Integration of toxicodynamic and toxicokinetic new approach methods into a weight-of-evidence analysis for pesticide developmental neurotoxicity assessment: A case-study with DL- and L-glufosinate.

DL-glufosinate ammonium (DL-GLF) is a registered herbicide for which a guideline Developmental Neurotoxicity (DNT) study has been conducted. Offspring effects included altered brain morphometrics, decreased body weight, and increased motor activity. Guideline DNT studies are not available for its enriched isomers L-GLF acid and L-GLF ammonium; conducting one would be time consuming, resource-intensive, and possibly redundant given the existing DL-GLF DNT. To support deciding whether to request a guideline DNT study for the L-GLF isomers, DL-GLF and the L-GLF isomers were screened using in vitro assays for network formation and neurite outgrowth. DL-GLF and L-GLF isomers were without effects in both assays. DL-GLF and L-GLF (1-100 µM) isomers increased mean firing rate of mature networks to 120-140% of baseline. In vitro toxicokinetic assessments were used to derive administered equivalent doses (AEDs) for the in vitro testing concentrations. The AED for L-GLF was ∼3X higher than the NOAEL from the DL-GLF DNT indicating that the available guideline study would be protective of potential DNT due to L-GLF exposure. Based in part on the results of these in vitro studies, EPA is not requiring L-GLF isomer guideline DNT studies, thereby providing a case study for a useful application of DNT screening assays.

Sensitivity of neuroprogenitor cells to chemical-induced apoptosis using a multiplexed assay suitable for high-throughput screening.

High-throughput methods are useful for rapidly screening large numbers of chemicals for biological activity, including the perturbation of pathways that may lead to adverse cellular effects. In vitro assays for the key events of neurodevelopment, including apoptosis, may be used in a battery of tests for detecting chemicals that could result in developmental neurotoxicity. Apoptosis contributes to nervous system development by regulating the size of the neuroprogenitor cell pool, and the balance between cellular proliferation and apoptosis during neuroprogenitor cell proliferation helps to determine the size and shape of the nervous system. Therefore, chemicals that affect apoptosis during neuronal development can have deleterious effects on the developing brain. The present study examined the utility of a high-throughput assay to detect chemical-induced apoptosis in mouse or human neuroprogenitor cells, as well as differentiated human neurons derived from induced pluripotent stem cells. Apoptosis was assessed using an assay that measures enzymatic activity of caspase-3/7 in a rapid and cost efficient manner. The results show that all three commercially available models generated a robust source of proliferating neuroprogenitor cells, and that the assay was sensitive and reproducible when used in a multi-well plate format. There were differences in the response of rodent and human neuroprogenitor cells to a set of chemicals previously shown to induce apoptosis in vitro. Neuroprogenitor cells were more sensitive to chemical-induced apoptosis than differentiated neurons, suggesting that neuroprogenitor cells are one of the cell models that should be considered for use in a developmental neurotoxicity screening battery.

Media formulation influences chemical effects on neuronal growth and morphology.

Screening for developmental neurotoxicity using in vitro, cell-based systems has been proposed as an efficient alternative to performing in vivo studies. One tool currently used for developmental neurotoxicity screening is automated high-content imaging of neuronal morphology. While high-content imaging (HCI) has been demonstrated to be useful in detection of potential developmental neurotoxicants, comparison of results between laboratories or assays can be complicated due to methodological differences. In order to determine whether high-content imaging-based developmental neurotoxicity assays can be affected by differences in media formulation, a systematic comparison of serum-supplemented (Dulbecco's modified Eagle's media (DMEM) + 10% serum) and serum-free (Neurobasal A + B27) culture media on neuronal morphology was performed using primary rat cortical neurons. Concentration-response assays for neuritogenesis, axon and dendrite outgrowth, and synaptogenesis were performed in each media type using chemicals with previously demonstrated effects. Marked qualitative and quantitative differences in the characteristics of neurons cultured in the two media types were observed, with increased neuronal growth and less basal cell death in Neurobasal A + B27. Media formulation also affected assay sensitivity and selectivity. Increases in assay sensitivity were observed in Neurobasal A + B27 media as compared to serum-supplemented DMEM. In some instances, a greater difference between effective concentrations for cell death and neurodevelopmental-specific endpoints was also observed in Neurobasal A + B27 media as compared to serum-supplemented DMEM. These data show that media formulation must be considered when comparing data for similar endpoints between studies. Neuronal culture maintained in Neurobasal A + B27 media had several features advantageous for HCI applications including less basal cell death, less cell clustering and neurite fasciculation, and a tendency towards increased sensitivity and selectivity in chemical concentration-response studies.

