Timothy J. Shafer

Developmental Neurotoxicity of Pyrethroid Insecticides: Critical Review and Future Research Needs

Pyrethroid insecticides have been used for more than 40 years and account for 25% of the worldwide insecticide market. Although their acute neurotoxicity to adults has been well characterized, information regarding the potential developmental neurotoxicity of this class of compounds is limited. There is a large age dependence to the acute toxicity of pyrethroids in which neonatal rats are at least an order of magnitude more sensitive than adults to two pyrethroids. There is no information on age-dependent toxicity for most pyrethroids. In the present review we examine the scientific data related to potential for age-dependent and developmental neurotoxicity of pyrethroids. As a basis for understanding this neurotoxicity, we discuss the heterogeneity and ontogeny of voltage-sensitive sodium channels, a primary neuronal target of pyrethroids. We also summarize 22 studies of the developmental neurotoxicity of pyrethroids and review the strengths and limitations of these studies. These studies examined numerous end points, with changes in motor activity and muscarinic acetylcholine receptor density the most common. Many of the developmental neurotoxicity studies suffer from inadequate study design, problematic statistical analyses, use of formulated products, and/or inadequate controls. These factors confound interpretation of results. To better understand the potential for developmental exposure to pyrethroids to cause neurotoxicity, additional, well-designed and well-executed developmental neurotoxicity studies are needed. These studies should employ state-of-the-science methods to promote a greater understanding of the mode of action of pyrethroids in the developing nervous system.

Microelectrode arrays: A physiologically based neurotoxicity testing platform for the 21st century §

Microelectrode arrays (MEAs) have been in use over the past decade and a half to study multiple aspects of electrically excitable cells. In particular, MEAs have been applied to explore the pharmacological and toxicological effects of numerous compounds on spontaneous activity of neuronal and cardiac cell networks. The MEA system enables simultaneous extracellular recordings from multiple sites in the network in real time, increasing spatial resolution and thereby providing a robust measure of network activity. The simultaneous gathering of action potential and field potential data over long periods of time allows the monitoring of network functions that arise from the interaction of all cellular mechanisms responsible for spatio-temporal pattern generation. In these functional, dynamic systems, physical, chemical, and pharmacological perturbations are holistically reflected by the tissue responses. Such features make MEA technology well suited for the screening of compounds of interest, and also allow scaling to high throughput systems that can record from multiple, separate cell networks simultaneously in multi-well chips or plates. This article is designed to be useful to newcomers to this technology as well as those who are currently using MEAs in their research. It explains how MEA systems operate, summarizes what systems are available, and provides a discussion of emerging mathematical schemes that can be used for a rapid classification of drug or chemical effects. Current efforts that will expand this technology to an influential, high throughput, electrophysiological approach for reliable determinations of compound toxicity are also described and a comprehensive review of toxicological publications using MEAs is provided as an appendix to this publication. Overall, this article highlights the benefits and promise of MEA technology as a high throughput, rapid screening method for toxicity testing.

In vitro assessment of developmental neurotoxicity: use of microelectrode arrays to measure functional changes in neuronal network ontogeny.

Because the Developmental Neurotoxicity Testing Guidelines require large numbers of animals and is expensive, development of in vitro approaches to screen chemicals for potential developmental neurotoxicity is a high priority. Many proposed approaches for screening are biochemical or morphological, and do not assess function of neuronal networks. In this study, microelectrode arrays (MEAs) were used to determine if chemical-induced changes in function could be detected by assessing the development of spontaneous network activity. MEAs record individual action potential spikes as well as groups of spikes (bursts) in neuronal networks, and activity can be assessed repeatedly over days in vitro (DIV). Primary cultures of rat cortical neurons were prepared on MEAs and spontaneous activity was assessed on DIV 2, 6, 9, 13, and 20 to determine the in vitro developmental profile of spontaneous spiking and bursting in cortical networks. In addition, 5 μM of the protein kinase C inhibitor bisindolylmaleamide-1 (Bis-1) was added to MEAs (n = 9–18) on DIV 5 to determine if changes in spontaneous activity could be detected in response to inhibition of neurite outgrowth. A clear profile of in vitro activity development occurred in control MEAs, with the number of active channels increasing from 0/MEA on DIV 2 to 37 ± 5/MEA by DIV 13; the rate of increase was most rapid between DIV 6 and 9, and activity declined by DIV 20. A similar pattern was observed for the number of bursting channels, as well as the total number of bursts. Bis-1 decreased the number of active channels/MEA and the number of bursting channels/MEA. Burst characteristics, such as burst duration and the number of spikes in a burst, were unchanged by Bis-1. These results demonstrate that MEAs can be used to assess the development of functional neuronal networks in vitro, as well as chemical-induced dysfunction.

