RESUMO
Approaches for extrapolating in vitro toxicity testing results for prediction of human in vivo outcomes are needed. The purpose of this case study was to employ in vitro toxicokinetics and PBPK modeling to perform in vitro to in vivo extrapolation (IVIVE) of lindane neurotoxicity. Lindane cell and media concentrations in vitro, together with in vitro concentration-response data for lindane effects on neuronal network firing rates, were compared to in vivo data and model simulations as an exercise in extrapolation for chemical-induced neurotoxicity in rodents and humans. Time- and concentration-dependent lindane dosimetry was determined in primary cultures of rat cortical neurons in vitro using "faux" (without electrodes) microelectrode arrays (MEAs). In vivo data were derived from literature values, and physiologically based pharmacokinetic (PBPK) modeling was used to extrapolate from rat to human. The previously determined EC50 for increased firing rates in primary cultures of cortical neurons was 0.6µg/ml. Media and cell lindane concentrations at the EC50 were 0.4µg/ml and 7.1µg/ml, respectively, and cellular lindane accumulation was time- and concentration-dependent. Rat blood and brain lindane levels during seizures were 1.7-1.9µg/ml and 5-11µg/ml, respectively. Brain lindane levels associated with seizures in rats and those predicted for humans (average=7µg/ml) by PBPK modeling were very similar to in vitro concentrations detected in cortical cells at the EC50 dose. PBPK model predictions matched literature data and timing. These findings indicate that in vitro MEA results are predictive of in vivo responses to lindane and demonstrate a successful modeling approach for IVIVE of rat and human neurotoxicity.
Assuntos
Hexaclorocicloexano/farmacocinética , Hexaclorocicloexano/toxicidade , Modelos Biológicos , Neurotoxinas/farmacocinética , Neurotoxinas/toxicidade , Convulsões/induzido quimicamente , Animais , Encéfalo/metabolismo , Células Cultivadas , Feminino , Hexaclorocicloexano/sangue , Humanos , Masculino , Microeletrodos , Neocórtex/citologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurotoxinas/sangue , Ratos Long-Evans , Convulsões/metabolismo , ToxicocinéticaRESUMO
The US EPA's Toxicity Forecaster (ToxCast) is a suite of high-throughput in vitro assays to screen environmental toxicants and predict potential toxicity of uncharacterized chemicals. This work examines the relevance of ToxCast assay intended gene targets to putative molecular initiating events (MIEs) of neurotoxicants. This effort is needed as there is growing interest in the regulatory and scientific communities about developing new approach methodologies (NAMs) to screen large numbers of chemicals for neurotoxicity and developmental neurotoxicity. Assay gene function (GeneCards, NCBI-PUBMED) was used to categorize gene target neural relevance (1 = neural, 2 = neural development, 3 = general cellular process, 3â¯A = cellular process critical during neural development, 4 = unlikely significance). Of 481 unique gene targets, 80 = category 1 (16.6â¯%); 16 = category 2 (3.3â¯%); 303 = category 3 (63.0â¯%); 97 = category 3â¯A (20.2â¯%); 82 = category 4 (17.0â¯%). A representative list of neurotoxicants (548) was researched (ex. PUBMED, PubChem) for neurotoxicity associated MIEs/Key Events (KEs). MIEs were identified for 375 compounds, whereas only KEs for 173. ToxCast gene targets associated with MIEs were primarily neurotransmitter (ex. dopaminergic, GABA)receptors and ion channels (calcium, sodium, potassium). Conversely, numerous MIEs associated with neurotoxicity were absent. Oxidative stress (OS) mechanisms were 79.1â¯% of KEs. In summary, 40â¯% of ToxCast assay gene targets are relevant to neurotoxicity mechanisms. Additional receptor and ion channel subtypes and increased OS pathway coverage are identified for potential future assay inclusion to provide more complete coverage of neural and developmental neural targets in assessing neurotoxicity.
