High-Content Screening Assay for Identification of Chemicals Impacting Spontaneous Activity in Zebrafish Embryos
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Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, South Carolina 29208 United States
*Phone: (803) 777-0218; fax: (803) 777-3391; e-mail: [email protected]
Copyright © 2013 American Chemical Society
Abstract
Although cell-based assays exist, rapid and cost-efficient high-content screening (HCS) assays within intact organisms are needed to support prioritization for developmental neurotoxicity testing in rodents. During zebrafish embryogenesis, spontaneous tail contractions occur from late-segmentation (∼19 h postfertilization, hpf) through early pharyngula (∼29 hpf) and represent the first sign of locomotion. Using transgenic zebrafish (fli1:egfp) that stably express eGFP beginning at ∼14 hpf, we have developed and optimized a 384-well-based HCS assay that quantifies spontaneous activity within single zebrafish embryos after exposure to test chemicals in a concentration–response format. Following static exposure of one embryo per well from 5 to 25 hpf, automated image acquisition procedures and custom analysis protocols were used to quantify total body area and spontaneous activity in live embryos. Survival and imaging success rates across control plates ranged from 87.5 to 100% and 93.3–100%, respectively. Using our optimized procedures, we screened 16 chemicals within the US EPA’s ToxCast Phase-I library, and found that exposure to abamectin and emamectin benzoate—both potent avermectins—abolished spontaneous activity in the absence of gross malformations. Overall, compared to existing locomotion-based zebrafish assays conducted later in development, this method provides a simpler discovery platform for identifying potential developmental neurotoxicants.
Introduction
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The developing nervous system is a sensitive target for chemical exposure in both humans and animal models, (1, 2) and early life-stage exposures can lead to long-term effects on motor activity, sensory function, and cognition. (3-5) Developed and issued by the Organization for Economic Co-operation and Development (OECD), the developmental neurotoxicity (DNT) test guideline (http://www.oecd-ilibrary.org/environment/test-no-426-developmental-neurotoxicity-study_9789264067394-en) is used to assess the potential effects of pre- and postnatal chemical (mainly pesticide) exposure on the morphology and function of the developing nervous system within preweaning, adolescent, and young adult rodents. Commonly used strains of rats are preferred for this guideline, and a minimum of 20 litters per treatment group and 3–4 dose levels plus a vehicle control are needed for successful study completion. Therefore, assuming an average litter size of 8–12 pups, (6) each DNT study would require 640 to 1200 rats (excluding dams) per chemical.
Currently, there are minimal to no DNT data available for thousands of chemicals used in commerce. (1, 7, 8) In addition to animal use considerations, it is impractical to screen these chemicals using the existing DNT test guideline, as this test is costly, time-intensive, and low-throughput. Although significant advancements in development and application of high-throughput screening (HTS) and high-content screening (HCS) assays have occurred over the last 5–10 years, a large majority of assays relevant to DNT are focused on key molecular and cellular events in vitro and, as such, do not address the potential for chemically induced DNT within an intact organism. (9, 10) Therefore, there is a recognized need to use alternative nonmammalian models to support screening and prioritization of chemicals for DNT testing. (9, 10)
Zebrafish have long been used as a model for understanding vertebrate neurodevelopment (11, 12) and, as a result, the nervous system circuitry within zebrafish is well characterized and understood. (13, 14) Due to the small size, transparency, and ex utero development during embryogenesis, the potential for adverse effects on the developing nervous system can be readily evaluated at both the morphologic- and functional-level. For example, several zebrafish-based locomotion assays have been used to examine spontaneous tail contractions, touch-response, and/or swimming patterns in response to chemical and/or light stimuli in microplate format. (15-18) Although valuable for providing information on behavioral responses following developmental exposure, these assays can be complex, requiring treatment solution renewals (and, therefore, more technical material), excessive embryo/larval handling, and cost-prohibitive infrared cameras needed for imaging under dark conditions. Moreover, typical zebrafish-based locomotive assays are conducted using a larval stage (6–7 days postfertilization (dpf)) that is protected by animal use regulations around the world, likely resulting in minimal animal use reductions for DNT testing.
Despite significant advancements in zebrafish locomotion assays, rapid and streamlined HCS assays are needed to facilitate screening and prioritization of chemicals for DNT testing within rodents. Therefore, for this study, we focused on spontaneous tail contractions—the first sign of motor activity—within zebrafish embryos as a behavioral end point amenable for identification of potential developmental neurotoxicants. Although other assays have focused on this end point, the HCS assay reported here provides a streamlined discovery platform with (1) increased sample sizes; (2) broad concentration–response format; (3) short assay duration (∼22 h total); and (4) minimal technical-grade test material needed for screening.
Materials and Methods
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Animals
For this assay, we relied on a robust line of transgenic zebrafish (fli1:egfp) that stably express enhanced green fluorescent protein (eGFP) within vascular endothelial cells. (19) This strain begins expressing eGFP at ∼14 h postfertilization (hpf), allowing us to accurately track spontaneous tail contractions in the absence of shading effects observed under transmitted light. Adult fli1:egfp zebrafish were maintained on a 14-h/10-h light/dark cycle within a Mini Mass Embryo Production System (mini-MEPS) (Aquatic Habitats, Inc., Apopka, FL) containing a photoperiod light cycle dome and recirculating conditioned reverse osmosis (RO) water (∼27–28 °C). Adult females and males were bred directly within the mini-MEPS, directly on-system using in-tank breeding traps suspended within 3 L tanks, or off-system within a light- and temperature-controlled incubator using breeding traps suspended within 1 L tanks. For all experiments described below, newly fertilized eggs were staged according to previously described methods. (20) All fish were handled and treated in accordance with approved Institutional Animal Care and Use Committee (IACUC) protocols at the University of South Carolina—Columbia.
Chemicals
Chemicals were purchased from ChemService, Inc. (West Chester, PA) and Sigma Aldrich (St. Louis, MO). Chemical names, chemical formulas, CAS registry numbers, vendor, and purities are provided within Supporting Information, SI, Table S1. Within 24 h of test initiation, stock solutions of each chemical were prepared by dissolving chemicals in high performance liquid chromatography (HPLC)-grade dimethyl sulfoxide (DMSO) (50 mM), and then performing 2-fold serial dilutions into DMSO to create stock solutions for each working solution. All stock solutions were stored at room temperature within 2-mL amber glass vials containing polytetrafluoroethylene (PTFE)-lined caps. For each individual plate, working solutions of all treatments were freshly prepared by spiking stock solutions into embryo media (EM) (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4), resulting in 0.1% DMSO within all vehicle control and treatment groups.
High-Content Screening (HCS) Assay
Exposure Setup
Black 384-well microplates containing 0.17-mm glass-bottom wells (Matrical Bioscience, Spokane, WA) were used for this assay. Newly fertilized eggs were collected immediately after spawning and placed in groups of approximately 50 per glass Petri dish within a light- and temperature-controlled incubator until 5 hpf. Although dechorionation may help decrease uncertainties regarding uptake across the chorion, previous attempts within our laboratory to dechorionate embryos prior to exposure resulted in unacceptable survival within control embryos (data not shown); therefore, chorionated embryos were used for all exposures. Vehicle control (0.1% DMSO) or treatment solution (50 μL per well) was added to odd columns of a 384-well plate at 5 hpf. Using clean forceps, 192 viable fli1:egfp embryos were then manually arrayed into the plate over a 20-min time period, resulting in one embryo per well and 16 initial embryos per column. Each plate was checked to ensure that all embryos were intact or undamaged following loading. Initial attempts to use 384 embryos were unsuccessful due to increased imaging duration, resulting in variation in spontaneous activity within vehicle control embryos across the plate (data not shown). For chemical screening assays, vehicle control groups occupied two columns flanking the left and right sides of the plate (columns 1 and 23) to account for potential control variance in spontaneous activity from initiation to termination of image acquisition. The plate was then incubated at 28 °C under a 14-h/10-h light/dark cycle and static conditions until 24 hpf.
Image Acquisition
At 24 hpf, the plate was removed from the incubator and allowed to acclimate in a second incubator at 25 °C in treatment solution for 1 h with the lid removed. This acclimation was done to ensure that changes in temperature from the incubator (28°C) to the imager (25°C) did not affect spontaneous activity, as previous experiments have demonstrated that temperature alterations can affect this behavior. (26) The plate was then centrifuged for 3 min at 200 rpm to ensure that the majority of embryos were near the bottom of each well. Using an automated image acquisition protocol (SI Figure S1) and parameters (SI Table S2) optimized for our ImageXpress Micro (IXM) Widefield High-Content Screening System (Molecular Devices, Sunnyvale, CA), each embryo was imaged in treatment solution over a 6-s time period to assess the presence or absence of spontaneous tail contractions. Image acquisition began within 5 to 10 min after centrifugation, commencing at well A01 and proceeding down each column across the plate from columns 1 to 23. Throughout this process, one 6-s stream was acquired at a time for each well. This time period was chosen to minimize variation in spontaneous activity across the plate due to increased imaging duration. Image acquisition procedures were identical for each independent plate. During the entire ∼1.4-h image acquisition period, internal temperature within the IXM system was maintained between 25 and 27 °C by removing panels on both sides of the IXM system and blowing air from left to right through the IXM with a portable fan; internal temperature was monitored and recorded at initiation and termination of imaging using a digital thermometer. In accordance with National Institutes of Health (NIH) guidelines, (21) embryos were then euthanized by placing the plate at −20 °C.
Data Extraction
Custom journal scripts for video generation were developed using MetaXpress 4.0.0.24 software (Molecular Devices, Sunnyvale, CA). Prior to data extraction, stream acquisitions within each well were inspected within MetaXpress to assess survival. As heart morphogenesis is not complete until ∼48 hpf, presence or absence of heartbeat was not used as a criterion for survival. Rather, coagulated embryos and embryos with undeveloped or absent tails were considered dead and were not analyzed for total body area or spontaneous activity. Fully automated journal scripts were used to generate videos of each well by converting each stream acquisition to an .AVI file. Video file sizes were reduced within Format Factory (http://www.pcfreetime.com/) by conversion to MPEG4 (DivX). Converted videos were then imported into EthoVision XT 9.0 (Noldus Information Technology, Leesburg, VA) to determine the mobility state of each embryo. On the basis of user-defined detection settings that identify the embryo as the subject, embryos were considered highly mobile (exhibiting spontaneous tail contractions) if the change in subject pixel area from one frame to another was above a previously optimized user-defined threshold. Initial attempts to use EthoVision to quantitatively assess the number or duration of spontaneous tail contractions were unsuccessful due to variation in this behavioral phenotype within individual control embryos (data not shown). Rather, EthoVision output was used to assign spontaneous activity values to each embryo. Inactive embryos were defined as embryos that were immobile during the entire 6-s stream acquisition and were assigned an activity value of 0. Active embryos were defined as embryos that moved for at least 0.1 s and were assigned an activity value of 1. Within EthoVision, duration is assessed in 0.1-s increments; therefore, we used this value as a criterion for activity in order to include embryos exhibiting partial tail contractions as active embryos.
Automated custom journal scripts within MetaXpress were used to analyze total embryo body area in order to (1) reliably identify treatment-related effects and (2) focus our analyses on targeted impacts on spontaneous activity in the absence of significant effects on embryonic growth. Treatments resulting in a significant decrease in total body area were not analyzed for spontaneous activity. Embryos exhibiting notochord malformations that were not detected by analysis of total body area were also excluded from analysis of spontaneous activity. Additional information about parameters used in EthoVision XT 9.0 is provided in SI Table S3. Additional details about the data extraction and analysis process are provided in SI Figure S1.
Statistical Analysis
All statistical procedures were performed using SPSS Statistics 20.0 (Chicago, IL). Nonparametric tests were used for analyzing spontaneous activity data, as these data were categorical and did not meet normality assumptions. A Kruskal–Wallis test was used (α = 0.05) to test for main effect of treatment, and Mann–Whitney pairwise comparisons were used to test for differences between vehicle control columns 1 and 23 and treatment columns relative to vehicle control columns (α = 0.05). For chemical screening assays, treatments were only considered significant if different from both control columns. For total body area analysis, a general linear model (GLM) analysis of variance (ANOVA) (α = 0.05) was used, as these data did not meet the equal variance assumption for non-GLM ANOVAs. Pair-wise Tukey-based multiple comparisons of least-squares means were performed to identify significant treatment-related effects. To estimate the median lethal concentration (LC50) after static exposure to each chemical from 5 to 25 hpf, a four-parameter concentration–response curve was fit to percent mortality data using log-transformed chemical concentrations within Prism 6.0 (GraphPad Software Inc., La Jolla, CA).
