Adverse developmental impacts in progeny of zebrafish exposed to the agricultural herbicide atrazine during embryogenesis.

Atrazine (ATZ) is an herbicide commonly used on crops in the Midwestern US and other select global regions. The US Environmental Protection Agency ATZ regulatory limit is 3 parts per billion (ppb; µg/L), but this limit is often exceeded. ATZ has a long half-life, is a common contaminant of drinking water sources, and is indicated as an endocrine disrupting chemical in multiple species. The zebrafish was used to test the hypothesis that an embryonic parental ATZ exposure alters protein levels leading to modifications in morphology and behavior in developing progeny. Zebrafish embryos (F1) were collected from adults (F0) exposed to 0, 0.3, 3, or 30 ppb ATZ during embryogenesis. Differential proteomics, morphology, and behavior assays were completed with offspring aged 120 or 144 h with no additional chemical treatment. Proteomic analysis identified differential expression of proteins associated with neurological development and disease; and organ and organismal morphology, development, and injury, specifically the skeletomuscular system. Head length and ratio of head length to total length was significantly increased in the F1 of 0.3 and 30 ppb ATZ groups (p < 0.05). Based on molecular pathway alterations, further craniofacial morphology assessment found decreased distance for cartilaginous structures, decreased surface area and distance between saccular otoliths, and a more posteriorly positioned notochord (p < 0.05), indicating delayed ossification and skeletal growth. The visual motor response assay showed hyperactivity in progeny of the 30 ppb treatment group for distance moved and of the 0.3 and 30 ppb treatment groups for time spent moving (p < 0.05). Due to the changes in saccular otoliths, an acoustic startle assay was completed and showed decreased response in the 0.3 and 30 ppb treatments (p < 0.05). These findings suggest that a single embryonic parental exposure alters cellular pathways in their progeny that lead to perturbations in craniofacial development and behavior.

Anxiety-related behavior and associated brain transcriptome and epigenome alterations in adult female zebrafish exposed to atrazine during embryogenesis.

Atrazine often contaminates drinking water sources, exceeding the maximum contaminant level established by the US Environmental Protection Agency at 3 parts per billion (ppb; µg/L). Atrazine is linked to endocrine disruption, neurotoxicity, and cancer, with delayed health effects observed after developmental exposure in line with the developmental origins of health and disease (DOHaD) hypothesis. To test the hypothesis that embryonic atrazine exposure induces delayed neurotoxicity in adult female zebrafish (Danio rerio), embryos were exposed to 0, 0.3, 3, or 30 ppb atrazine during embryogenesis (1-72 h post fertilization (hpf)) and raised to adults with no additional atrazine exposure. Behavioral outcomes were tested through a novel tank test, light-dark box, and open field test and indicated female zebrafish had more anxious phenotypes at 9 months post fertilization (mpf). Female brain transcriptomic analysis at 9 mpf found altered gene expression pathways related to organismal injury and cancer with beta-estradiol and estrogen receptor as top upstream regulators. These results were compared to 9 mpf male and 6 mpf female groups with the same atrazine embryonic exposures and showed differences in specific genes that were altered, but similarities in top molecular pathways. Molecular pathways associated with behavior were observed only in the 6 mpf transcriptomic profiles, suggesting prediction of observed behavioral outcomes at 9 mpf. The expression of genes associated with serotonin neurotransmission was also evaluated at 14 mpf to determine persistence; however, no significant changes were observed. Brain global methylation in 12 mpf zebrafish observed an increased percent 5 mC in females with embryonic 0.3 ppb atrazine exposure. Finally, the body length, body weight, and brain weight were determined at 14 mpf and were altered in all treatment groups. These results indicate that embryonic atrazine exposure does cause delayed neurotoxicity within the DOHaD framework, which is significant given atrazine's presence and persistence in the environment.

A Novel Neuraminidase Virus-Like Particle Vaccine Offers Protection Against Heterologous H3N2 Influenza Virus Infection in the Porcine Model.