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.

Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth.

There is a need for rapid, efficient and cost-effective alternatives to traditional in vivo developmental neurotoxicity testing. In vitro cell culture models can recapitulate many of the key cellular processes of nervous system development, including neurite outgrowth, and may be used as screening tools to identify potential developmental neurotoxicants. The present study compared primary rat cortical cultures and human embryonic stem cell-derived neural cultures in terms of: 1) reproducibility of high content image analysis based neurite outgrowth measurements, 2) dynamic range of neurite outgrowth measurements and 3) sensitivity to chemicals which have been shown to inhibit neurite outgrowth. There was a large increase in neurite outgrowth between 2 and 24h in both rat and human cultures. Image analysis data collected across multiple cultures demonstrated that neurite outgrowth measurements in rat cortical cultures were more reproducible and had higher dynamic range as compared to human neural cultures. Human neural cultures were more sensitive than rat cortical cultures to chemicals previously shown to inhibit neurite outgrowth. Parallel analysis of morphological (neurite count, neurite length) and cytotoxicity (neurons per field) measurements were used to detect selective effects on neurite outgrowth. All chemicals which inhibited neurite outgrowth in rat cortical cultures did so at concentrations which did not concurrently affect the number of neurons per field, indicating selective effects on neurite outgrowth. In contrast, more than half the chemicals which inhibited neurite outgrowth in human neural cultures did so at concentrations which concurrently decreased the number of neurons per field, indicating that effects on neurite outgrowth were secondary to cytotoxicity. Overall, these data demonstrate that the culture models performed differently in terms of reproducibility, dynamic range and sensitivity to neurite outgrowth inhibitors. While human neural cultures were more sensitive to neurite outgrowth inhibitors, they also had a lower dynamic range for detecting chemical-induced neurite outgrowth inhibition and greater variability from culture-to-culture as compared to rat primary cortical cultures.

Quantitative assessment of neurite outgrowth in human embryonic stem cell-derived hN2 cells using automated high-content image analysis.

Throughout development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxic chemicals that interfere with this process may result in permanent deficits in nervous system function. Traditionally, rodent primary neural cultures and immortalized human and non-human clonal cell lines have been used to investigate the molecular mechanisms controlling neurite outgrowth and examine chemical effects on this process. The present study characterizes the molecular phenotype of hN2 human embryonic stem cell (hESC)-derived neural cells and uses automated high-content image analysis to measure neurite outgrowth in vitro. At 24h post-plating hN2 cells express a number of protein markers indicative of a neuronal phenotype, including: nestin, beta(III)-tubulin, microtubule-associated protein 2 (MAP2) and phosphorylated neurofilaments. Neurite outgrowth in hN2 cells proceeded rapidly, with a majority of cells extending one to three neurites by 48h in culture. In addition, concentration-dependent decreases in neurite outgrowth and ATP-content were observed following treatment of hN2 cells with either bisindolylmaleimide I, U0126, lithium chloride, sodium orthovanadate and brefeldin A, all of which have previously been shown to inhibit neurite outgrowth in primary rodent neural cultures. Overall, the molecular phenotype, rate of neurite outgrowth and sensitivity of hN2 cells to neurite outgrowth inhibitors were comparable to other in vitro models previously characterized in the literature. hN2 cells provide a model in which to investigate chemical effects on neurite outgrowth in a non-transformed human-derived cells and provide an alternative to the use of primary rodent neural cultures or immortalized clonal cell lines.