Results
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HCS Assay Variability
To determine assay variability, we imaged three independent control plates containing embryos incubated in EM at 28 °C from 5 to 24 hpf, and then acclimated for 1 h at 25 °C prior to imaging. Parameters provided in SI Figure S1 and Tables S2 and S3 were used to analyze total body area and spontaneous activity for each control plate. Percent survival and image success rates for each plate were consistently above 95% and 99.5%, respectively (Table 1 and SI Figure S2A). When summarized by column, survival ranged from 87.5 to 100%, while image success rates ranged from 93.3 to 100% (SI Table S4). While total body area was consistent within and across control plates (SI Figure S2B), spontaneous activity within and across control plates was variable, with an average percent by column ranging from 36.3 to 63.4% across all three plates (Figures 1 and SI S2C).
Table 1. Image Success Rates for Analysis of Spontaneous Activity and Total Body Area within Control 25-hpf Zebrafish Embryos Across Three Independent Plates Containing an Initial Sample Size of 192 Embryos Per Platea
| live embryos | spontaneous activity | total body area | ||||
|---|---|---|---|---|---|---|
| control plate | total (no.) | total (%) | analyzed (no.) | analyzed (%) | analyzed (no.) | analyzed (%) |
| 1 | 192 | 100.0 | 192 | 100.0 | 192 | 100.0 |
| 2 | 185 | 96.4 | 185 | 100.0 | 184 | 99.5 |
| 3 | 192 | 100.0 | 191 | 99.5 | 191 | 99.5 |
a
The percentage of live embryos was relative to an initial sample size of 192 embryos per plate, whereas the percentage of analyzed embryos was relative to the number of live embryos.
Figure 1
Figure 1. Spontaneous activity (%) following exposure to 50 μL embryo media from 5 to 25 hpf in a 384-well plate containing 192 initial embryos. Spontaneous activity (%) data are presented as mean ± SD across three independent control plates. Numbers above each bar denote percent spontaneous activity within that column.
On the basis of performance within control plates, we then developed decision criteria for assay success (SI Figure S3). The percent of embryos with spontaneous activity per column ranged from 18.75% to 75% across all three plates (36 columns total), with the majority of columns ranging from 25 to 74% (Figure S4). Therefore, based on these data, we concluded that spontaneous activity within each control group (columns 1 and 23 for chemical screening assays) must be >18% in order to be considered a successful plate (SI Figure S3). Additionally, spontaneous activity within columns 1 and 23 must not be significantly different (p > 0.05) from each other based on a Mann–Whitney test. If both criteria were met, then the entire plate was then analyzed for total body area and spontaneous activity (SI Figure S3).
HCS Assay Reproducibility
On the basis of previously published data, nicotine and paraoxon were selected as reference chemicals to evaluate assay reproducibility. (22, 23) Nicotine exposure resulted in no effect on survival, total body area, or spontaneous activity (SI Figure S5). However, in the absence of an effect on survival and total body area at all concentrations tested (SI Figures S6A and S6B), paraoxon exposure resulted in a concentration-dependent decrease in the percent of embryos with spontaneous activity at 12.5, 25, and 50 μM (Figures 2A and SI S6C). On the basis of this concentration–response test, three reference plates containing 96 initial embryos per treatment were then exposed to vehicle or 25 μM paraoxon. Percent survival, total body area, and image success rates of paraoxon-treated embryos were similar to vehicle controls (SI Figures S7A and S7B and Table 2), and the hypoactive effect of paraoxon on spontaneous activity was reproducible within and across all three reference plates (Figures 2B and SI S7C). On the basis of results of control and reference chemical plates, we concluded that this assay is reproducible based on sample sizes of 16 initial embryos per column.
Table 2. Image Success Rates for Analysis of Spontaneous Activity and Total Body Area within Reference Chemical Plates Consisting of 25-hpf Zebrafish Exposed to Vehicle or 25 μM Paraoxona
| live embryos | spontaneous activity | total body area | |||||
|---|---|---|---|---|---|---|---|
| plate no. | treatment | total (no.) | total (%) | analyzed (no.) | analyzed (%) | analyzed (no.) | analyzed (%) |
| 1 | vehicle (0.1% DMSO) | 93 | 96.9 | 93 | 100.0 | 92 | 98.9 |
| 25 μM paraoxon | 93 | 96.9 | 93 | 100.0 | 91 | 97.8 | |
| 2 | vehicle (0.1% DMSO) | 95 | 99.0 | 95 | 100.0 | 95 | 100.0 |
| 25 μM paraoxon | 93 | 96.9 | 93 | 100.0 | 91 | 97.8 | |
| 3 | vehicle (0.1% DMSO) | 95 | 99.0 | 95 | 100.0 | 92 | 96.8 |
| 25 μM paraoxon | 95 | 99.0 | 95 | 100.0 | 94 | 98.9 | |
a
Three independent plates contained an initial sample size of 96 embryos per treatment per plate. The percentage of live embryos was relative to an initial sample size of 96 embryos per treatment per plate, whereas the percentage of analyzed embryos was relative to the number of live embryos.
Figure 2
Figure 2. Spontaneous activity (%) following (A) exposure to paraoxon in a concentration response-format or (B) exposure to vehicle (0.1% DMSO) or 25 μM paraoxon (reference plates) from 5 to 25 hpf in a 384-well plate containing 192 initial embryos. Paraoxon significantly decreased spontaneous activity in the absence of effects on survival and total body area (SI Figures S6A, S6B, S7A, and S7B). Spontaneous activity (%) data in Panel B are presented as mean ± SD across three independent reference plates. Numbers above each bar denote percent spontaneous activity within that column. Asterisk denotes significant difference from both vehicle control columns 1 and 23 (p < 0.05).
Chemical Screening
Following assay development and optimization, we tested a small chemical library to evaluate the potential utility of this HCS assay for screening and prioritizing chemicals for DNT testing. On the basis of data available from previous teratogenesis screens using zebrafish embryos, (24) acute toxicity benchmarks (AC50) for chemicals within the U.S. Environmental Protection Agency’s (EPA’s) ToxCast phase-I chemical library were ranked from most to least potent based on survival and gross malformations present at 6 dpf following a 8-hpf to 5-dpf exposure. Using protocols described in Figure 1, the most potent 18 chemicals (SI Table S1, excluding nicotine and paraoxon) were selected to evaluate the ability of this assay to detect targeted effects on spontaneous tail contractions. Two chemicals (tefluthrin and milbemectin) were not commercially available (as of August 2013), and fentin was insoluble in DMSO at concentrations as low as 2.5 mM. Therefore, these three chemicals were not included in the final screen, resulting in a total of 15 chemicals tested in a concentration–response format.
A summary of statistical results and overall effects on spontaneous activity for all chemicals screened is provided within SI Table S5. Of the chemicals tested, rotenone was the most acutely toxic based on survival and total body area, as 0.09 μM was the highest concentration analyzed due to a significant decrease in total body area at 0.19 μM and 0% survival in all other concentrations (SI Figure S8). On the contrary, butafenacil, flumetralin, propargite, tribufos, and pyraflufen-ethyl showed no evidence of acute toxicity in this assay, with no effect on survival or total body area across all concentrations tested (0.09–50 μM) (SI Figures S11, S13, S16, S19, S22). Approximately one-third of the chemicals screened (thiram, fluthiacet-methyl, pyraclostrobin, (Z,E)-fenpyroximate, and trifloxystrobin) resulted in steep concentration–response curves based on survival, where only a 2-fold increase in concentration was required to decrease survival from >85% to 0% (SI Figures S9, S14, S17, S18, S23).
To determine the potential for DNT, spontaneous activity was assessed within treatment groups with >85% survival and no significant decrease in mean total body area relative to vehicle controls. On the basis of our chemical screen, exposure to abamectin—a widely used insecticide and chloride channel activator belonging to the avermectin class—resulted in a significant decrease in spontaneous activity at concentrations as low as 3.125 μM (Figures 3A and SI S15C). Out of all 15 chemicals screened, abamectin was the only chemical that affected spontaneous activity in the absence of effects on gross embryonic development (SI Figures S15A and S15B). On the basis of these data, the entire ToxCast phase-I chemical library was then searched for other commercially available avermectins to determine whether exposure to chemicals within the same class resulted in similar adverse effects on spontaneous activity. On the basis of this search, emamectin benzoate was screened and, similar to abamectin, significantly decreased the percent of embryos with spontaneous activity (at 25 and 50 μM) in the absence of effects on gross embryonic development, albeit with approximately 8-fold less potency than abamectin (Figures 3B and SI S24A-C).
Figure 3
Figure 3. Spontaneous activity (%) following exposure to (A) abamectin or (B) emamectin benzoate from 5 to 25 hpf in a 384-well plate containing 192 initial embryos. Both chemicals significantly decreased spontaneous activity in the absence of effects on survival and total body area (SI Figures S15A, S15B, S24A, and S24B). Numbers above each bar denote percent spontaneous activity within that column. Asterisk denotes significant difference from both vehicle control columns 1 and 23 (p < 0.05).
Discussion
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Although assays quantifying zebrafish locomotion have been developed, the HCS assay reported here provides a simpler discovery platform for identifying potential developmental neurotoxicants. Using 384-well plates, we were able to (1) increase sample size to 16 individual embryos per treatment (192 embryos total), (2) examine a broad concentration–response (vehicle control and 10 chemical concentrations), (3) decrease assay duration and test material, and (4) identify chemicals that target the developing nervous system at nonteratogenic concentrations. After confirming assay variability and reproducibility using control plates and reference chemicals known to affect spontaneous activity, we then screened 16 chemicals within the US EPA’s ToxCast phase-I chemical library and found that exposure to abamectin and emamectin benzoate—both potent avermectins—abolished spontaneous activity in the absence of effects on survival and total body area.
Contrary to other vertebrates, zebrafish neural tube formation and central nervous system development overlaps with the segmentation period (10–24 hpf). (20) By 18 hpf (18-somite stage), the anterior neural tube develops into ten distinct brain neuromeres while the posterior neural tube develops into the spinal cord. (20) Within each brain neuromere and spinal cord segment, primary interneurons differentiate and extend axons to form a simple scaffold along the anteroposterior and dorsoventral axis. Within the spinal cord, the axons of primary motoneurons innervate target axial muscles in a highly stereotyped, error-free manner, (13) resulting in initiation of weak, spontaneous muscle contractions within the developing embryo. (20) This form of locomotion is controlled by neural networks within the spinal cord, as brain lesions nor isolation of the spinal cord from descending brain inputs do not affect this behavior. (25) Although spontaneous activity has been suggested to occur in the absence of external cues such as touch, (26, 27) there is evidence that spontaneous activity may be affected by high-intensity light stimuli. (15) Despite controlling for developmental stage, temperature, and light conditions, we observed a high degree of natural variability in the percent of control embryos exhibiting spontaneous activity. We attempted to minimize this variation by loading two embryos per well and extending the imaging duration per embryo; however, these changes had no impact on minimizing variation of this behavioral phenotype (data not shown). Therefore, since parameters were tightly controlled within our assay, the environmental or biological factors responsible for variation observed in our assay are currently unclear.
Despite the natural variation in control spontaneous activity, our assay detected a significant decrease in spontaneous activity after exposure to paraoxon, and we demonstrated that this effect was reproducible across reference plates. However, contrary to previous studies in zebrafish, (23) nicotine did not affect spontaneous activity, a discrepancy that was likely due to differences in experimental design and exposure duration. In a study conducted by Thomas et al., (23) chorionated zebrafish embryos were incubated from fertilization to 23 hpf in 50-mm Petri dishes and placed in groups of three embryos per dish. Embryos were then transferred to 30 μM nicotine for 5 min, and an increase in the number of spontaneous tail contractions was observed over this 5-min imaging period under transmitted light. In our assay, chorionated embryos were individually exposed to treatment solution from 5 to 25 hpf within single wells of a 384-well plate, and then imaged for 6 sec each under fluorescence. Therefore, the absence of a nicotine-induced effect on spontaneous activity within our assay may be a result of different rearing conditions, extended exposure duration, decreased image acquisition duration, and/or the use of fluorescence rather than transmitted light.
Out of 16 ToxCast phase-I chemicals screened, exposure to 14 of these chemicals resulted in no impact on spontaneous activity using this assay. Out of these 14 chemicals, five chemicals had no impact on survival, total body area, or spontaneous activity up to 50 μM (the highest concentration tested), suggesting that these chemicals exhibited limited embryonic uptake—due to high rates of chemical absorption to microplate wells or low rates of chemical transport across the chorion—and/or negligible toxicity during this stage of development. However, exposure to nine out of these 14 chemicals resulted in significant effects on survival or total body area at one or more concentrations yet did not affect spontaneous activity at nonteratogenic concentrations, suggesting that the primitive nervous system during this stage of development was not a target organ for these nine chemicals.