Assuntos
Ensaios de Triagem em Larga Escala , Síndromes Neurotóxicas , Ensaios de Triagem em Larga Escala/métodos , Animais , Humanos , Síndromes Neurotóxicas/genética , Síndromes Neurotóxicas/etiologia , Testes de Toxicidade/métodos , Neurônios/efeitos dos fármacos , Neurônios/metabolismoRESUMO
Aging adults are a growing segment of the U.S. population and are likely to exhibit increased susceptibility to many environmental toxicants. However, there is little information on the susceptibility of the aged to toxicants. The toxicity of toluene has been well characterized in young adult rodents but there is little information in the aged. Three approaches were used: (1) pharmacokinetic (PK), (2) cardiac biomarkers, and (3) whole-animal physiology to assess whether aging increases susceptibility to toluene in the Brown Norway (BN) rat. Three life stages, young adult, middle aged, and aged (4, 12, and 24 mo, respectively), were administered toluene orally at doses of 0, 0.3, 0.65, or 1 g/kg and subjected to the following: terminated at 45 min or 4 h post dosing, and blood and brain toluene concentration were measured; terminated at 4 h post dosing, and biomarkers of cardiac function were measured; or monitor heart rate (HR), core temperature (Tc), and motor activity (MA) by radiotelemetry before and after dosing. Brain toluene concentration was significantly elevated in aged rats at 4 h after dosing with either 0.3 or 1 g/kg. Blood toluene concentrations were unaffected by age. There were various interactions between aging and toluene-induced effects on cardiac biomarkers. Most notably, toluene exposure led to reductions in mRNA markers for oxidative stress in aged but not younger animals. Toluene also produced a reduction in cardiac endothelin-1 in aged rats. Higher doses of toluene led to tachycardia, hypothermia, and a transient elevation in MA. Aged rats were less sensitive to the tachycardic effects of toluene but showed a prolonged hypothermic response. Elevated brain levels of toluene in aged rats may be attributed to their suppressed cardiovascular and respiratory responses. The expression of several cardiac biochemical markers of toluene exposure in the aged may also reflect differential susceptibility to this toxicant.
Assuntos
Envelhecimento/fisiologia , Tolueno/farmacocinética , Tolueno/toxicidade , Animais , Biomarcadores , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Coração/efeitos dos fármacos , Masculino , Miocárdio/metabolismo , Ratos , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Tolueno/sangueRESUMO
The Hard-Soft Acid and Base hypothesis can be used to predict the potential bio-reactivity (electrophilicity) of a chemical with intracellular proteins, resulting in neurotoxicity. Twelve chemicals predicted to be neurotoxic were evaluated in vitro in rat dorsal root ganglia (DRG) for effects on cytotoxicity (%LDH), neuronal structure (total neurite length/neuron, NLPN), and neurophysiology (mean firing rate, MFR). DRGs were treated acutely on days in vitro (DIV) 7 (1-100⯵M) with test chemical; %LDH and NLPN were measured after 48â¯h. 4-cyclohexylhexanone (4-C) increased %LDH release at 50 (29%) and 100⯵M (56%), citronellal (Cit) and 1-bromopropane increased %LDH at 100⯵M (22% and 26%). 4-C, Cit, 2,5 Hexanedione (2,5Hex), phenylacetylaldehyde (PAA) and 2-ethylhexanal decreased mean NLPN at 48â¯h; 50 and 100⯵M for 4-C (28% and 60%), 100⯵M Cit (52%), 100⯵M 2,5- Hex (37%) 100⯵M PAA (41%) and 100⯵M for 2-ethylhexanal (23%). Separate DRG cultures were treated on DIV 14 and changes in MFR measured. Four compounds decreased MFR at 50 or 100⯵M: Acrylamide (-83%), 3,4-dichloro-1-butene (-93%), 4-C (-89%) and hexane (-79%, 50⯵M). Changes in MFR and NLPN occurred in absence of cytotoxicity. While the current study showed little cytotoxicity, it gave insight to initial changes in MFR. Results provide insight for future chronic exposure experiments to evaluate neurotoxicity.