Neuronal models for evaluation of proliferation in vitro using high content screening.

In vitro test methods can provide a rapid approach for the screening of large numbers of chemicals for their potential to produce toxicity (hazard identification). In order to identify potential developmental neurotoxicants, a battery of in vitro tests for neurodevelopmental processes such as cell proliferation, differentiation, growth, and synaptogenesis has been proposed. The development of in vitro approaches for toxicity testing will require choosing a model system that is appropriate to the endpoint of concern. This study compared several cell lines as models for neuronal proliferation. The sensitivities of neuronal cell lines derived from three species (PC12, rat; N1E-115, mouse; SH-SY5Y, human) to chemicals known to affect cell proliferation were assessed using a high content screening system. After optimizing conditions for cell growth in 96-well plates, proliferation was measured as the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into replicating DNA during S phase. BrdU-labeled cells were detected by immunocytochemistry and cell counts were obtained using automated image acquisition and analysis. The three cell lines showed approximately 30-40% of the population in S phase after a 4h pulse of BrdU. Exposure to the DNA polymerase inhibitor aphidicolin for 20 h prior to the 4h pulse of BrdU significantly decreased proliferation in all three cell lines. The sensitivities of the cell lines were compared by exposure to eight chemicals known to affect proliferation (positive controls) and determination of the concentration inhibiting proliferation by 50% of control (I(50)). PC12 cells were the most sensitive to chemicals; 6 out of 8 chemicals (aphidicolin, cadmium, cytosine arabinoside, dexamethasone, 5-fluorouracil, and methylmercury) inhibited proliferation at the concentrations tested. SH-SY5Y cells were somewhat less sensitive to chemical effects, with five out of eight chemicals inhibiting proliferation; dexamethasone had no effect, and cadmium inhibited proliferation only at concentrations that decreased cell viability. Data from the N1E-115 cell line was extremely variable between experiments, and only 4 out of 8 chemicals resulted in inhibition of proliferation. Chemicals that had not been previously shown to alter proliferation (negative controls) did not affect proliferation or cell viability in any cell line. The results show that high content screening can be used to rapidly assess chemical effects on proliferation. Three neuronal cell lines exhibited differential sensitivity to the effect of chemicals on this endpoint, with PC12 cells being the most sensitive to inhibition of proliferation.

Comparison of PC12 and cerebellar granule cell cultures for evaluating neurite outgrowth using high content analysis.

Development of high-throughput assays for chemical screening and hazard identification is a pressing priority worldwide. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite outgrowth is one such critical cellular process underlying nervous system development that can be quantified using automated microscopy and image analysis (high content analysis). The present study characterized and compared the PC-12 cell line (NS-1) and primary cultures of cerebellar granular cells (CGC), as models for assessing chemical effects on neurite outgrowth. High content analysis of neurite outgrowth was performed using the Cellomics ArrayScan V(Ti) automated epifluorescent imaging system to acquire and analyze images of beta-tubulin immunostained cells in 96-well plates. Cell viability was assessed using the CellTiter-Glo assay. Culture of NS-1 or CGC in nerve growth factor or serum respectively, rapidly induced neurite outgrowth that increased over four days in vitro. Seven compounds previously shown to affect neurite outgrowth in vitro were tested in both models for changes in total neurite length and cell viability. In NS-1 cells, four chemicals (PKC inhibitor Bis-I, MEK inhibitor U0126, trans-Retinoic acid, methylmercury) inhibited neurite outgrowth, while lead, amphetamine and valproic acid had no effect. In CGC, five chemicals inhibited neurite outgrowth (Bis-I, U0126, lead, methylmercury, and amphetamine), while trans-Retinoic acid decreased cell viability but not neurite outgrowth. Valproic acid was without effect. The sensitivity of the two models was chemical specific: NS-1 cells were more sensitive to Bis-I, methylmercury and trans-Retinoic acid, while CGC were more sensitive to U0126, lead, and amphetamine. For every chemical (except trans-Retinoic acid), neurite outgrowth was equal to or more sensitive than cell viability. In comparison, out of seven chemicals without prior evidence for effects on neurite outgrowth, only one decreased neurite outgrowth (diphenhydramine in CGC). These findings demonstrate that the effects of chemicals on neurite outgrowth may be cell type specific.