Using this HCS assay, we discovered that exposure to abamectin and emamectin benzoate—both potent avermectins—significantly decreased spontaneous activity in the absence of effects on survival and total body area. Avermectins are primarily used for control of insects and helminthes (28) and are thought to induce neurotoxicity by activation of either glutamate- or gamma aminobutyric acid (GABA)-gated chloride channels, interfering with signal transmission between nerve cells. (29, 30) Although registered in the U.S. since the 1980s, (30) to our knowledge very few studies have examined the neurotoxic effects of avermectin exposure on mammalian cells and model organisms, particularly during early development. In mouse neuroblastoma N2a cells, exposure to abamectin and doramectin resulted in a significant concentration-dependent inhibition of neurite growth. (31) In zebrafish, semistatic exposure to abamectin resulted in a decrease in swimming ability at 96-hpf. (32) Most importantly, within a DNT study similar to OECD Test Guideline 426, (6) exposure of mated female Sprague–Dawley rats to emamectin benzoate from gestation day 6 through lactation day 20 resulted in a dose-dependent decrease in motor activity in male and female offspring on postnatal day (PND) 17 but not PND 21; this decrease was also observed on PND 59 in females only. (33) Therefore, on the basis of our results and limited data available within the published literature, exposure to avermectins appears to result in disruption of nervous system function during early development.
In summary, this HCS assay provides a streamlined platform with sufficient replication and exposure concentrations to screen and identify potential developmental neurotoxicants. Given that we relied on an embryonic stage (25 hpf) with a less developed nervous system compared to later larval stages (6–7 dpf), we recognize that our assay has the potential to be biased toward identifying chemicals with specific neurotoxic modes-of-action. However, compared to existing zebrafish-based locomotion assays conducted within 96-well plates, (34) this assay provides shorter exposure duration (1 day vs 6 days) and decreased treatment solution volumes needed for screening (50 μL vs 250 μL per well for static exposures). As a result, within a tiered testing framework, this assay has the potential for rapid, cost-effective prioritization of chemicals for DNT assays that rely on later stages of zebrafish and rodent development. Moreover, in the future, this HCS assay can be readily coupled with reverse genetics approaches to identify the potential role of aberrant neurotransmitter signaling in chemically induced effects on early neurodevelopment within zebrafish embryos. Over the long-term, we envision that this HCS assay will help uncover mechanisms of DNT for pesticides and understudied high-production volume chemicals in commerce.
Supporting Information
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Supplemental File 1: Chemical names, chemical formulas, CAS registry numbers, vendors, and purities (Table S1); imaging and analysis parameters (Tables S2–S3); control image success rates (Table S4); a summary of chemical screening results (Table S5); figures for assay optimization (Figures S1–S7); and figures for chemical screening (Figures S8–S24). Microsoft Excel spreadsheets containing raw data for all assays are provided within Supplemental File 2. This information is available free of charge via the Internet at http://pubs.acs.org.
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Acknowledgment
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Funding was provided by a U.S. Environmental Protection Agency Science to Achieve Results (STAR) Grant No. R835169 to D.C.V. The contents of this manuscript are solely the responsibility of D.C.V. and do not necessarily represent the official views of the US EPA. Further, the US EPA does not endorse the purchase of any commercial products or services mentioned in the publication. We gratefully thank Dr. Robert Tanguay (Oregon State University) for providing founder fish to establish our fli1:egfp zebrafish colony, Sylvia de Bruin (Molecular Devices) for providing technical assistance during custom journal script development, and Wilant van Giessen for providing assistance during optimization of EthoVision XT 9.0 analysis parameters.
References
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This article references 34 other publications.
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- 2Giordano, G.; Costa, L. G. Developmental neurotoxicity: Some old and new issues ISRN Toxicol. 2012, 814795[CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XpsFymsL4%253D&md5=a6f815ab935b92f12fddd27ddd3d8a90Developmental neurotoxicity: some old and new issuesGiordano, Gennaro; Costa, Lucio G.ISRN Toxicology (2012), (), 814795, 12 pp.CODEN: ITSOCH; ISSN:2090-6196. (International Scholarly Research Network)The developing central nervous system is often more vulnerable to injury than the adult one. Of the almost 200 chems. known to be neurotoxic, many are developmental neurotoxicants. Exposure to these compds. in utero or during childhood can contribute to a variety of neurodevelopmental and neurol. disorders. Two established developmental neurotoxicants, methylmercury and lead, and two classes of chems., the polybrominated di-Ph ether flame retardants and the organophosphorus insecticides, which are emerging as potential developmental neurotoxicants, are discussed in this paper. Developmental neurotoxicants may also cause silent damage, which would manifest itself only as the individual ages, and may contribute to neurodegenerative diseases such as Parkinson's or Alzheimer's diseases. Guidelines for developmental neurotoxicity testing have been implemented, but there is still room for their improvement and for searching and validating alternative testing approaches.
- 3Icenogle, L. M.; Christopher, N. C.; Blackwelder, W. P.; Caldwell, D. P.; Qiao, D.; Seidler, F. J.; Slotkin, T. A.; Levin, E. D. Behavioral alterations in adolescent and adult rats caused by a brief subtoxic exposure to chlorpyrifos during neurulation Neurotoxicol. Teratol. 2004, 26 (1) 95– 101[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhslWmu7s%253D&md5=73c947bcbc582b8e6efc884dcb0881f4Behavioral alterations in adolescent and adult rats caused by a brief subtoxic exposure to chlorpyrifos during neurulationIcenogle, Laura M.; Christopher, N. Channelle; Blackwelder, W. Paul; Caldwell, D. Patrick; Qiao, Dan; Seidler, Frederic J.; Slotkin, Theodore A.; Levin, Edward D.Neurotoxicology and Teratology (2004), 26 (1), 95-101CODEN: NETEEC; ISSN:0892-0362. (: Elsevier Inc.)The widely used organophosphate insecticide, chlorpyrifos (CPF), elicits neurobehavioral abnormalities after apparently subtoxic neonatal exposures. In the current study, the authors administered 1 or 5 mg/kg/day of CPF to pregnant rats on gestational days 9-12, the embryonic phase spanning the formation and closure of the neural tube. Although there were no effects on growth or viability, offspring showed behavioral abnormalities when tested in adolescence and adulthood. In the CPF-exposed groups, locomotor hyperactivity was noted in early T-maze trials, and in the elevated plus-maze; alterations in the rate of habituation were also identified. Learning and memory were adversely affected, as assessed using the 16-arm radial maze. Although all CPF-exposed animals eventually learned the task, ref. and working memory were impaired in the early training sessions. After training, rats in the CPF group did not show the characteristic amnestic effect of scopolamine, a muscarinic acetylcholine antagonist, suggesting that, unlike the situation in the control group, muscarinic pathways were not used to solve the maze. These results indicate that apparently subtoxic CPF exposure during neurulation adversely affects brain development, leading to behavioral anomalies that selectively include impairment of cholinergic circuits used in learning and memory. The resemblance of these findings to those of late gestational or neonatal CPF exposure indicates a prolonged window of vulnerability of brain development to CPF.
- 4Burbacher, T. M.; Rodier, P. M.; Weiss, B. Methylmercury developmental neurotoxicity: A comparison of effects in humans and animals Neurotoxicol. Teratol. 1990, 12 (3) 191– 202[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXkslamtb0%253D&md5=078b7d96d95d88961bcd81870117700fMethylmercury developmental neurotoxicity: a comparison of effects in humans and animalsBurbacher, Thomas M.; Rodier, Patricia M.; Weiss, BernardNeurotoxicology and Teratology (1990), 12 (3), 191-202CODEN: NETEEC; ISSN:0892-0362.A review with 74 refs. of qual. and quant. comparison of the neuropathol. and neurobehavioral effects of early methylmercury (MeHg) exposure is presented. The focus of the qual. comparison is the examn. of how specific end-points (and categories of behavioral functions) compare across species. The focus of the quant. comparison is the investigation of the relationship between MeHg exposure, target-organ dose, and effects in humans and animals. The results of the comparisons are discussed in the context of the adequacy of the proposed EPA neurotoxicity battery to characterize the risk of MeHg to humans.
- 5Burns, C. J.; McIntosh, L. J.; Mink, P. J.; Jurek, A. M.; Li, A. A. Pesticide exposure and neurodevelopmental outcomes: Review of the epidemiologic and animal studies J. Toxicol. Environ. Health B Crit. Rev. 2013, 16 (3–4) 127– 283
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- 7Bjorling-Poulsen, M.; Andersen, H. R.; Grandjean, P. Potential developmental neurotoxicity of pesticides used in Europe Environ. Health 2008, 7, 50[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1cjhtlamsg%253D%253D&md5=e4c20bfdce1480f3ef82e026e1a561e2Potential developmental neurotoxicity of pesticides used in EuropeBjorling-Poulsen Marina; Andersen Helle Raun; Grandjean PhilippeEnvironmental health : a global access science source (2008), 7 (), 50 ISSN:.Pesticides used in agriculture are designed to protect crops against unwanted species, such as weeds, insects, and fungus. Many compounds target the nervous system of insect pests. Because of the similarity in brain biochemistry, such pesticides may also be neurotoxic to humans. Concerns have been raised that the developing brain may be particularly vulnerable to adverse effects of neurotoxic pesticides. Current requirements for safety testing do not include developmental neurotoxicity. We therefore undertook a systematic evaluation of published evidence on neurotoxicity of pesticides in current use, with specific emphasis on risks during early development. Epidemiologic studies show associations with neurodevelopmental deficits, but mainly deal with mixed exposures to pesticides. Laboratory experimental studies using model compounds suggest that many pesticides currently used in Europe--including organophosphates, carbamates, pyrethroids, ethylenebisdithiocarbamates, and chlorophenoxy herbicides--can cause neurodevelopmental toxicity. Adverse effects on brain development can be severe and irreversible. Prevention should therefore be a public health priority. The occurrence of residues in food and other types of human exposures should be prevented with regard to the pesticide groups that are known to be neurotoxic. For other substances, given their widespread use and the unique vulnerability of the developing brain, the general lack of data on developmental neurotoxicity calls for investment in targeted research. While awaiting more definite evidence, existing uncertainties should be considered in light of the need for precautionary action to protect brain development.
- 8Crofton, K. M.; Mundy, W. R.; Shafer, T. J. Developmental neurotoxicity testing: A path forward Congenit. Anom. (Kyoto) 2012, 52 (3) 140– 146[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC38bhsF2gsQ%253D%253D&md5=646889dd21cb1ab77c7d7c358f5ee37eDevelopmental neurotoxicity testing: a path forwardCrofton Kevin M; Mundy William R; Shafer Timothy JCongenital anomalies (2012), 52 (3), 140-6 ISSN:.Great progress has been made over the past 40 years in understanding the hazards of exposure to a small number of developmental neurotoxicants. Lead, polychlorinated biphenyls, and methylmercury are all good examples of science-based approaches to characterizing the hazard to the developing nervous systems from environmental contaminants. However, very little effort has been spent to address the challenge of assessing the potential developmental neurotoxic hazard of the thousands of other chemicals in common commercial use. The extensive time, financial and animal resource requirements for current regulatory testing guideline methods make this an untenable solution to this challenge. A new testing paradigm is needed that uses time and cost-efficient methods to screen large numbers of chemicals for developmental neurotoxicity (DNT). In silico models are needed to provide rapid chemical structure-based screening. In vitro techniques are being developed to provide rapid and efficient testing in cell-free and cell-based systems. In addition, the use of alternative species, such as zebrafish, will provide efficient models for testing the effects of chemicals in organisms with intact developing nervous systems. Finally, these methods and models need to be used in an integrated fashion to provide the data needs for hazard assessment in a manner that is problem-driven and cost-efficient. This paper summarizes discussions on these issues from the symposium 'Developmental neurotoxicity testing: Scientific approaches towards the next generation to protecting the developing nervous system of children' held at the 2011 annual meeting of the Japanese Teratology Society.
- 9Coecke, S.; Goldberg, A. M.; Allen, S.; Buzanska, L.; Calamandrei, G.; Crofton, K.; Hareng, L.; Hartung, T.; Knaut, H.; Honegger, P.; Jacobs, M.; Lein, P.; Li, A.; Mundy, W.; Owen, D.; Schneider, S.; Silbergeld, E.; Reum, T.; Trnovec, T.; Monnet-Tschudi, F.; Bal-Price, A. Workgroup report: Incorporating in vitro alternative methods for developmental neurotoxicity into international hazard and risk assessment strategies Environ. Health Perspect. 2007, 115 (6) 924– 931
- 10Crofton, K. M.; Mundy, W. R.; Lein, P. J.; Bal-Price, A.; Coecke, S.; Seiler, A. E.; Knaut, H.; Buzanska, L.; Goldberg, A. Developmental neurotoxicity testing: Recommendations for developing alternative methods for the screening and prioritization of chemicals ALTEX 2011, 28 (1) 9– 15Google ScholarThere is no corresponding record for this reference.