Influenza A viruses (IAVs) pose a global health threat, contributing to hundreds of thousands of deaths and millions of hospitalizations annually. The two major surface glycoproteins of IAVs, hemagglutinin (HA) and neuraminidase (NA), are important antigens in eliciting neutralizing antibodies and protection against disease. However, NA is generally ignored in the formulation and development of influenza vaccines. In this study, we evaluate the immunogenicity and efficacy against challenge of a novel NA virus-like particles (VLPs) vaccine in the porcine model. We developed an NA2 VLP vaccine containing the NA protein from A/Perth/16/2009 (H3N2) and the matrix 1 (M1) protein from A/MI/73/2015, formulated with a water-in-oil-in-water adjuvant. Responses to NA2 VLPs were compared to a commercial adjuvanted quadrivalent whole inactivated virus (QWIV) swine IAV vaccine. Animals were prime boost vaccinated 21 days apart and challenged four weeks later with an H3N2 swine IAV field isolate, A/swine/NC/KH1552516/2016. Pigs vaccinated with the commercial QWIV vaccine demonstrated high hemagglutination inhibition (HAI) titers but very weak anti-NA antibody titers and subsequently undetectable NA inhibition (NAI) titers. Conversely, NA2 VLP vaccinated pigs demonstrated undetectable HAI titers but high anti-NA antibody titers and NAI titers. Post-challenge, NA2 VLPs and the commercial QWIV vaccine showed similar reductions in virus replication, pulmonary neutrophilic infiltration, and lung inflammation compared to unvaccinated controls. These data suggest that anti-NA immunity following NA2 VLP vaccination offers comparable protection to QWIV swine IAV vaccines inducing primarily anti-HA responses.

Atrazine exposure in zebrafish induces aberrant genome-wide methylation.

Atrazine (ATZ) is the second most common agricultural herbicide used in the United States and is an endocrine disrupting chemical (EDC). Developmental exposure to ATZ can lead to significant behavioral and morphological alterations in exposed animals and their progeny suggesting the involvement of an epigenetic mechanism. Specific epigenetic mechanisms responsible for these alterations, however, are yet to be elucidated. In this study, we exposed zebrafish embryos to 0, 0.3, 3, or 30 ppb (µg/L) of ATZ from 1 to 72 h post fertilization (hpf). Chemical exposure was ceased and zebrafish maintained until 9 months post fertilization (mpf), when whole-genome bisulfite sequencing (WGBS) was performed to assess the effects of embryonic ATZ exposure on DNA methylation in female fish brains. The number of differentially methylated genes (DMGs) increased with increasing treatment concentration. DMGs were enriched in neurological pathways with extensive methylation changes consistently observed in neuroendocrine pathways. Specifically, DMGs with methylation changes in promoter regions showed hypomethylation in estrogen receptor signaling and hypermethylation in androgen signaling. DMGs with methylation changes in genebody were primarily enriched for mitochondrion-related pathways associated with healthy aging. Integrated analysis with transcriptomic data at 9 mpf exhibited a similar trend identifying CABLES1 and NDUFA4 as shared targets at all concentrations. We then compared the predicted upstream regulators of transcriptomic changes with DMGs and identified CALML3 as a common upstream regulator at both 0.3 and 30 ppb that exhibit significant methylation changes. Collectively, our study identified long-lasting DNA methylation changes in genome after embryonic ATZ exposure and elucidated potential gene targets whose aberrant methylation features may drive alterations in gene transcription in long-term.

Developmental atrazine exposure in zebrafish produces the same major metabolites as mammals along with altered behavioral outcomes.