Protein biomarkers associated with growth and synaptogenesis in a cell culture model of neuronal development.

Cerebellar granule cells (CGC) provide a homogenous population of cells which can be used as an in vitro model for studying the cellular processes involved in the normal development of the CNS. They may also be useful for hazard identification as in vitro screens for developmental neurotoxicity. The present study examined morphologic and biochemical markers of CGC neurite outgrowth and synaptogenesis in vitro using both qualitative and quantitative approaches. CGC exhibit a rapid outgrowth of neurites over 14 days in vitro, concomitant with the expression of the synaptic protein Synapsin 1 that was observed as puncta associated with cell bodies and neurites. The expression of neurotypic proteins associated with the cytoskeleton (NF68, MAP2), growth cones (GAP-43) and the synapse (Synapsin I) present an ontogeny that reflects the morphological growth of CGC. The utility of these neurotypic proteins as biomarkers was examined by inhibiting CGC growth using pharmacologic inhibitors of PKC activity and the MAP kinase pathway. Quantitative analysis of neurite outgrowth was performed using an automated image acquisition and analysis system. Treatment of CGC with the MAP kinase pathway inhibitor U0126 significantly decreased total neurite outgrowth, while the inhibitor of classic PKC isoforms Bis I had no effect on this measure. The ontogenetic expression of neurotypic proteins was reduced after treatment with both inhibitors. In particular, Synapsin 1 and GAP-43 expression were both significantly reduced by chemical treatment. These data demonstrate that neurotypic proteins can be used as biomarkers of neuronal development in vitro, and in some cases, may detect changes that are not apparent using morphologic measures.

Apoptosis of cerebellar granule cells induced by organotin compounds found in drinking water: involvement of MAP kinases.

Mono- and dialkyl organotin compounds are used primarily as heat stabilizers in polyvinyl chloride (PVC) plastics. Recently, monomethyltin (MMT), dimethyltin (DMT), monobutyltin (MBT), and dibutyltin (DBT) have been detected in water from homes and businesses served by PVC pipes. While trialkyl organotins such as trimethyltin (TMT) and triethyltin (TET) are well known neurotoxicants, the toxicity of the mono- and dialkyl organotins is not well described. The present study compared the cytotoxicity of organotins found in drinking water with the known neurotoxicant TMT in primary cultures of cerebellar granule cells, and examined the role of MAP kinase signaling in organotin-induced cell death. Twenty-four hour exposure to TMT resulted in a concentration-dependent decrease in cell viability with an EC(50) of 3 microM. Exposure to MMT, DMT, and MBT at concentrations up to 10 microM had no effect. DBT, however, was very potent, and decreased cell viability with an EC(50) of 0.3 microM. Staining of organotin-treated cerebellar granule cells with the nuclear dye Syto-13 revealed that TMT and DBT, but not MMT, DMT, or MBT, produced condensation and fragmentation of chromatin characteristic of apoptosis. TMT- and DBT-induced apoptosis was confirmed using TUNEL staining and measurement of PARP cleavage. Activation of MAP kinase pathways was examined after 6 h of exposure to the organotins which induced apoptosis. Both TMT and DBT activated ERK1/2, but only TMT activated the JNK/c-Jun and p38 pathways. Pharmacologic blockade of JNK/c-Jun and p38 activation significantly decreased apoptosis produced by TMT, but not by DBT. These results show that DBT is a potent neurotoxicant in vitro, but unlike TMT, does not induce cell death via activation of MAP kinase signaling.

Accumulation of PBDE-47 in primary cultures of rat neocortical cells.