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- 13Myers, P. Z.; Eisen, J. S.; Westerfield, M. Development and axonal outgrowth of identified motoneurons in the zebrafish J. Neurosci. 1986, 6 (8) 2278– 2289[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaL28zgslSitA%253D%253D&md5=64596422be71d49e5736ae868c4708a7Development and axonal outgrowth of identified motoneurons in the zebrafishMyers P Z; Eisen J S; Westerfield MThe Journal of neuroscience : the official journal of the Society for Neuroscience (1986), 6 (8), 2278-89 ISSN:0270-6474.We have observed the development of live, fluorescently labeled motoneurons in the spinal cord of embryonic and larval zebrafish. There are 2 classes of motoneurons: primary and secondary. On each side of each spinal segment there are 3 individually identifiable primary motoneurons, named CaP, MiP, and RoP. The motoneurons of the embryo and larva are similar in morphology and projection pattern to those of the adult. During initial development, axons of primary motoneurons make cell-specific, divergent pathway choices and grow without error to targets appropriate for their adult functions. We observed no period of cell death, and except for one consistently observed case, there was no remodeling of peripheral arbors. We have observed a consistent temporal sequence of axonal outgrowth within each spinal segment. The CaP motor axon is the first to leave the spinal cord, followed by the axons of the other primary motoneurons. The Mauthner growth cone enters the spinal cord after all the primary motoneurons of the trunk spinal cord have begun axonal outgrowth. Secondary motor growth cones appear only after the Mauthner growth cone has passed by. Our results suggest that this stereotyped temporal sequence of axonal outgrowth may play a role in defining the contacts between the Mauthner axon and the motoneurons; the behavior of growth cones in the periphery suggests that interactions with the environment, not timing, may determine path-finding and peripheral connectivity of the motoneurons.
- 14Westerfield, M.; McMurray, J. V.; Eisen, J. S. Identified motoneurons and their innervation of axial muscles in the zebrafish J. Neurosci. 1986, 6 (8) 2267– 2277[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaL28zgslSitw%253D%253D&md5=0ecb023850154e0bafbbd344137471fbIdentified motoneurons and their innervation of axial muscles in the zebrafishWesterfield M; McMurray J V; Eisen J SThe Journal of neuroscience : the official journal of the Society for Neuroscience (1986), 6 (8), 2267-77 ISSN:0270-6474.The organization of spinal cord motoneurons and their innervation of axial (white) muscles in the zebrafish were studied. Motoneurons can be divided into 2 classes, primary and secondary, on the basis of their cell-body sizes and positions. Each side of each spinal segment contains 3 primary motoneurons that are uniquely identifiable as individuals by their stereotyped cell-body positions and peripheral branching patterns. Moreover, these motoneurons precisely innervate cell-specific subsets of contiguous muscle fibers in mutually exclusive regions of their own body segment. Individual muscle fibers receive inputs from a single primary motoneuron and, in addition, from up to 3 secondary motoneurons. The results demonstrate that the precision of innervation previously described in invertebrates is also present in some vertebrates.
- 15Kokel, D.; Bryan, J.; Laggner, C.; White, R.; Cheung, C. Y. J.; Mateus, R.; Healey, D.; Kim, S.; Werdich, A. A.; Haggarty, S. J.; MacRae, C. A.; Shoichet, B.; Peterson, R. T. Rapid behavior-based identification of neuroactive small molecules in the zebrafish Nat. Chem. Biol. 2010, 6 (3) 231– 237[Crossref], [PubMed], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXntVCnuw%253D%253D&md5=0e9b9d4778ea8443dde1d2b860382616Rapid behavior-based identification of neuroactive small molecules in the zebrafishKokel, David; Bryan, Jennifer; Laggner, Christian; White, Rick; Cheung, Chung Yan J.; Mateus, Rita; Healey, David; Kim, Sonia; Werdich, Andreas A.; Haggarty, Stephen J.; MacRae, Calum A.; Shoichet, Brian; Peterson, Randall T.Nature Chemical Biology (2010), 6 (3), 231-237CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Neuroactive small mols. are indispensable tools for treating mental illnesses and dissecting nervous system function. However, it has been difficult to discover novel neuroactive drugs. Here, we describe a high-throughput, behavior-based approach to neuroactive small mol. discovery in the zebrafish. We used automated screening assays to evaluate thousands of chem. compds. and found that diverse classes of neuroactive mols. caused distinct patterns of behavior. These 'behavioral barcodes' can be used to rapidly identify new psychotropic chems. and to predict their mol. targets. For example, we identified new acetylcholinesterase and monoamine oxidase inhibitors using phenotypic comparisons and computational techniques. By combining high-throughput screening technologies with behavioral phenotyping in vivo, behavior-based chem. screens can accelerate the pace of neuroactive drug discovery and provide small-mol. tools for understanding vertebrate behavior.
- 16Irons, T. D.; MacPhail, R. C.; Hunter, D. L.; Padilla, S. Acute neuroactive drug exposures alter locomotor activity in larval zebrafish Neurotoxicol. Teratol. 2010, 32 (1) 84– 90[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXnvFGlsw%253D%253D&md5=e64855a825d03093aefe6117454f9dadAcute neuroactive drug exposures alter locomotor activity in larval zebrafishIrons, T. D.; MacPhail, R. C.; Hunter, D. L.; Padilla, S.Neurotoxicology and Teratology (2010), 32 (1), 84-90CODEN: NETEEC; ISSN:0892-0362. (Elsevier)As part of the development of a rapid in vivo screen for prioritization of toxic chems., we have begun to characterize the locomotor activity of zebrafish (Danio rerio) larvae by assessing the acute effects of prototypic drugs that act on the central nervous system. Initially, we chose ethanol, d-amphetamine, and cocaine, which are known, in mammals, to increase locomotion at low doses and decrease locomotion at higher doses. Wild-type larvae were individually maintained in 96-well microtiter plates at 26 °C, under a 14:10 h light:dark cycle, with lights on at 0830 h. At 6 days post-fertilization, ethanol (1-4% vol./vol.), d-amphetamine sulfate (0.1-20.0 μM) or cocaine hydrochloride (0.2-50.0 μM) were administered to the larvae by immersion. Beginning 20 min into the exposure, locomotion was assessed for each animal for 70 min using 10-min, alternating light (visible light) and dark (IR light) periods. Low concns. of ethanol and d-amphetamine increased activity, while higher concns. of all three drugs decreased activity. Because ethanol effects occurred predominately during the light periods, whereas the d-amphetamine and cocaine effects occurred during the dark periods, alternating lighting conditions proved to be advantageous. These results indicate that zebrafish larvae are sensitive to neuroactive drugs, and their locomotor response is similar to that of mammals.
- 17Stanley, K. A.; Curtis, L. R.; Simonich, S. L. M.; Tanguay, R. L. Endosulfan I and endosulfan sulfate disrupts zebrafish embryonic development Aquat. Toxicol. 2009, 95 (4) 355– 361[Crossref], [PubMed], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsV2gsrjK&md5=85258996885a59d11fe76f36bd43251bEndosulfan I and endosulfan sulfate disrupts zebrafish embryonic developmentStanley, Kerri A.; Curtis, Lawrence R.; Massey Simonich, Staci L.; Tanguay, Robert L.Aquatic Toxicology (2009), 95 (4), 355-361CODEN: AQTODG; ISSN:0166-445X. (Elsevier B.V.)Fish in agricultural and remote areas may be exposed to endosulfan and its degrdn. products as a result of direct runoff, atm. transport and deposition. The following study used the zebrafish developmental model to investigate the responses to endosulfan I and endosulfan sulfate, the major degrdn. product of endosulfan I and II. Embryos were dechorionated and waterborne exposed to the endosulfan I or endosulfan sulfate from 6 to 120 h post-fertilization (hpf). Endosulfan I exposure concns. ranged from 0.01 to 10 μg/L and endosulfan sulfate from 1 to 100 μg/L. Water solns. were renewed every 24 h and fish were scored for overt developmental and behavioral abnormalities. Chem. anal. was performed on water, whole embryo, and larvae samples to det. waterborne exposure concns. and tissue concns. throughout the 5-day period. The most sensitive toxicity endpoint for both endosulfan I and endosulfan sulfate was an abnormal response of the embryo/larvae to touch, suggesting that endosulfan I and sulfate are developmentally neurotoxic. The waterborne exposure EC50s for inhibition of touch response for endosulfan I and endosulfan sulfate were 2.2 μg/L and 23 μg/L, resp. The endosulfans were highly concd. by the organisms, and the inhibition of touch response tissue EC50, detd. from the measured tissue concns., was 367 ng/g for endosulfan I and 4552 ng/g for endosulfan sulfate.
- 18Selderslaghs, I. W. T.; Hooyberghs, J.; Blust, R.; Witters, H. E. Assessment of the developmental neurotoxicity of compounds by measuring locomotor activity in zebrafish embryos and larvae Neurotoxicol. Teratol. 2013, 37, 44– 56[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXotlyqsLo%253D&md5=f6ce2ae4fdfd5a66897c08521c9abffdAssessment of the developmental neurotoxicity of compounds by measuring locomotor activity in zebrafish embryos and larvaeSelderslaghs, Ingrid W. T.; Hooyberghs, Jef; Blust, Ronny; Witters, Hilda E.Neurotoxicology and Teratology (2013), 37 (), 44-56CODEN: NETEEC; ISSN:0892-0362. (Elsevier Inc.)The developmental neurotoxic potential of the majority of environmental chems. and drugs is currently undetd. Specific in vivo studies provide useful data for hazard assessment but are not amenable to screen thousands of untested compds. In this study, methods which use zebrafish embryos, eleutheroembryos and larvae as model organisms, were proposed as alternatives for developmental neurotoxicity (DNT) testing. The evaluation of spontaneous tail coilings in zebrafish embryos aged 24-26 h post fertilization (hpf) and the swimming activity of eleutheroembryos at 120 and larvae at 144 hpf, i.e. parameters for locomotor activity, were investigated as potential endpoints for DNT testing, according to available std. protocols. The overall performance and predictive value of these methods was then examd. by testing a training set of 10 compds., including known developmental neurotoxicants and compds. not considered to be neurotoxic. The classification of the selected compds. as either neurotoxic or non-neurotoxic, based on the effects obsd. in zebrafish embryos and larvae, was compared to available mammalian data and an overall concordance of 90% was achieved. Furthermore, the specificity of the selected endpoints for DNT was evaluated as well as the potential similarities between zebrafish and mammals with regard to mechanisms of action for the selected compds. Although further studies, including the screening of a large testing set of compds. are required, we suggest that the proposed methods with zebrafish embryos and larvae might be valuable alternatives for animal testing for the screening and prioritization of compds. for DNT.
- 19Lawson, N. D.; Weinstein, B. M. In vivo imaging of embryonic vascular development using transgenic zebrafish Dev. Biol. 2002, 248 (2) 307– 318
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- 21NIH (National Institutes of Health). Guidelines for Use of Zebrafish in the NIH Intramural Research Program; Office of Animal Care and Use (OACU): Bethesda, MD, 2013; http://oacu.od.nih.gov/ARAC/documents/Zebrafish.pdf.Google ScholarThere is no corresponding record for this reference.
- 22Yozzo, K. L.; McGee, S. P.; Volz, D. C. Adverse outcome pathways during zebrafish embryogenesis: A case study with paraoxon Aquat. Toxicol. 2013, 126, 346– 354[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsVKrur%252FL&md5=4d1173501dc86473d00076e0e6f0cc7eAdverse outcome pathways during zebrafish embryogenesis: A case study with paraoxonYozzo, Krystle L.; McGee, Sean P.; Volz, David C.Aquatic Toxicology (2013), 126 (), 346-354CODEN: AQTODG; ISSN:0166-445X. (Elsevier B.V.)Using paraoxon as a ref. acetylcholinesterase (AChE) inhibitor, the objective of this study was to develop an adverse outcome pathway (AOP) that provided quant. linkages across levels of biol. organization during zebrafish embryogenesis. Within normal zebrafish embryos, we first demonstrated that ache transcripts and AChE activity increased in a stage-dependent manner following segmentation. We then showed that static exposure of embryos to paraoxon (31.2-500 nM) from 5 to 96 hpf resulted in significant stage- and concn.-dependent AChE inhibition, albeit these effects were fully reversible within 48 h following transfer to clean water. However, even in the presence of significant AChE inhibition, exposure to non-teratogenic paraoxon concns. (≤250 nM) did not adversely impact secondary motoneuron development at 96 hpf. Therefore, we investigated the potential effects of paraoxon exposure on spontaneous tail contractions at 26 hpf - an early locomotor behavior that results from innervation of primary (not secondary) motoneuron axons to target axial muscles. Based on these studies, the frequency of spontaneous tail contractions at 26 hpf - a developmental stage with minimal AChE expression and activity - was significantly higher following exposure to paraoxon concns. as low as 31.2 nM. Overall, our data suggest that (1) normal AChE activity is not required for secondary motoneuron development and (2) spontaneous tail contractions at 26 hpf are sensitive to paraoxon exposure, an effect that may be independent of AChE inhibition. Using a well-studied ref. chem., this study highlights the potential challenges in developing quant. AOPs to support chem. screening and prioritization strategies.