Assuntos
Gânglios Espinais/fisiologia , Neuritos/fisiologia , Síndromes Neurotóxicas , Testes de Toxicidade/métodos , Animais , Sobrevivência Celular , Simulação por Computador , Embrião de Mamíferos , Feminino , Gravidez , Ratos Long-EvansRESUMO
Organophosphate (OP) and carbamate (CB) insecticides inhibit cholinesterase (ChE) activity and induce acute hypothermia in adult rats. Studies showed that juveniles are generally more susceptible to neurotoxic insult than adults. However, little is known concerning the effects of OP and CB pesticides on thermoregulation in developing animals. Thus, alterations in core body temperature (Tc) in juvenile animals exposed to an OP and CB insecticide were investigated. Male rat pups were anesthetized on postnatal day (PND) 15 with metofane and a radio transmitter (Data Sciences) was implanted in the abdominal cavity to monitor Tc and motor activity (MA). Two days were allowed for recovery. The PND 17 pups were then dosed by oral gavage with the OP chlorpyrifos (CHP) (1, 5, 10, or 15 mg/kg) or the CB carbaryl (CAR) (10, 20, 80, 120, or 160 mg/kg) or the corn oil vehicle. Pups were returned to their dams and littermates immediately after dosing and monitored for the next several days. CHP doses of 10 and 15 mg/kg resulted in 1.0 degrees C and 2.4 degrees C reductions in Tc, respectively. Tc recovered to control levels by approximately 16 h after dosing. There was significant mortality in rats dosed with 15 mg/kg CHP (6 of 11). CAR doses of 10 to 80 mg/kg had little effect on Tc. The highest dose of CAR (160 mg/kg) resulted in a 1.3 degrees C reduction in Tc that recovered in 9 h. In contrast, past studies found that adult male rats become hypothermic at CHP doses of >25 mg/kg, whereas a CAR dose of 50 mg/kg is effective at inducing hypothermia. Overall, it appears that during the development from preweanling to adult rat, there is a progressive attenuation in CHP-induced hypothermia. Conversely, CAR-induced hypothermia increases as a function of development.
Assuntos
Regulação da Temperatura Corporal/efeitos dos fármacos , Carbaril/toxicidade , Clorpirifos/toxicidade , Inibidores da Colinesterase/toxicidade , Inseticidas/toxicidade , Análise de Variância , Animais , Animais Recém-Nascidos , Carbaril/administração & dosagem , Clorpirifos/administração & dosagem , Inibidores da Colinesterase/administração & dosagem , Inseticidas/administração & dosagem , Masculino , Ratos , Ratos Long-EvansRESUMO
There is a need for methods to screen and prioritize chemicals for potential hazard, including neurotoxicity. Microelectrode array (MEA) systems enable simultaneous extracellular recordings from multiple sites in neural networks in real time and thereby provide a robust measure of network activity. In this study, spontaneous activity measurements from primary neuronal cultures treated with three neurotoxic or three non-neurotoxic compounds was evaluated across four different laboratories. All four individual laboratories correctly identifed the neurotoxic compounds chlorpyrifos oxon (an organophosphate insecticide), deltamethrin (a pyrethroid insecticide) and domoic acid (an excitotoxicant). By contrast, the other three compounds (glyphosate, dimethyl phthalate and acetaminophen) considered to be non-neurotoxic ("negative controls"), produced only sporadic changes of the measured parameters. The results were consistent across the different laboratories, as all three neurotoxic compounds caused concentration-dependent inhibition of mean firing rate (MFR). Further, MFR appeared to be the most sensitive parameter for effects of neurotoxic compounds, as changes in electrical activity measured by mean frequency intra burst (MFIB), and mean burst duration (MBD) did not result in concentration-response relationships for some of the positive compounds, or required higher concentrations for an effect to be observed. However, greater numbers of compounds need to be tested to confirm this. The results obtained indicate that measurement of spontaneous electrical activity using MEAs provides a robust assessment of compound effects on neural network function.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inseticidas/farmacologia , Neurônios/efeitos dos fármacos , Neurotoxinas/farmacologia , Toxicologia/métodos , Animais , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/instrumentação , Concentração Inibidora 50 , Microeletrodos , Neurônios/fisiologia , RatosRESUMO