Neural progenitor cells as models for high-throughput screens of developmental neurotoxicity: State of the science

In vitro, high-throughput methods have been widely recommended as an approach to screen chemicals for the potential to cause developmental neurotoxicity and prioritize them for additional testing. The choice of cellular models for such an approach will have important ramifications for the accuracy, predictivity and sensitivity of the screening assays. In recent years neuroprogenitor cells from rodents and humans have become more widely available and may offer useful models having advantages over primary neuronal cultures and/or transformed cell lines. To date, these models have been utilized in only a limited number of toxicity studies. This review summarizes the state of the science regarding stem and neuroprogenitor models that could be used for screening assays, provides researchers in this field with examples of how these cells have been utilized to date, and discusses the advantages, limitations and knowledge gaps regarding these models. Data are available from both rodent and human stem and neuroprogenitor cell models that indicate that these models will be a valid and useful tool for developmental neurotoxicity testing. Full potential of these models will only be achieved following advances in neurobiology that elucidate differentiation pathways more clearly, and following further evaluation of larger sets of developmentally neurotoxic and non-toxic chemicals to define the sensitivity and predictivity of assays based on stem or progenitor cell models.

Development of micro-electrode array based tests for neurotoxicity: assessment of interlaboratory reproducibility with neuroactive chemicals

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 (IC50s) 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.

CAN THE MECHANISMS OF ALUMINUM NEUROTOXICITY BE INTEGRATED INTO A UNIFIED SCHEME

Regardless of the host, the route of administration, or the speciation, aluminum is a potent neurotoxicant. In the young adult or developmentally mature host, the neuronal response to Al exposure can be dichotomized on morphological grounds. In one, intraneuronal neurofilamentous aggregates are formed, whereas in the other, significant neurochemical and neurophysiological perturbations are induced without neurofilamentous aggregate formation. Evidence is presented that the induction of neurofilamentous aggregates is a consequence of alterations in the posttranslational processing of neurofilament (NF), particularly with regard to phosphorylation state. Although Al has been reported to impact on gene expression, this does not appear to be critical to the induction of cytoskeletal pathology. In hosts responding to Al exposure without the induction of cytoskeletal pathology, impairments in glucose utilization, agonist-stimulated inositol phosphate accumulation, free radical-mediated cytotoxicity, lipid peroxidation, reduced cholinergic function, and altered protein phosphorylation have been described. The extent to which these neurochemical modifications correlate with the induction of a characteristic neurobehavioral state is unknown. In addition to these paradigms, Al is toxic in the immediate postnatal interval. Whether unique mechanisms of toxicity are involved during development remains to be determined. In this article, the mechanisms of Al neurotoxicity are reviewed and recommendations are put forth with regard to future research. Primary among these is the determination of the molecular site of Al toxicity, and whether this is based on Al substitution for divalent metals in a number of biological processes. Encompassed within this is the need to further understand the genesis of host- and developmental-specific responses.

Complete inhibition of spontaneous activity in neuronal networks in vitro by deltamethrin and permethrin

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.

Mechanisms of Pyrethroid Insecticide-Induced Stimulation of Calcium Influx in Neocortical Neurons

Pyrethroid insecticides bind to voltage-gated sodium channels (VGSCs) and modify their gating kinetics, thereby disrupting neuronal function. Pyrethroids have also been reported to alter the function of other channel types, including activation of voltage-gated calcium channels. Therefore, the present study compared the ability of 11 structurally diverse pyrethroids to evoke Ca2+ influx in primary cultures of mouse neocortical neurons. Nine pyrethroids (tefluthrin, deltamethrin, λ-cyhalothrin, β-cyfluthrin, esfenvalerate, S-bioallethrin, fenpropathrin, cypermethrin, and bifenthrin) produced concentration-dependent elevations in intracellular calcium concentration ([Ca2+]i) in neocortical neurons. Permethrin and resmethrin were without effect on [Ca2+]i. These pyrethroids displayed a range of efficacies on Ca2+ influx; however, the EC50 values for active pyrethroids all were within one order of magnitude. Tetrodotoxin blocked increases in [Ca2+]i caused by all nine active pyrethroids, indicating that the effects depended on VGSC activation. The pathways for deltamethrin- and tefluthrin-induced Ca2+ influx include N-methyl-d-aspartic acid receptors, L-type Ca2+ channels, and reverse mode of operation of the Na+/Ca2+ exchanger inasmuch as antagonists of these sites blocked deltamethrin-induced Ca2+ influx. These data demonstrate that pyrethroids stimulate Ca2+ entry into neurons subsequent to their actions on VGSCs.