Atrazine (ATZ) is the second most commonly applied agricultural herbicide in the United States. Due to contamination concerns, the U.S. EPA has set the maximum contaminant level in potable water sources at 3 parts per billion (ppb; µg/l). Depending on the time of year and sampling location, water sources often exceed this limit. ATZ is an endocrine disrupting chemical in multiple species observed to target the neuroendocrine system. In this study the zebrafish vertebrate model was used to test the hypothesis that a developmental ATZ exposure generates metabolites similar to those found in mammals and alters morphology and behavior in developing larvae. Adult AB zebrafish were bred, embryos were collected, and exposed to 0, 0.3, 3, or 30 ppb ATZ from 1 to 120 h post fertilization (hpf). Targeted metabolomic analysis found that zebrafish produce the same major ATZ metabolites as mammals: desethyl atrazine (DEA), deisopropyl atrazine (DIA), and diaminochloroatrazine (DACT). The visual motor response test at 120 hpf detected hyperactivity in larvae in the 0.3 ppb treatment group and hypoactivity in the 30 ppb treatment group (p < 0.05). Further analysis into behavior during the dark and light phases showed zebrafish larvae exposed to 0.3 ppb ATZ had an increase in total distance moved in the first light phase and time spent moving in the first dark and light phases (p < 0.05). Alternatively, a decrease in total distance moved was observed in the second and third dark phases in zebrafish exposed to 30 ppb ATZ (p < 0.05). No significant differences were observed for any of the morphological measurements following ATZ exposure from 1 to 120 hpf (p > 0.05). These findings suggest that a ATZ exposure during early development generates metabolite profiles similar to mammals and leads to behavioral alterations supporting ATZ as a neurodevelopmental toxicant.

Embryonic atrazine exposure and later in life behavioral and brain transcriptomic, epigenetic, and pathological alterations in adult male zebrafish.

Atrazine (ATZ), a commonly used pesticide linked to endocrine disruption, cancer, and altered neurochemistry, frequently contaminates water sources at levels above the US Environmental Protection Agency's 3 parts per billion (ppb; µg/L) maximum contaminant level. Adult male zebrafish behavior, brain transcriptome, brain methylation status, and neuropathology were examined to test the hypothesis that embryonic ATZ exposure causes delayed neurotoxicity, according to the developmental origins of health and disease paradigm. Zebrafish (Danio rerio) embryos were exposed to 0 ppb, 0.3 ppb, 3 ppb, or 30 ppb ATZ during embryogenesis (1-72 h post fertilization (hpf)), then rinsed and raised to maturity. At 9 months post fertilization (mpf), males had decreased locomotor parameters during a battery of behavioral tests. Transcriptomic analysis identified altered gene expression in organismal development, cancer, and nervous and reproductive system development and function pathways and networks. The brain was evaluated histopathologically for morphometric differences, and decreased numbers of cells were identified in raphe populations. Global methylation levels were evaluated at 12 mpf, and the body length, body weight, and brain weight were measured at 14 mpf to evaluate effects of ATZ on mature brain size. No significant difference in genome methylation or brain size was observed. The results demonstrate that developmental exposure to ATZ does affect neurodevelopment and neural function in adult male zebrafish and raises concern for possible health effects in humans due to ATZ's environmental presence and persistence. Graphical abstract.

Developmental toxicity of trichloroethylene in zebrafish (Danio rerio).

Trichloroethylene (TCE), an industrial solvent and degreaser, is an environmental toxicant that contaminates over half of Superfund sites, is a known carcinogen, and is linked to congenital defects and neurodegenerative disease. The developmental toxicity of TCE near ecologically relevant levels needs further characterization in order to better assess health risks of exposure. In this study, the toxicodynamics of TCE in the zebrafish (Danio rerio) model was investigated through the establishment of a LC50 concentration and by monitoring the acute developmental toxicity of ecologically relevant concentrations (0, 5, 50, and 500 parts per billion; ppb) of TCE during two different exposure lengths (1-72 hours post fertilization (hpf) and 1-120 hpf). Acute developmental toxicity was assessed by monitoring survival and hatching, larval morphology, larval heart rate, and behavioral responses during an embryonic photomotor response test and a larval visual motor response test. Embryonic exposure to TCE was associated with decreased percent hatch at 48 hpf, altered larval morphology, increased heart rate, and altered behavioral responses during the photomotor response test and visual motor response test. Larval morphology and behavioral alterations were more pronounced in the 1-120 hpf exposure length trials. The observed alterations suggest developmental TCE toxicity is still a concern at regulatory concentrations and that timing of exposure influences developmental toxicity.

Microarray, IPA and GSEA Analysis in Mice Models.

This protocol details a method to analyze two tissue samples at the transcriptomic level using microarray analysis, ingenuity pathway analysis (IPA) and gene set enrichment analysis (GSEA). Methods such as these provide insight into the mechanisms underlying biological differences across two samples and thus can be applied to interrogate a variety of processes across different tissue samples, conditions, and the like. The full method detailed below can be applied to determine the effects of muscle-specific Notch1 activation in the mdx mouse model and to analyze previously published microarray data of human liposarcoma cell lines.