- 23Thomas, L. T.; Welsh, L.; Galvez, F.; Svoboda, K. R. Acute nicotine exposure and modulation of a spinal motor circuit in embryonic zebrafish Toxicol. Appl. Pharmacol. 2009, 239 (1) 1– 12[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXptVCju74%253D&md5=4cdf61c877d7320ddfd2daa25d1636adAcute nicotine exposure and modulation of a spinal motor circuit in embryonic zebrafishThomas, Latoya T.; Welsh, Lillian; Galvez, Fernando; Svoboda, Kurt R.Toxicology and Applied Pharmacology (2009), 239 (1), 1-12CODEN: TXAPA9; ISSN:0041-008X. (Elsevier B.V.)The zebrafish model system is ideal for studying nervous system development. Ultimately, one would like to link the developmental biol. to various aspects of behavior. The authors are studying the consequences of nicotine exposure on nervous system development in zebrafish and have previously shown that chronic nicotine exposure produces paralysis. The authors also have made observations that the embryos moved in the initial minutes of the exposure as the bend rates of the musculature increased. This nicotine-induced behavior manifests as an increase in the rate of spinal musculature bends, which spontaneously begin at ∼18 h postfertilization. The behavioral observations prompted the systematic characterization of nicotine-induced modulation of zebrafish embryonic motor output; bends of the trunk musculature. The authors first characterized embryonic motor output in zebrafish embryos with and without their chorions. They then characterized the motor output in embryos raised at 28° and 25°. The act of dechorionation along with temp. influenced the embryonic bend rate. Nicotine exposure increased embryonic motor output. Nicotine exposure caused the musculature bends to alternate in a left-right-left fashion. Nicotine was able to produce this phenotype in embryos lacking supraspinal input. The authors then characterized the kinetics of nicotine influx and efflux and demonstrated that nicotine ≥1 μM can disrupt embryonic physiol. Taken together, these results indicate the presence of nicotinic acetylcholine receptors (nAChRs) assocd. with embryonic spinal motor circuits early in embryogenesis.
- 24Padilla, S.; Corum, D.; Padnos, B.; Hunter, D. L.; Beam, A.; Houck, K. A.; Sipes, N.; Kleinstreuer, N.; Knudsen, T.; Dix, D. J.; Reif, D. M. Zebrafish developmental screening of the ToxCast (TM) Phase I chemical library Reprod. Toxicol. 2012, 33 (2) 174– 187[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xkt1Ggtrg%253D&md5=27fa06df73fd73941a8fc10865e7ce67Zebrafish developmental screening of the ToxCast Phase I chemical libraryPadilla, S.; Corum, D.; Padnos, B.; Hunter, D. L.; Beam, A.; Houck, K. A.; Sipes, N.; Kleinstreuer, N.; Knudsen, T.; Dix, D. J.; Reif, D. M.Reproductive Toxicology (2012), 33 (2), 174-187CODEN: REPTED; ISSN:0890-6238. (Elsevier Inc.)Zebrafish (Danio rerio) is an emerging toxicity screening model for both human health and ecol. As part of the Computational Toxicol. Research Program of the U. S. EPA, the toxicity of the 309 ToxCast Phase I chems. was assessed using a zebrafish screen for developmental toxicity. All exposures were by immersion from 6-8 h post fertilization (hpf) to 5 days post fertilization (dpf); nominal concn. range of 1 nM-80 μM. On 6 dpf larvae were assessed for death and overt structural defects. Results revealed that the majority (62%) of chems. were toxic to the developing zebrafish; both toxicity incidence and potency was correlated with chem. class and hydrophobicity (logP); and inter-and intra-plate replicates showed good agreement. The zebrafish embryo screen, by providing an integrated model of the developing vertebrate, compliments the ToxCast assay portfolio and has the potential to provide information relative to overt and organismal toxicity.
- 25Downes, G. B.; Granato, M. Supraspinal input is dispensable to generate glycine-mediated locomotive behaviors in the zebrafish embryo J. Neurobiol. 2006, 66 (5) 437– 451[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XlsFCqsLo%253D&md5=18741cd2cba91d3b3049c4379be24f32Supraspinal input is dispensable to generate glycine-mediated locomotive behaviors in the zebrafish embryoDownes, Gerald B.; Granato, MichaelJournal of Neurobiology (2006), 66 (5), 437-451CODEN: JNEUBZ; ISSN:0022-3034. (John Wiley & Sons, Inc.)The anatomy of the developing zebrafish spinal cord is relatively simple but, despite this simplicity, it generates a sequence of three patterns of locomotive behaviors. The first behavior exhibited is spontaneous movement, then touch-evoked coiling, and finally swimming. Previous studies in zebrafish have suggested that spontaneous movements occur independent of supraspinal input and do not require chem. neurotransmission, while touch-evoked coiling and swimming depend on glycinergic neurotransmission as well as supraspinal input. In contrast, studies in other vertebrate prepns. have shown that spontaneous movement requires glycine and other neurotransmitters and that later behaviors do not require supraspinal input. Here, the authors use lesion anal. combined with high-speed kinematic anal. to re-examine the role of glycine and supraspinal input in each of the three behaviors. The authors find that, similar to other vertebrate prepns., supraspinal input is not essential for spontaneous movement, touch-evoked coiling, or swimming behavior. Moreover, the authors find that blockade of glycinergic neurotransmission decreases the rate of spontaneous movement and impairs touch-evoked coiling and swimming, suggesting that glycinergic neurotransmission plays crit. yet distinct roles for individual patterns of locomotive behaviors.
- 26Saint-Amant, L.; Drapeau, P. Time course of the development of motor behaviors in the zebrafish embryo J. Neurobiol. 1998, 37 (4) 622– 632[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK1M%252Fntl2ktg%253D%253D&md5=e5fca7135b1e9d9fca2798f33851495cTime course of the development of motor behaviors in the zebrafish embryoSaint-Amant L; Drapeau PJournal of neurobiology (1998), 37 (4), 622-32 ISSN:0022-3034.The development and properties of locomotor behaviors in zebrafish embryos raised at 28.5 degrees C were examined. When freed from the chorion, embryonic zebrafish showed three sequential stereotyped behaviors: a transient period of alternating, coiling contractions followed by touch-evoked rapid coils, then finally, organized swimming. The three different behaviors were characterized by video microscopy. Spontaneous, alternating contractions of the trunk appeared suddenly at 17 h postfertilization (hpf), with a frequency of 0.57 Hz, peaked at 19 hpf at 0.96 Hz, and gradually decreased to <0.1 Hz by 27 hpf. Starting at 21 hpf, touching either the head or the tail of the embryos resulted in vigorous coils. The coils accelerated with development, reaching a maximum speed of contraction before 48 hpf, which is near the time of hatching. After 27 hpf, touching the embryos, particularly on the tail, could induce partial coils (instead of full coils). At this time, embryos started to swim in response to a touch, preferentially to the tail. The swim cycle frequency gradually increased with age from 7 Hz at 27 hpf to 28 Hz at 36 hpf. Lesions of the central nervous system rostral to the hindbrain had no effect on the three behaviors. Lesioning the hindbrain eliminated swimming and touch responses, but not the spontaneous contractions. Our observations suggest that the spontaneous contractions result from activation of a primitive spinal circuit, while touch and swimming require additional hindbrain inputs to elicit mature locomotor behaviors.
- 27Brustein, E.; Saint-Amant, L.; Buss, R.; Chong, M.; McDearmid, J.; Drapeau, P. Steps during the development of the zebrafish locomotor network J. Physiol.-Paris 2003, 97 (1) 77– 86[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2c%252FhtVejsg%253D%253D&md5=1b231f84692577877699e1b0c995a77fSteps during the development of the zebrafish locomotor networkBrustein Edna; Saint-Amant Louis; Buss Robert R; Chong Mabel; McDearmid Jonathan R; Drapeau PierreJournal of physiology, Paris (2003), 97 (1), 77-86 ISSN:0928-4257.This review summarizes recent data from our lab concerning the development of motor activities in the developing zebrafish. The zebrafish is a leading model for studies of vertebrate development because one can obtain a large number of transparent, externally and rapidly developing embryos with motor behaviors that are easy to assess (e.g. for mutagenic screens). The emergence of embryonic motility was studied behaviorally and at the cellular level. The embryonic behaviors appear sequentially and include an early, transient period of spontaneous, alternating tail coilings, followed by responses to touch, and swimming. Patch clamp recording in vivo revealed that an electrically coupled network of a subset of spinal neurons generates spontaneous tail coiling, whereas a chemical (glutamatergic and glycinergic) synaptic drive underlies touch responses and swimming and requires input from the hindbrain. Swimming becomes sustained in larvae once serotonergic neuromodulatory effects are integrated. We end with a brief overview of the genetic tools available for the study of the molecular determinants implicated in locomotor network development in the zebrafish. Combining genetic, behavioral and cellular experimental approaches will advance our understanding of the general principles of locomotor network assembly and function.
- 28Taylor, M. A. Recent developments in ectoparasiticides Vet. J. 2001, 161 (3) 253– 268[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXjslSlt7g%253D&md5=eff972ab22d8d5bde11ad60a335d3f4dRecent developments in ectoparasiticidesTaylor, M. A.Veterinary Journal (2001), 161 (3), 253-268CODEN: VTJRFP; ISSN:1090-0233. (Bailliere Tindall Ltd.)A review with many refs. The sales and use of ectoparasiticides for the control of arthropod parasites of domestic animals constitute a major sector of the global animal health market. Animals are infected by a no. of parasitic insect and acarine species causing major economic losses in prodn. livestock, intense irritation and skin disease in companion animals, or public health issues, including bites of humans or zoonotic disease transmission. Dog and cat fleas, for example, can be a serious source of both animal and human irritation, which has led to a rapid expansion in the development of flea control products. The control of ectoparasite infections of veterinary importance still relies heavily on the use of chems. that target the arthropod nervous system. Such compds. have suffered from a no. of drawbacks, including the development of resistance and concerns over human and environmental safety. The search for safer technologies has, however, been hindered by the limited no. of active target sites present in arthropods and, to some degree, by the ever-increasing costs of research and development of compds. with novel modes of action. This review provides a background to the currently available groups of ectoparasiticide compds. used in veterinary medicine and highlights some of the more recent developments including the introduction of insect growth regulators and new and improved methods of product application.
- 29Bloomquist, J. R. Ion channels as targets for insecticides Annu. Rev. Entomol. 1996, 41, 163– 190[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XksVyrsQ%253D%253D&md5=2e9f9d8ebf2dba6e9fe54fe7a787d44aIon channels as targets for insecticidesBloomquist, Jeffrey R.Annual Review of Entomology (1996), 41 (), 163-90CODEN: ARENAA; ISSN:0066-4170. (Annual Reviews)A review with 120 refs. Ion channels are the primary target sites for several classes of natural and synthetic insecticidal compds. The voltage-sensitive sodium channel is the major target site for DDT and pyrethroids, the veratrum alkaloids, and N-alkyl-amides. Recently, neurotoxic proteins from arthropod venoms, some of which specifically attack insect sodium channels, have been engineered into baculoviruses to act as biopesticides. The synthetic pyrazolines also primarily affect the sodium channel, although some members of this group target neuronal calcium channels as well. The ryanoids have also found use as insecticides, and these materials induce muscle contracture by irreversible activation of the calcium-release channel of the sarcoplasmic reticulum. The arylheterocycles (e.g. endosulfan and fipronil) are potent convulsants and insecticides that block the GABA-gated chloride channel. In contrast, the avermectins activate both ligand- and voltage-gated chloride channels, which leads to paralysis. At field-use rates, a neurotoxic effect of the ecdysteroid agonist RH-5849 is obsd. that involves blockage of both muscle and neuronal potassium channels. The future use of ion channels as targets for chem. and genetically engineered insecticides is also discussed.
- 30US EPA. Ecological Risk Assessment for Abamectin; Office of Prevention, Environmental Fate and Effects Division: Washington, D.C., 2004.Google ScholarThere is no corresponding record for this reference.
