8 ppm mercury after 1 and 3 h exposure to 0.33 ppm MeHg. For comparison, mercury accumulation in rat fetal and pup brain tissue after maternal exposure [0, 0.1, 1.0, or 2.0 mg/kg/day MeHg from gestational day (GD) 6-15] ranged from 0.05 to 7.89 ppm in 0.1 mg/kg dose animals on postnatal day 10 and 2.0 mg/kg dose animals on GD16, respectively. These results demonstrate that accumulation of PCBs and MeHg in vitro is tissue-, time-, and concentration-dependent and indicates that tissue levels rather than exposure concentrations are a more appropriate metric for comparison of in vitro to in vivo effects.
Assuntos
/metabolismo , Hipocampo/metabolismo , Compostos de Metilmercúrio/metabolismo , Neocórtex/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , /toxicidade , Meios de Cultura , Relação Dose-Resposta a Droga , Embrião de Mamíferos/metabolismo , Feminino , Técnicas In Vitro , Masculino , Exposição Materna/efeitos adversos , Compostos de Metilmercúrio/administração & dosagem , Compostos de Metilmercúrio/toxicidade , Modelos Biológicos , Células PC12 , Gravidez , Ratos , Ratos Long-Evans , Fatores de TempoRESUMO
Methylmercury (CH3Hg+) is a potent neurotoxicant in humans and laboratory animals, and both epidemiological and laboratory data suggest that the developing nervous system is more susceptible to CH3Hg+ neurotoxicity than is the adult nervous system. While it is recognized that the developing nervous system is more susceptible to CH3Hg+ neurotoxicity compared to the adult nervous system, it is presently not clear what level of exposure, if any, is without effect on the developing human nervous system. A better understanding of mechanisms of action of CH3Hg+ for developmental neurotoxicity would be useful in defining risks associated with CH3Hg+ exposure. While alterations in a variety of processes may contribute to the neurotoxicity of CH3Hg+, changes in ion channel function and electrical activity in neuronal cells is a consistent observation following acute exposure in a variety of preparations. Additional research, however, is needed to clarify the relationship between alterations in neuronal electrophysiological function and developmental neurotoxicity of CH3Hg+. This article suggests several issues to be considered in order to address the relationship between in vitro acute effects of CH3Hg+ on ion channels and electrophysiological function in neurons and developmental neurotoxicity. Future studies need: 1) to examine effects on ion channel function and neuronal electrophysiology following subacute and chronic in vitro exposure to CH3Hg+; 2) to utilize model systems which consider developmental changes in neuronal function; 3) to consider direct vs. indirect effects of CH3Hg+; 4) to compare in vitro to ex vivo and in vivo effects; 5) to utilize in vitro dose levels which reflect in vivo exposure, and 6) to consider interactions between CH3Hg+ and other potential neurotoxicants found in environmental mixtures. Ultimately, it may be possible to develop biologically-based dose-response models of CH3Hg+ neurotoxicity which will be useful in assessing the risks of developmental neurotoxicity of this metal.