Toluene inhibits voltage-sensitive calcium channels expressed in pheochromocytoma cells.

Commercial solvents such as toluene are commonly used as drugs of abuse by children and adolescents. The cellular and molecular sites and mechanisms of actions of these compounds are not well studied but their effects on behavior resemble those of central nervous system depressants such as alcohol, barbiturates and benzodiazepines. In this study, the effects of toluene on voltage-sensitive calcium channels (VSCCs) were measured in pheochromocytoma cells. The KCl-induced rise in intracellular calcium as measured by calcium imaging was almost completely blocked by the dihydropyridine calcium channel antagonist nifedipine verifying that undifferentiated pheochromocytoma cells express mainly the L-type of calcium channel. Toluene (0.3-3000 microM) by itself did not affect intracellular calcium levels in resting cells but dose-dependently inhibited the KCl-induced rise in calcium. This inhibition was substantially reversed upon washout of the toluene-containing solution. KCl-dependent increases in intracellular calcium in cells differentiated with nerve growth factor (NGF) were largely insensitive to nifedipine. Toluene produced a greater inhibition of the KCl response in NGF treated cells as compared with undifferentiated cells. A similar finding was obtained when whole-cell patch-clamp-electrophysiology was used to directly monitor the effects of toluene on voltage-activated calcium currents in undifferentiated and differentiated cells. These results show that dihydropyridine sensitive and insensitive calcium channels are inhibited by toluene and may represent important sites of action for this compound.

Comparison of chemical-induced changes in proliferation and apoptosis in human and mouse neuroprogenitor cells

There is a need to develop rapid and efficient models to screen chemicals for their potential to cause developmental neurotoxicity. Use of in vitro neuronal models, including human cells, is one approach that allows for timely, cost-effective toxicity screening. The present study compares the sensitivity of human (ReN CX) and mouse (mCNS) neuroprogenitor cell lines to chemicals using a multiplex assay for proliferation and apoptosis, endpoints that are critical for neural development. Cells were exposed to 0.001-100 μM concentrations of 11 chemicals (cadmium, chlorpyrifos oxon, dexamethasone, dieldrin, ketamine, lead, maneb, methylmercury, nicotine, trans-retinoic acid, and trimethyltin) reported in the literature to affect proliferation and/or apoptosis, and 5 chemicals (dimethyl pthalate, glyphosate, omeprazole, saccharin, and d-sorbitol) with no reports of effects on either endpoint. High-content screening of markers for proliferation (BrdU incorporation) and apoptosis (activated caspase 3 and p53) was used to assess the effect of chemicals in both cell lines. Of the chemicals tested, methylmercury, cadmium, dieldrin, chlorpyrifos oxon, trans-retinoic acid, and trimethyltin decreased proliferation by at least 50% of control in either the ReN CX or mCNS cells. None of the chemicals tested activated caspase 3 or p53 in the ReN CX cells, while methylmercury, cadmium, dieldrin, chlorpyrifos oxon, trimethyltin, and glyphosate all induced at least a doubling in these apoptotic markers in the mCNS cells. Compared to control, cadmium, trans-retinoic acid, and trimethyltin decreased cell viability (ATP levels) by at least 50% in the ReN CX cells, while cadmium, dieldrin, and methylmercury decreased viability by at least 50% in the mCNS cells. Based on these results, BrdU is an appropriate marker for assessing chemical effects on proliferation, and human cells are more sensitive than mouse cells for this endpoint. By contrast, caspase 3 and p53 were altered by environmental chemicals in mouse, but not in human cells. Therefore, these markers are not appropriate to assess the ability of environmental chemicals to induce apoptosis in the ReN CX cells.