A number of recent studies have examined the neurotoxic actions of polybrominated diphenyl ethers (PBDEs) using in vitro cell culture models. However, there are few data reporting the final concentration of PBDEs in cells after in vitro exposure to these compounds. To address this issue, the present study examined the concentration-dependent and time-dependent accumulation of 2,2',4,4'-tetrabromodiphenyl ether (PBDE-47) in primary cultures of rat neocortex. Mixed cultures of neuronal and glial cells were prepared from the neocortex of newborn rats and grown for 7 days in vitro. The cells were then exposed to freshly prepared serum-free culture medium containing (14)C-PBDE-47. Radiolabel associated with the cells or remaining in the medium was determined by liquid scintillation spectrometry. Exposure to 0.01-3.0 microM PBDE-47 for 60 min resulted in a concentration-dependent accumulation in cells. At each concentration, approximately 15% of the applied PBDE-47 was associated with the cells, resulting in a 100-fold magnification of the applied concentration (e.g., a 60-min exposure to 1 microM resulted in an approximate 100 microM concentration in the cells); 55% of the PBDE remained in the medium and 30% was associated with the plastic culture dish. Exposure to 1 microM PBDE-47 resulted in a linear increase in PBDE-47 in cells with time for the first 60 min, which began to saturate at 120 min. Addition of serum proteins to the medium decreased accumulation; at 10% serum in the medium, only 3% of the applied PBDE-47 was associated with the cells and 96% remained in the media after 60 min. The total volume of exposure also influenced accumulation of PBDE-47. Doubling the volume of serum-free exposure medium (from 2 ml to 4 ml) but leaving the concentration constant (1 microM) resulted in a 1.5-fold increase in PBDE-47 concentration in the cells. These data show that a number of factors, including duration of exposure, volume of exposure, and concentration of serum proteins in the medium, can influence the accumulation of PBDE-47 in cells in vitro. For this highly lipophilic compound, use of medium concentration underestimates tissue concentration by up to two orders of magnitude. Thus, accurate information on the tissue concentration for in vitro experiments should be determined empirically.

Assessment of PC12 cell differentiation and neurite growth: a comparison of morphological and neurochemical measures.

In vitro techniques are used increasingly to screen for and characterize neurotoxicants. In many cases, chemical-induced injury to developing neurons has been examined in vitro by assessing morphological changes in differentiation and neurite growth. This research evaluated the use of proteins associated with axonal growth and synaptogenesis as surrogates for morphological measurement of neuronal differentiation. PC12 cells, which differentiate upon nerve growth factor (NGF) stimulation, were used as the in vitro model. NGF-induced (50 ng/ml) differentiation (cells with at least one neurite with a length equal to the cell body diameter) and neurite growth (length of longest neurite) were determined using light microscopy and computer-based quantitative image analysis. PC12 cell differentiation and neurite growth reached a plateau after 6 days in culture. Expression of the axonal growth associated protein 43 (GAP-43) and the synaptic protein synapsin I were assessed simultaneously by Western blot during cell differentiation. Expression of GAP-43 was low on Culture Day 0 and increased progressively to maximum levels on Culture Day 5. Likewise, synapsin I expression increased slowly on Days 0-4, and then rapidly on Days 5-7 of culture. Pharmacologic inhibitors of NGF-induced signaling were used to test the sensitivity of the proteins to chemical disruption of differentiation. The MAP kinase inhibitor, U0126 (5-30 microM) and the PKC inhibitor, bisindolylmaleimide I (Bis I; 1.25-5 microM) inhibited differentiation and neurite outgrowth in a concentration-dependent manner. U0126 and Bis I significantly decreased GAP-43, but not synapsin I expression. Interestingly, the PI-PLC inhibitor edelfosine (ET-18; 5-30 microM) stimulated differentiation at early times of exposure followed by a significant decrease in neurite length at later time points. However, ET-18 did not alter the expression of GAP-43 or synapsin I. These data suggest that GAP-43 may be a useful indicator of the status of PC12 cell differentiation.