Most toxicity data are based on studies using single compounds. This study assessed if there is an interaction between mixtures of the anticholinesterase insecticides chlorpyrifos (CHP) and carbaryl (CAR) using hypothermia and cholinesterase (ChE) inhibition as toxicological endpoints. Core temperature (T(c)) was continuously monitored by radiotelemetry in adult Long-Evans rats administered CHP at doses ranging from 0 to 50mg/kg and CAR doses of 0-150 mg/kg. The temperature index (TI), an integration of the change in T(c) over a 12h period, was quantified. Effects of mixtures of CHP and CAR in 2:1 and 1:1 ratios on the TI were examined and the data analyzed using a statistical model designed to assess significant departures from additivity for chemical mixtures. CHP and CAR elicited a marked hypothermia and dose-related decrease in the TI. The TI response to a 2:1 ratio of CHP:CAR was significantly less than that predicted by additivity. The TI response to a 1:1 ratio of CHP and CAR was not significantly different from the predicted additivity. Plasma and brain ChE activity were measured 4h after dosing with CHP, CAR, and mixtures in separate groups of rats. There was a dose-additive interaction for the inhibition of brain ChE for the 2:1 ratio, but an antagonistic effect for the 1:1 ratio. The 2:1 and 1:1 mixtures had an antagonistic interaction on plasma ChE. Overall, the departures from additivity for the physiological (i.e., temperature) and biochemical (i.e., ChE inhibition) endpoints for the 2:1 and 1:1 mixtures studies did not coincide as expected. An interaction between CHP and CAR appears to depend on the ratio of compounds in the mixture as well as the biological endpoint.
Assuntos
Regulação da Temperatura Corporal/efeitos dos fármacos , Carbaril/toxicidade , Organofosfatos/toxicidade , Administração Oral , Animais , Regulação da Temperatura Corporal/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Carbaril/administração & dosagem , Clorpirifos/administração & dosagem , Clorpirifos/toxicidade , Inibidores da Colinesterase/administração & dosagem , Inibidores da Colinesterase/toxicidade , Colinesterases/metabolismo , Diarreia/induzido quimicamente , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Eritrócitos/efeitos dos fármacos , Eritrócitos/enzimologia , Inseticidas/administração & dosagem , Inseticidas/toxicidade , Masculino , Mastigação/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Debilidade Muscular/induzido quimicamente , Organofosfatos/administração & dosagem , Ratos , Ratos Long-Evans , Sialorreia/induzido quimicamente , Lágrimas/efeitos dos fármacos , Lágrimas/metabolismo , Fatores de TempoRESUMO
Diazinon is an organophosphate (OP)-based, anticholinesterase insecticide that irreversibly inhibits acetylcholinesterase activity and produces cholinergic stimulation in central nervous system (CNS) and peripheral tissues. Our laboratory has found that OPs administered orally in rats induce a transient period of hypothermia followed by a delayed fever that persists for several days after exposure. There is little information on the thermoregulatory effects of diazinon. Core temperature (Tc) and motor activity (MA) were monitored by radiotelemetry in male and female rats of the Long-Evans strain dosed orally with diazinon (0 [corn-oil vehicle], 100, 200, or 300 mg/kg in males and 0, 50, 100, or 200 mg/kg in females). There was a dose-dependent decrease in Tc during the first night after treatment, with females exhibiting slightly greater sensitivity than males. MA was unaffected in females exposed to diazinon at doses of 50 to 200 mg/kg; MA of males was reduced during the first night after dosing with 300 mg/kg. There was a delayed elevation in Tc of males dosed with 200 and 300 mg/kg and females dosed with 50, 100, and 200 mg/kg diazinon. The elevated Tc was only manifested during d 2 and 3 after diazinon. Administration of 200 mg/kg sodium salicylate to females 48 h after being treated with 200 mg/kg diazinon led to a rapid abatement of the fever. Diazinon doses of 50 to 300 mg/kg led to 40% to 50% inhibition in plasma cholinesterase (ChE) activity 4 h after dosing, and females displayed a significantly slower recovery of ChE activity compared to males. When compared on a molar basis, the hypothermic response to diazinon was relatively small compared to other OPs such as chlorpyrifos. The delayed fever and efficacy of sodium salicylate to block diazinon-induced fever are similar to the effects of OPs chlorpyrifos and diisopropyl fluoro-phosphate (DFP).