- 31Sun, Y. J.; Long, D. X.; Li, W.; Hou, W. Y.; Wu, Y. J.; Shen, J. Z. Effects of avermectins on neurite outgrowth in differentiating mouse neuroblastoma N2a cells Toxicol. Lett. 2010, 192 (2) 206– 211[Crossref], [PubMed], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXotVCjtw%253D%253D&md5=6dc6e48b88d8fa9d17e1e98582113b28Effects of avermectins on neurite outgrowth in differentiating mouse neuroblastoma N2a cellsSun, Ying-Jian; Long, Ding-Xin; Li, Wei; Hou, Wei-Yuan; Wu, Yi-Jun; Shen, Jian-ZhongToxicology Letters (2010), 192 (2), 206-211CODEN: TOLED5; ISSN:0378-4274. (Elsevier Ireland Ltd.)Avermectins (AVMs) are macrocyclic lactone compds. that have been widely used as parasiticides in veterinary and human medicine and as pesticides in agriculture and horticulture. The multidrug resistance transporter, P-glycoprotein (P-gp), is assocd. with the efflux transport of AVMs and other drugs across the blood-brain and placental barrier, and plays an important role in attenuating the neurotoxicity and developmental toxicity of AVMs. In this study, the mouse neuroblastoma N2a cell line was used to investigate the neurotoxicity of two AVM derivs.: abamectin (ABM) and doramectin (DOR). We found that both these compds. caused significant dose-dependent inhibition of neurite growth in differentiating N2a cells. In addn., Western blotting anal. showed that ABM and DOR significantly inhibited the expression of not only P-gp but also the cytoskeletal proteins, β-actin and β-tubulin. This suggests ABM and DOR may inhibit neurite growth by down-regulating the expression of P-gp and cytoskeletal proteins. Furthermore, knockdown of P-gp expression by RNA interference in N2a cells reduced neurite growth even in the absence of ABM and DOR, and reduced it even more in the presence of low levels of these compds. These results suggest that even subcytotoxic levels of ABM and DOR can be neurotoxic in differentiating cells and that this neurotoxicity may, at least in part, be the result of the down-regulation of P-gp and cytoskeletal proteins.
- 32Tisler, T.; Erzen, N. K. Abamectin in the aquatic environment Ecotoxicology 2006, 15 (6) 495– 502
- 33Wise, L. D.; Allen, H. L.; Hoe, C. M. L.; Verbeke, D. R.; Gerson, R. J. Developmental neurotoxicity evaluation of the avermectin pesticide, emamectin benzoate, in Sprague-Dawley rats Neurotoxicol. Teratol. 1997, 19 (4) 315– 326[Crossref], [PubMed], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXkvFGkt7Y%253D&md5=fa9ebd4b3f5f323a09b4c72226d1aad5Developmental neurotoxicity evaluation of the avermectin pesticide, emamectin benzoate, in Sprague-Dawley ratsWise, L. David; Allen, Henry L.; Hoe, Chao-Min L.; Verbeke, David R.; Gerson, Ronald J.Neurotoxicology and Teratology (1997), 19 (4), 315-326CODEN: NETEEC; ISSN:0892-0362. (Elsevier)The potential of emamectin benzoate (EB) to cause developmental neurotoxicity in Sprague-Dawley rats was assessed using a study design proposed by the US EPA. Dosages of 0 (deionized water), 0.1, 0.6, or 3.6 mg/kg/day were administered at 5 mL/kg by oral gavage from gestational day (GD) 6 to lactational day (LD) 20 groups of 25 mated females each. Between GD 17 and 20 the high dose was reduced to 2.5 mg/kg/day because of pup tremors obsd. at this dose level in a concurrent two-generation study. Females were allowed to deliver and the young were evaluated for survival, growth, development, behavior, and histol. changes to brain, spinal cord, peripheral nerve, and skeletal muscle. Behavioral assessment of the offspring consisted of open field motor activity, auditory startle habituation, and passive avoidance tests; each was conducted on weanling and adult animals (one animal/sex/litter). Histopathol. examn. of the CNS and PNS was conducted on one animal/sex/litter on postnatal days (PND) 11 and 60. There were significant increases in av. F0 maternal body wt. gains during gestation in the 0.6 and 3.6/2.5 mg/kg/day groups, but no other effects were obsd. in pregnant females of these or the low-dose groups during the study. Beginning on PND 6, tremors were obsd. in high-dose pups, and this was followed by hindlimb splay in all high-dose pups by PND 15-26. Both of these phys. signs disappeared by PND 34 (i.e., 10-11 days after weaning). There were no compd.-related deaths in F1 offspring. Beginning on PND 11, progressive decreases in preweaning av. wts. were obsd. in the high-dose group (to 42% below control in females on PND 21). Av. wt. gain during the postweaning period was significantly decreased in the 3.6/2.5 mg/kg/day group. There were EB-related effects in behavioral tests only in the high-dose group. A significant increase in PND 13 av. horizontal motor activity was due to stereotypical movements. Av. horizontal activity was decreased on PND 17 and in adult females, but there were no effects on PND 21. Av. peak auditory startle response amplitude was decreased on PND 22 and in adults. There were no EB-related effects in the passive avoidance test, relative brain wts., or in the histol. examn. (including morphometry) of the nervous system. These results demonstrate that the high-dose EB exposure during gestation and lactation to rats produced evidence of neurotoxicity in the F1 offspring, and a clear no obsd. adverse effect level (NOAEL) for developmental neurotoxicity of EB was detd. to be 0.6 mg/kg/day.
- 34Padilla, S.; Hunter, D. L.; Padnos, B.; Frady, S.; MacPhail, R. C. Assessing locomotor activity in larval zebrafish: Influence of extrinsic and intrinsic variables Neurotoxicol. Teratol. 2011, 33 (6) 624– 630[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsFegsrrN&md5=9dacd7bade934f2cc1e92db6305ef870Assessing locomotor activity in larval zebrafish: Influence of extrinsic and intrinsic variablesPadilla, S.; Hunter, D. L.; Padnos, B.; Frady, S.; MacPhail, R. C.Neurotoxicology and Teratology (2011), 33 (6), 624-630CODEN: NETEEC; ISSN:0892-0362. (Elsevier B.V.)The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large nos. of chems. for developmental toxicity. We are exploring methods to detect developmentally neurotoxic chems. using zebrafish behavior at 6 days of age. The behavioral paradigm simultaneously tests individual larval zebrafish under both light and dark conditions in a 96-well plate using a video tracking system. We have found that many variables affect the level or pattern of locomotor activity, including age of the larvae, size of the well, and the presence of malformations. Some other variables, however, do not appear to affect larval behavior including type of rearing soln. (10% Hank's vs. 1:3 Danieau vs 60 mg/kg Instant Ocean vs 1× and 1:10× EPA Moderately Hard Water). Zebrafish larval behavior using a microtiter plate format may be an ideal endpoint for screening developmentally neurotoxic chems., but it is imperative that many test variables be carefully specified and controlled.
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Abstract
Figure 1
Figure 1. Spontaneous activity (%) following exposure to 50 μL embryo media from 5 to 25 hpf in a 384-well plate containing 192 initial embryos. Spontaneous activity (%) data are presented as mean ± SD across three independent control plates. Numbers above each bar denote percent spontaneous activity within that column.
Figure 2
Figure 2. Spontaneous activity (%) following (A) exposure to paraoxon in a concentration response-format or (B) exposure to vehicle (0.1% DMSO) or 25 μM paraoxon (reference plates) from 5 to 25 hpf in a 384-well plate containing 192 initial embryos. Paraoxon significantly decreased spontaneous activity in the absence of effects on survival and total body area (SI Figures S6A, S6B, S7A, and S7B). Spontaneous activity (%) data in Panel B are presented as mean ± SD across three independent reference plates. Numbers above each bar denote percent spontaneous activity within that column. Asterisk denotes significant difference from both vehicle control columns 1 and 23 (p < 0.05).
Figure 3
Figure 3. Spontaneous activity (%) following exposure to (A) abamectin or (B) emamectin benzoate from 5 to 25 hpf in a 384-well plate containing 192 initial embryos. Both chemicals significantly decreased spontaneous activity in the absence of effects on survival and total body area (SI Figures S15A, S15B, S24A, and S24B). Numbers above each bar denote percent spontaneous activity within that column. Asterisk denotes significant difference from both vehicle control columns 1 and 23 (p < 0.05).
References
ARTICLE SECTIONSThis article references 34 other publications.
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- 14Westerfield, M.; McMurray, J. V.; Eisen, J. S. Identified motoneurons and their innervation of axial muscles in the zebrafish J. Neurosci. 1986, 6 (8) 2267– 2277[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaL28zgslSitw%253D%253D&md5=0ecb023850154e0bafbbd344137471fbIdentified motoneurons and their innervation of axial muscles in the zebrafishWesterfield M; McMurray J V; Eisen J SThe Journal of neuroscience : the official journal of the Society for Neuroscience (1986), 6 (8), 2267-77 ISSN:0270-6474.The organization of spinal cord motoneurons and their innervation of axial (white) muscles in the zebrafish were studied. Motoneurons can be divided into 2 classes, primary and secondary, on the basis of their cell-body sizes and positions. Each side of each spinal segment contains 3 primary motoneurons that are uniquely identifiable as individuals by their stereotyped cell-body positions and peripheral branching patterns. Moreover, these motoneurons precisely innervate cell-specific subsets of contiguous muscle fibers in mutually exclusive regions of their own body segment. Individual muscle fibers receive inputs from a single primary motoneuron and, in addition, from up to 3 secondary motoneurons. The results demonstrate that the precision of innervation previously described in invertebrates is also present in some vertebrates.
- 15Kokel, D.; Bryan, J.; Laggner, C.; White, R.; Cheung, C. Y. J.; Mateus, R.; Healey, D.; Kim, S.; Werdich, A. A.; Haggarty, S. J.; MacRae, C. A.; Shoichet, B.; Peterson, R. T. Rapid behavior-based identification of neuroactive small molecules in the zebrafish Nat. Chem. Biol. 2010, 6 (3) 231– 237[Crossref], [PubMed], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXntVCnuw%253D%253D&md5=0e9b9d4778ea8443dde1d2b860382616Rapid behavior-based identification of neuroactive small molecules in the zebrafishKokel, David; Bryan, Jennifer; Laggner, Christian; White, Rick; Cheung, Chung Yan J.; Mateus, Rita; Healey, David; Kim, Sonia; Werdich, Andreas A.; Haggarty, Stephen J.; MacRae, Calum A.; Shoichet, Brian; Peterson, Randall T.Nature Chemical Biology (2010), 6 (3), 231-237CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Neuroactive small mols. are indispensable tools for treating mental illnesses and dissecting nervous system function. However, it has been difficult to discover novel neuroactive drugs. Here, we describe a high-throughput, behavior-based approach to neuroactive small mol. discovery in the zebrafish. We used automated screening assays to evaluate thousands of chem. compds. and found that diverse classes of neuroactive mols. caused distinct patterns of behavior. These 'behavioral barcodes' can be used to rapidly identify new psychotropic chems. and to predict their mol. targets. For example, we identified new acetylcholinesterase and monoamine oxidase inhibitors using phenotypic comparisons and computational techniques. By combining high-throughput screening technologies with behavioral phenotyping in vivo, behavior-based chem. screens can accelerate the pace of neuroactive drug discovery and provide small-mol. tools for understanding vertebrate behavior.
- 16Irons, T. D.; MacPhail, R. C.; Hunter, D. L.; Padilla, S. Acute neuroactive drug exposures alter locomotor activity in larval zebrafish Neurotoxicol. Teratol. 2010, 32 (1) 84– 90[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXnvFGlsw%253D%253D&md5=e64855a825d03093aefe6117454f9dadAcute neuroactive drug exposures alter locomotor activity in larval zebrafishIrons, T. D.; MacPhail, R. C.; Hunter, D. L.; Padilla, S.Neurotoxicology and Teratology (2010), 32 (1), 84-90CODEN: NETEEC; ISSN:0892-0362. (Elsevier)As part of the development of a rapid in vivo screen for prioritization of toxic chems., we have begun to characterize the locomotor activity of zebrafish (Danio rerio) larvae by assessing the acute effects of prototypic drugs that act on the central nervous system. Initially, we chose ethanol, d-amphetamine, and cocaine, which are known, in mammals, to increase locomotion at low doses and decrease locomotion at higher doses. Wild-type larvae were individually maintained in 96-well microtiter plates at 26 °C, under a 14:10 h light:dark cycle, with lights on at 0830 h. At 6 days post-fertilization, ethanol (1-4% vol./vol.), d-amphetamine sulfate (0.1-20.0 μM) or cocaine hydrochloride (0.2-50.0 μM) were administered to the larvae by immersion. Beginning 20 min into the exposure, locomotion was assessed for each animal for 70 min using 10-min, alternating light (visible light) and dark (IR light) periods. Low concns. of ethanol and d-amphetamine increased activity, while higher concns. of all three drugs decreased activity. Because ethanol effects occurred predominately during the light periods, whereas the d-amphetamine and cocaine effects occurred during the dark periods, alternating lighting conditions proved to be advantageous. These results indicate that zebrafish larvae are sensitive to neuroactive drugs, and their locomotor response is similar to that of mammals.
- 17Stanley, K. A.; Curtis, L. R.; Simonich, S. L. M.; Tanguay, R. L. Endosulfan I and endosulfan sulfate disrupts zebrafish embryonic development Aquat. Toxicol. 2009, 95 (4) 355– 361[Crossref], [PubMed], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsV2gsrjK&md5=85258996885a59d11fe76f36bd43251bEndosulfan I and endosulfan sulfate disrupts zebrafish embryonic developmentStanley, Kerri A.; Curtis, Lawrence R.; Massey Simonich, Staci L.; Tanguay, Robert L.Aquatic Toxicology (2009), 95 (4), 355-361CODEN: AQTODG; ISSN:0166-445X. (Elsevier B.V.)Fish in agricultural and remote areas may be exposed to endosulfan and its degrdn. products as a result of direct runoff, atm. transport and deposition. The following study used the zebrafish developmental model to investigate the responses to endosulfan I and endosulfan sulfate, the major degrdn. product of endosulfan I and II. Embryos were dechorionated and waterborne exposed to the endosulfan I or endosulfan sulfate from 6 to 120 h post-fertilization (hpf). Endosulfan I exposure concns. ranged from 0.01 to 10 μg/L and endosulfan sulfate from 1 to 100 μg/L. Water solns. were renewed every 24 h and fish were scored for overt developmental and behavioral abnormalities. Chem. anal. was performed on water, whole embryo, and larvae samples to det. waterborne exposure concns. and tissue concns. throughout the 5-day period. The most sensitive toxicity endpoint for both endosulfan I and endosulfan sulfate was an abnormal response of the embryo/larvae to touch, suggesting that endosulfan I and sulfate are developmentally neurotoxic. The waterborne exposure EC50s for inhibition of touch response for endosulfan I and endosulfan sulfate were 2.2 μg/L and 23 μg/L, resp. The endosulfans were highly concd. by the organisms, and the inhibition of touch response tissue EC50, detd. from the measured tissue concns., was 367 ng/g for endosulfan I and 4552 ng/g for endosulfan sulfate.
- 18Selderslaghs, I. W. T.; Hooyberghs, J.; Blust, R.; Witters, H. E. Assessment of the developmental neurotoxicity of compounds by measuring locomotor activity in zebrafish embryos and larvae Neurotoxicol. Teratol. 2013, 37, 44– 56[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXotlyqsLo%253D&md5=f6ce2ae4fdfd5a66897c08521c9abffdAssessment of the developmental neurotoxicity of compounds by measuring locomotor activity in zebrafish embryos and larvaeSelderslaghs, Ingrid W. T.; Hooyberghs, Jef; Blust, Ronny; Witters, Hilda E.Neurotoxicology and Teratology (2013), 37 (), 44-56CODEN: NETEEC; ISSN:0892-0362. (Elsevier Inc.)The developmental neurotoxic potential of the majority of environmental chems. and drugs is currently undetd. Specific in vivo studies provide useful data for hazard assessment but are not amenable to screen thousands of untested compds. In this study, methods which use zebrafish embryos, eleutheroembryos and larvae as model organisms, were proposed as alternatives for developmental neurotoxicity (DNT) testing. The evaluation of spontaneous tail coilings in zebrafish embryos aged 24-26 h post fertilization (hpf) and the swimming activity of eleutheroembryos at 120 and larvae at 144 hpf, i.e. parameters for locomotor activity, were investigated as potential endpoints for DNT testing, according to available std. protocols. The overall performance and predictive value of these methods was then examd. by testing a training set of 10 compds., including known developmental neurotoxicants and compds. not considered to be neurotoxic. The classification of the selected compds. as either neurotoxic or non-neurotoxic, based on the effects obsd. in zebrafish embryos and larvae, was compared to available mammalian data and an overall concordance of 90% was achieved. Furthermore, the specificity of the selected endpoints for DNT was evaluated as well as the potential similarities between zebrafish and mammals with regard to mechanisms of action for the selected compds. Although further studies, including the screening of a large testing set of compds. are required, we suggest that the proposed methods with zebrafish embryos and larvae might be valuable alternatives for animal testing for the screening and prioritization of compds. for DNT.
- 19Lawson, N. D.; Weinstein, B. M. In vivo imaging of embryonic vascular development using transgenic zebrafish Dev. Biol. 2002, 248 (2) 307– 318
- 20Kimmel, C. B.; Ballard, W. W.; Kimmel, S. R.; Ullmann, B.; Schilling, T. F. Stages of embryonic-development of the zebrafish Dev. Dyn. 1995, 203 (3) 253– 310
- 21NIH (National Institutes of Health). Guidelines for Use of Zebrafish in the NIH Intramural Research Program; Office of Animal Care and Use (OACU): Bethesda, MD, 2013; http://oacu.od.nih.gov/ARAC/documents/Zebrafish.pdf.Google ScholarThere is no corresponding record for this reference.
- 22Yozzo, K. L.; McGee, S. P.; Volz, D. C. Adverse outcome pathways during zebrafish embryogenesis: A case study with paraoxon Aquat. Toxicol. 2013, 126, 346– 354[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsVKrur%252FL&md5=4d1173501dc86473d00076e0e6f0cc7eAdverse outcome pathways during zebrafish embryogenesis: A case study with paraoxonYozzo, Krystle L.; McGee, Sean P.; Volz, David C.Aquatic Toxicology (2013), 126 (), 346-354CODEN: AQTODG; ISSN:0166-445X. (Elsevier B.V.)Using paraoxon as a ref. acetylcholinesterase (AChE) inhibitor, the objective of this study was to develop an adverse outcome pathway (AOP) that provided quant. linkages across levels of biol. organization during zebrafish embryogenesis. Within normal zebrafish embryos, we first demonstrated that ache transcripts and AChE activity increased in a stage-dependent manner following segmentation. We then showed that static exposure of embryos to paraoxon (31.2-500 nM) from 5 to 96 hpf resulted in significant stage- and concn.-dependent AChE inhibition, albeit these effects were fully reversible within 48 h following transfer to clean water. However, even in the presence of significant AChE inhibition, exposure to non-teratogenic paraoxon concns. (≤250 nM) did not adversely impact secondary motoneuron development at 96 hpf. Therefore, we investigated the potential effects of paraoxon exposure on spontaneous tail contractions at 26 hpf - an early locomotor behavior that results from innervation of primary (not secondary) motoneuron axons to target axial muscles. Based on these studies, the frequency of spontaneous tail contractions at 26 hpf - a developmental stage with minimal AChE expression and activity - was significantly higher following exposure to paraoxon concns. as low as 31.2 nM. Overall, our data suggest that (1) normal AChE activity is not required for secondary motoneuron development and (2) spontaneous tail contractions at 26 hpf are sensitive to paraoxon exposure, an effect that may be independent of AChE inhibition. Using a well-studied ref. chem., this study highlights the potential challenges in developing quant. AOPs to support chem. screening and prioritization strategies.
- 23Thomas, L. T.; Welsh, L.; Galvez, F.; Svoboda, K. R. Acute nicotine exposure and modulation of a spinal motor circuit in embryonic zebrafish Toxicol. Appl. Pharmacol. 2009, 239 (1) 1– 12[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXptVCju74%253D&md5=4cdf61c877d7320ddfd2daa25d1636adAcute nicotine exposure and modulation of a spinal motor circuit in embryonic zebrafishThomas, Latoya T.; Welsh, Lillian; Galvez, Fernando; Svoboda, Kurt R.Toxicology and Applied Pharmacology (2009), 239 (1), 1-12CODEN: TXAPA9; ISSN:0041-008X. (Elsevier B.V.)The zebrafish model system is ideal for studying nervous system development. Ultimately, one would like to link the developmental biol. to various aspects of behavior. The authors are studying the consequences of nicotine exposure on nervous system development in zebrafish and have previously shown that chronic nicotine exposure produces paralysis. The authors also have made observations that the embryos moved in the initial minutes of the exposure as the bend rates of the musculature increased. This nicotine-induced behavior manifests as an increase in the rate of spinal musculature bends, which spontaneously begin at ∼18 h postfertilization. The behavioral observations prompted the systematic characterization of nicotine-induced modulation of zebrafish embryonic motor output; bends of the trunk musculature. The authors first characterized embryonic motor output in zebrafish embryos with and without their chorions. They then characterized the motor output in embryos raised at 28° and 25°. The act of dechorionation along with temp. influenced the embryonic bend rate. Nicotine exposure increased embryonic motor output. Nicotine exposure caused the musculature bends to alternate in a left-right-left fashion. Nicotine was able to produce this phenotype in embryos lacking supraspinal input. The authors then characterized the kinetics of nicotine influx and efflux and demonstrated that nicotine ≥1 μM can disrupt embryonic physiol. Taken together, these results indicate the presence of nicotinic acetylcholine receptors (nAChRs) assocd. with embryonic spinal motor circuits early in embryogenesis.
- 24Padilla, S.; Corum, D.; Padnos, B.; Hunter, D. L.; Beam, A.; Houck, K. A.; Sipes, N.; Kleinstreuer, N.; Knudsen, T.; Dix, D. J.; Reif, D. M. Zebrafish developmental screening of the ToxCast (TM) Phase I chemical library Reprod. Toxicol. 2012, 33 (2) 174– 187[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xkt1Ggtrg%253D&md5=27fa06df73fd73941a8fc10865e7ce67Zebrafish developmental screening of the ToxCast Phase I chemical libraryPadilla, S.; Corum, D.; Padnos, B.; Hunter, D. L.; Beam, A.; Houck, K. A.; Sipes, N.; Kleinstreuer, N.; Knudsen, T.; Dix, D. J.; Reif, D. M.Reproductive Toxicology (2012), 33 (2), 174-187CODEN: REPTED; ISSN:0890-6238. (Elsevier Inc.)Zebrafish (Danio rerio) is an emerging toxicity screening model for both human health and ecol. As part of the Computational Toxicol. Research Program of the U. S. EPA, the toxicity of the 309 ToxCast Phase I chems. was assessed using a zebrafish screen for developmental toxicity. All exposures were by immersion from 6-8 h post fertilization (hpf) to 5 days post fertilization (dpf); nominal concn. range of 1 nM-80 μM. On 6 dpf larvae were assessed for death and overt structural defects. Results revealed that the majority (62%) of chems. were toxic to the developing zebrafish; both toxicity incidence and potency was correlated with chem. class and hydrophobicity (logP); and inter-and intra-plate replicates showed good agreement. The zebrafish embryo screen, by providing an integrated model of the developing vertebrate, compliments the ToxCast assay portfolio and has the potential to provide information relative to overt and organismal toxicity.
- 25Downes, G. B.; Granato, M. Supraspinal input is dispensable to generate glycine-mediated locomotive behaviors in the zebrafish embryo J. Neurobiol. 2006, 66 (5) 437– 451[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XlsFCqsLo%253D&md5=18741cd2cba91d3b3049c4379be24f32Supraspinal input is dispensable to generate glycine-mediated locomotive behaviors in the zebrafish embryoDownes, Gerald B.; Granato, MichaelJournal of Neurobiology (2006), 66 (5), 437-451CODEN: JNEUBZ; ISSN:0022-3034. (John Wiley & Sons, Inc.)The anatomy of the developing zebrafish spinal cord is relatively simple but, despite this simplicity, it generates a sequence of three patterns of locomotive behaviors. The first behavior exhibited is spontaneous movement, then touch-evoked coiling, and finally swimming. Previous studies in zebrafish have suggested that spontaneous movements occur independent of supraspinal input and do not require chem. neurotransmission, while touch-evoked coiling and swimming depend on glycinergic neurotransmission as well as supraspinal input. In contrast, studies in other vertebrate prepns. have shown that spontaneous movement requires glycine and other neurotransmitters and that later behaviors do not require supraspinal input. Here, the authors use lesion anal. combined with high-speed kinematic anal. to re-examine the role of glycine and supraspinal input in each of the three behaviors. The authors find that, similar to other vertebrate prepns., supraspinal input is not essential for spontaneous movement, touch-evoked coiling, or swimming behavior. Moreover, the authors find that blockade of glycinergic neurotransmission decreases the rate of spontaneous movement and impairs touch-evoked coiling and swimming, suggesting that glycinergic neurotransmission plays crit. yet distinct roles for individual patterns of locomotive behaviors.
- 26Saint-Amant, L.; Drapeau, P. Time course of the development of motor behaviors in the zebrafish embryo J. Neurobiol. 1998, 37 (4) 622– 632[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK1M%252Fntl2ktg%253D%253D&md5=e5fca7135b1e9d9fca2798f33851495cTime course of the development of motor behaviors in the zebrafish embryoSaint-Amant L; Drapeau PJournal of neurobiology (1998), 37 (4), 622-32 ISSN:0022-3034.The development and properties of locomotor behaviors in zebrafish embryos raised at 28.5 degrees C were examined. When freed from the chorion, embryonic zebrafish showed three sequential stereotyped behaviors: a transient period of alternating, coiling contractions followed by touch-evoked rapid coils, then finally, organized swimming. The three different behaviors were characterized by video microscopy. Spontaneous, alternating contractions of the trunk appeared suddenly at 17 h postfertilization (hpf), with a frequency of 0.57 Hz, peaked at 19 hpf at 0.96 Hz, and gradually decreased to <0.1 Hz by 27 hpf. Starting at 21 hpf, touching either the head or the tail of the embryos resulted in vigorous coils. The coils accelerated with development, reaching a maximum speed of contraction before 48 hpf, which is near the time of hatching. After 27 hpf, touching the embryos, particularly on the tail, could induce partial coils (instead of full coils). At this time, embryos started to swim in response to a touch, preferentially to the tail. The swim cycle frequency gradually increased with age from 7 Hz at 27 hpf to 28 Hz at 36 hpf. Lesions of the central nervous system rostral to the hindbrain had no effect on the three behaviors. Lesioning the hindbrain eliminated swimming and touch responses, but not the spontaneous contractions. Our observations suggest that the spontaneous contractions result from activation of a primitive spinal circuit, while touch and swimming require additional hindbrain inputs to elicit mature locomotor behaviors.