Assuntos
Regulação da Temperatura Corporal/efeitos dos fármacos , Colinesterases/sangue , Diazinon/toxicidade , Inseticidas/toxicidade , Análise de Variância , Animais , Feminino , Masculino , Atividade Motora/efeitos dos fármacos , Ratos , Ratos Long-Evans , Fatores SexuaisRESUMO
Microelectrode arrays (MEAs) can be used to detect drug and chemical induced changes in neuronal network function and have been used for neurotoxicity screening. As a proof-of-concept, the current study assessed the utility of analytical "fingerprinting" using principal components analysis (PCA) and chemical class prediction using support vector machines (SVMs) to classify chemical effects based on MEA data from 16 chemicals. Spontaneous firing rate in primary cortical cultures was increased by bicuculline (BIC), lindane (LND), RDX and picrotoxin (PTX); not changed by nicotine (NIC), acetaminophen (ACE), and glyphosate (GLY); and decreased by muscimol (MUS), verapamil (VER), fipronil (FIP), fluoxetine (FLU), chlorpyrifos oxon (CPO), domoic acid (DA), deltamethrin (DELT) and dimethyl phthalate (DMP). PCA was performed on mean firing rate, bursting parameters and synchrony data for concentrations above each chemical's EC50 for mean firing rate. The first three principal components accounted for 67.5, 19.7, and 6.9% of the data variability and were used to identify separation between chemical classes visually through spatial proximity. In the PCA, there was clear separation of GABAA antagonists BIC, LND, and RDX from other chemicals. For the SVM prediction model, the experiments were classified into the three chemical classes of increasing, decreasing or no change in activity with a mean accuracy of 83.8% under a radial kernel with 10-fold cross-validation. The separation of different chemical classes through PCA and high prediction accuracy in SVM of a small dataset indicates that MEA data may be useful for separating chemicals into effects classes using these or other related approaches.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Acetaminofen/farmacologia , Potenciais de Ação/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/fisiologia , Interpretação Estatística de Dados , Fluoxetina/farmacologia , Antagonistas GABAérgicos/farmacologia , Ácido Caínico/análogos & derivados , Ácido Caínico/farmacologia , Microeletrodos , Muscimol/farmacologia , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Neurônios/fisiologia , Nicotina/farmacologia , Praguicidas/farmacologia , Análise de Componente Principal , Ratos , Ratos Long-Evans , Verapamil/farmacologiaRESUMO
Glufosinate (GLF) at high levels in mammals causes convulsions and amnesia through a mechanism that is not completely understood. The structural similarity of GLF to glutamate (GLU) implicates the glutamatergic system as a target for GLF neurotoxicity. The current work examined in vitro GLF interaction with N-methyl-D-aspartate subtype GLU receptors (NMDARs) and GLT-1 transporters via [(3)H]CGP 39653 binding experiments and [(3)H]GLU uptake assays, respectively. GLF effects on neuronal network activity were assessed using microelectrode array (MEA) recordings in primary cultures of cortical neurons. GLF and its primary metabolite N-acetylglufosinate (NAcGLF) bind to the NMDAR; the IC50 value for GLF was 668 µM and for NAcGLF was about 100 µM. Concentrations of GLF greater than 1000 µM were needed to decrease GLU uptake through GLT-1. In MEA recordings from networks of rat primary cortical neurons, the concentration-responses for NMDA, GLF and NAcGLF on network mean firing rates (MFR) were biphasic, increasing at lower concentrations and decreasing below control levels at higher concentrations. Increases in MFR occurred between 3-10 µM NMDA (290% control, maximum), 100-300 µM NAcGLF (190% control, maximum) and 10-1000 µM GLF (340% control, maximum). The NMDAR antagonist MK801 attenuated both NMDA and GLF increases in MFR. The GLF concentration required to alter GLU transport through GLT-1 is not likely to be attained in vivo, and therefore not relevant to the neurotoxic mode of action. However, toxicokinetic data from reports of intentional human poisonings indicate that GLF concentrations in the CNS after acute exposure could reach levels high enough to lead to effects mediated via NMDARs. Furthermore, the newly characterized action of NAcGLF at the NMDAR suggests that both the parent compound and metabolite could contribute to neurotoxicity via this pathway.