- 27Brustein, E.; Saint-Amant, L.; Buss, R.; Chong, M.; McDearmid, J.; Drapeau, P. Steps during the development of the zebrafish locomotor network J. Physiol.-Paris 2003, 97 (1) 77– 86[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2c%252FhtVejsg%253D%253D&md5=1b231f84692577877699e1b0c995a77fSteps during the development of the zebrafish locomotor networkBrustein Edna; Saint-Amant Louis; Buss Robert R; Chong Mabel; McDearmid Jonathan R; Drapeau PierreJournal of physiology, Paris (2003), 97 (1), 77-86 ISSN:0928-4257.This review summarizes recent data from our lab concerning the development of motor activities in the developing zebrafish. The zebrafish is a leading model for studies of vertebrate development because one can obtain a large number of transparent, externally and rapidly developing embryos with motor behaviors that are easy to assess (e.g. for mutagenic screens). The emergence of embryonic motility was studied behaviorally and at the cellular level. The embryonic behaviors appear sequentially and include an early, transient period of spontaneous, alternating tail coilings, followed by responses to touch, and swimming. Patch clamp recording in vivo revealed that an electrically coupled network of a subset of spinal neurons generates spontaneous tail coiling, whereas a chemical (glutamatergic and glycinergic) synaptic drive underlies touch responses and swimming and requires input from the hindbrain. Swimming becomes sustained in larvae once serotonergic neuromodulatory effects are integrated. We end with a brief overview of the genetic tools available for the study of the molecular determinants implicated in locomotor network development in the zebrafish. Combining genetic, behavioral and cellular experimental approaches will advance our understanding of the general principles of locomotor network assembly and function.
- 28Taylor, M. A. Recent developments in ectoparasiticides Vet. J. 2001, 161 (3) 253– 268[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXjslSlt7g%253D&md5=eff972ab22d8d5bde11ad60a335d3f4dRecent developments in ectoparasiticidesTaylor, M. A.Veterinary Journal (2001), 161 (3), 253-268CODEN: VTJRFP; ISSN:1090-0233. (Bailliere Tindall Ltd.)A review with many refs. The sales and use of ectoparasiticides for the control of arthropod parasites of domestic animals constitute a major sector of the global animal health market. Animals are infected by a no. of parasitic insect and acarine species causing major economic losses in prodn. livestock, intense irritation and skin disease in companion animals, or public health issues, including bites of humans or zoonotic disease transmission. Dog and cat fleas, for example, can be a serious source of both animal and human irritation, which has led to a rapid expansion in the development of flea control products. The control of ectoparasite infections of veterinary importance still relies heavily on the use of chems. that target the arthropod nervous system. Such compds. have suffered from a no. of drawbacks, including the development of resistance and concerns over human and environmental safety. The search for safer technologies has, however, been hindered by the limited no. of active target sites present in arthropods and, to some degree, by the ever-increasing costs of research and development of compds. with novel modes of action. This review provides a background to the currently available groups of ectoparasiticide compds. used in veterinary medicine and highlights some of the more recent developments including the introduction of insect growth regulators and new and improved methods of product application.
- 29Bloomquist, J. R. Ion channels as targets for insecticides Annu. Rev. Entomol. 1996, 41, 163– 190[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XksVyrsQ%253D%253D&md5=2e9f9d8ebf2dba6e9fe54fe7a787d44aIon channels as targets for insecticidesBloomquist, Jeffrey R.Annual Review of Entomology (1996), 41 (), 163-90CODEN: ARENAA; ISSN:0066-4170. (Annual Reviews)A review with 120 refs. Ion channels are the primary target sites for several classes of natural and synthetic insecticidal compds. The voltage-sensitive sodium channel is the major target site for DDT and pyrethroids, the veratrum alkaloids, and N-alkyl-amides. Recently, neurotoxic proteins from arthropod venoms, some of which specifically attack insect sodium channels, have been engineered into baculoviruses to act as biopesticides. The synthetic pyrazolines also primarily affect the sodium channel, although some members of this group target neuronal calcium channels as well. The ryanoids have also found use as insecticides, and these materials induce muscle contracture by irreversible activation of the calcium-release channel of the sarcoplasmic reticulum. The arylheterocycles (e.g. endosulfan and fipronil) are potent convulsants and insecticides that block the GABA-gated chloride channel. In contrast, the avermectins activate both ligand- and voltage-gated chloride channels, which leads to paralysis. At field-use rates, a neurotoxic effect of the ecdysteroid agonist RH-5849 is obsd. that involves blockage of both muscle and neuronal potassium channels. The future use of ion channels as targets for chem. and genetically engineered insecticides is also discussed.
- 30US EPA. Ecological Risk Assessment for Abamectin; Office of Prevention, Environmental Fate and Effects Division: Washington, D.C., 2004.Google ScholarThere is no corresponding record for this reference.
- 31Sun, Y. J.; Long, D. X.; Li, W.; Hou, W. Y.; Wu, Y. J.; Shen, J. Z. Effects of avermectins on neurite outgrowth in differentiating mouse neuroblastoma N2a cells Toxicol. Lett. 2010, 192 (2) 206– 211[Crossref], [PubMed], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXotVCjtw%253D%253D&md5=6dc6e48b88d8fa9d17e1e98582113b28Effects of avermectins on neurite outgrowth in differentiating mouse neuroblastoma N2a cellsSun, Ying-Jian; Long, Ding-Xin; Li, Wei; Hou, Wei-Yuan; Wu, Yi-Jun; Shen, Jian-ZhongToxicology Letters (2010), 192 (2), 206-211CODEN: TOLED5; ISSN:0378-4274. (Elsevier Ireland Ltd.)Avermectins (AVMs) are macrocyclic lactone compds. that have been widely used as parasiticides in veterinary and human medicine and as pesticides in agriculture and horticulture. The multidrug resistance transporter, P-glycoprotein (P-gp), is assocd. with the efflux transport of AVMs and other drugs across the blood-brain and placental barrier, and plays an important role in attenuating the neurotoxicity and developmental toxicity of AVMs. In this study, the mouse neuroblastoma N2a cell line was used to investigate the neurotoxicity of two AVM derivs.: abamectin (ABM) and doramectin (DOR). We found that both these compds. caused significant dose-dependent inhibition of neurite growth in differentiating N2a cells. In addn., Western blotting anal. showed that ABM and DOR significantly inhibited the expression of not only P-gp but also the cytoskeletal proteins, β-actin and β-tubulin. This suggests ABM and DOR may inhibit neurite growth by down-regulating the expression of P-gp and cytoskeletal proteins. Furthermore, knockdown of P-gp expression by RNA interference in N2a cells reduced neurite growth even in the absence of ABM and DOR, and reduced it even more in the presence of low levels of these compds. These results suggest that even subcytotoxic levels of ABM and DOR can be neurotoxic in differentiating cells and that this neurotoxicity may, at least in part, be the result of the down-regulation of P-gp and cytoskeletal proteins.
- 32Tisler, T.; Erzen, N. K. Abamectin in the aquatic environment Ecotoxicology 2006, 15 (6) 495– 502
- 33Wise, L. D.; Allen, H. L.; Hoe, C. M. L.; Verbeke, D. R.; Gerson, R. J. Developmental neurotoxicity evaluation of the avermectin pesticide, emamectin benzoate, in Sprague-Dawley rats Neurotoxicol. Teratol. 1997, 19 (4) 315– 326[Crossref], [PubMed], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXkvFGkt7Y%253D&md5=fa9ebd4b3f5f323a09b4c72226d1aad5Developmental neurotoxicity evaluation of the avermectin pesticide, emamectin benzoate, in Sprague-Dawley ratsWise, L. David; Allen, Henry L.; Hoe, Chao-Min L.; Verbeke, David R.; Gerson, Ronald J.Neurotoxicology and Teratology (1997), 19 (4), 315-326CODEN: NETEEC; ISSN:0892-0362. (Elsevier)The potential of emamectin benzoate (EB) to cause developmental neurotoxicity in Sprague-Dawley rats was assessed using a study design proposed by the US EPA. Dosages of 0 (deionized water), 0.1, 0.6, or 3.6 mg/kg/day were administered at 5 mL/kg by oral gavage from gestational day (GD) 6 to lactational day (LD) 20 groups of 25 mated females each. Between GD 17 and 20 the high dose was reduced to 2.5 mg/kg/day because of pup tremors obsd. at this dose level in a concurrent two-generation study. Females were allowed to deliver and the young were evaluated for survival, growth, development, behavior, and histol. changes to brain, spinal cord, peripheral nerve, and skeletal muscle. Behavioral assessment of the offspring consisted of open field motor activity, auditory startle habituation, and passive avoidance tests; each was conducted on weanling and adult animals (one animal/sex/litter). Histopathol. examn. of the CNS and PNS was conducted on one animal/sex/litter on postnatal days (PND) 11 and 60. There were significant increases in av. F0 maternal body wt. gains during gestation in the 0.6 and 3.6/2.5 mg/kg/day groups, but no other effects were obsd. in pregnant females of these or the low-dose groups during the study. Beginning on PND 6, tremors were obsd. in high-dose pups, and this was followed by hindlimb splay in all high-dose pups by PND 15-26. Both of these phys. signs disappeared by PND 34 (i.e., 10-11 days after weaning). There were no compd.-related deaths in F1 offspring. Beginning on PND 11, progressive decreases in preweaning av. wts. were obsd. in the high-dose group (to 42% below control in females on PND 21). Av. wt. gain during the postweaning period was significantly decreased in the 3.6/2.5 mg/kg/day group. There were EB-related effects in behavioral tests only in the high-dose group. A significant increase in PND 13 av. horizontal motor activity was due to stereotypical movements. Av. horizontal activity was decreased on PND 17 and in adult females, but there were no effects on PND 21. Av. peak auditory startle response amplitude was decreased on PND 22 and in adults. There were no EB-related effects in the passive avoidance test, relative brain wts., or in the histol. examn. (including morphometry) of the nervous system. These results demonstrate that the high-dose EB exposure during gestation and lactation to rats produced evidence of neurotoxicity in the F1 offspring, and a clear no obsd. adverse effect level (NOAEL) for developmental neurotoxicity of EB was detd. to be 0.6 mg/kg/day.
- 34Padilla, S.; Hunter, D. L.; Padnos, B.; Frady, S.; MacPhail, R. C. Assessing locomotor activity in larval zebrafish: Influence of extrinsic and intrinsic variables Neurotoxicol. Teratol. 2011, 33 (6) 624– 630[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsFegsrrN&md5=9dacd7bade934f2cc1e92db6305ef870Assessing locomotor activity in larval zebrafish: Influence of extrinsic and intrinsic variablesPadilla, S.; Hunter, D. L.; Padnos, B.; Frady, S.; MacPhail, R. C.Neurotoxicology and Teratology (2011), 33 (6), 624-630CODEN: NETEEC; ISSN:0892-0362. (Elsevier B.V.)The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large nos. of chems. for developmental toxicity. We are exploring methods to detect developmentally neurotoxic chems. using zebrafish behavior at 6 days of age. The behavioral paradigm simultaneously tests individual larval zebrafish under both light and dark conditions in a 96-well plate using a video tracking system. We have found that many variables affect the level or pattern of locomotor activity, including age of the larvae, size of the well, and the presence of malformations. Some other variables, however, do not appear to affect larval behavior including type of rearing soln. (10% Hank's vs. 1:3 Danieau vs 60 mg/kg Instant Ocean vs 1× and 1:10× EPA Moderately Hard Water). Zebrafish larval behavior using a microtiter plate format may be an ideal endpoint for screening developmentally neurotoxic chems., but it is imperative that many test variables be carefully specified and controlled.
Supporting Information
Supporting Information
ARTICLE SECTIONSSupplemental File 1: Chemical names, chemical formulas, CAS registry numbers, vendors, and purities (Table S1); imaging and analysis parameters (Tables S2–S3); control image success rates (Table S4); a summary of chemical screening results (Table S5); figures for assay optimization (Figures S1–S7); and figures for chemical screening (Figures S8–S24). Microsoft Excel spreadsheets containing raw data for all assays are provided within Supplemental File 2. This information is available free of charge via the Internet at http://pubs.acs.org.
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