Embryonic atrazine exposure elicits proteomic, behavioral, and brain abnormalities with developmental time specific gene expression signatures.

Atrazine (ATZ), the second most commonly used herbicide in the United States, is an endocrine disrupting chemical linked to cancer and a common drinking water contaminant. This study further investigates ATZ-related developmental toxicity by testing the following hypotheses in zebrafish: the effects of embryonic ATZ exposure are dependent on timing of exposure; embryonic ATZ exposure alters brain development and function; and embryonic ATZ exposure changes protein abundance in carcinogenesis-related pathways. After exposing embryos to 0, 0.3, 3, or 30 parts per billion (ppb) ATZ, we monitored the expression of cytochrome P450 family 17 subfamily A member 1 (cyp17a1), glyoxalase I (glo1), ring finger protein 14 (rnf14), salt inducible kinase 2 (sik2), tetratricopeptide domain 3 (ttc3), and tumor protein D52 like 1 (tpd52l1) at multiple embryonic time points to determine normal expression and if ATZ exposure altered expression. Only cyp17a1 had normal dynamic expression, but ttc3 and tpd52l1 had ATZ-related expression changes before 72 h. Larvae exposed to 0.3 ppb ATZ had increased brain length, while larvae exposed to 30 ppb ATZ were hypoactive. Proteomic analysis identified altered protein abundance in pathways related to cellular function, neurodevelopment, and genital-tract cancer. The results indicate embryonic ATZ toxicity involves interactions of multiple pathways. SIGNIFICANCE: This is the first report of proteomic alterations following embryonic exposure to atrazine, an environmentally persistent pesticide and common water contaminant. Although the transcriptomic alterations in larval zebrafish with embryonic atrazine exposure have been reported, neither the time at which gene expression changes occur nor the resulting proteomic changes have been investigated. This study seeks to address these knowledge gaps by evaluating atrazine's effect on gene expression through multiple time points during embryogenesis, and correlating changes in gene expression to pathological alterations in brain length and functional changes in behavior. Finally, pathway analysis of the proteomic alterations identifies connections between the molecular changes and functional outcomes associated with embryonic atrazine exposure.

Making Waves: New Developments in Toxicology With the Zebrafish.

The laboratory zebrafish (Danio rerio) is now an accepted model in toxicologic research. The zebrafish model fills a niche between in vitro models and mammalian biomedical models. The developmental characteristics of the small fish are strategically being used by scientists to study topics ranging from high-throughput toxicity screens to toxicity in multi- and transgenerational studies. High-throughput technology has increased the utility of zebrafish embryonic toxicity assays in screening of chemicals and drugs for toxicity or effect. Additionally, advances in behavioral characterization and experimental methodology allow for observation of recognizable phenotypic changes after xenobiotic exposure. Future directions in zebrafish research are predicted to take advantage of CRISPR-Cas9 genome editing methods in creating models of disease and interrogating mechanisms of action with fluorescent reporters or tagged proteins. Zebrafish can also model developmental origins of health and disease and multi- and transgenerational toxicity. The zebrafish has many advantages as a toxicologic model and new methodologies and areas of study continue to expand the usefulness and application of the zebrafish.

An embryonic 100µg/L lead exposure results in sex-specific expression changes in genes associated with the neurological system in female or cancer in male adult zebrafish brains.

Developmental lead (Pb) exposure is linked to neurological health issues. Results from non-human primate and rodent studies suggest detrimental effects of an early life Pb exposure, showing transcriptional disturbances and pathological evidence of Alzheimer's disease in the adult animal brain. To elucidate the impacts of an embryonic Pb exposure on the adult brain, transcriptomic analysis was completed on the brain of zebrafish aged 12months exposed to a control treatment or to an embryonic 100µg/L Pb exposure by sex. In the adult female zebrafish brain, significant changes in expression profiles occurred in a number of genes involved in neurological disease and nervous system development and function. On the other hand, in adult males, a number of genes with significant expression alterations were found to be associated with cancer and tumors. p38 mitogen-activated protein kinase (p38 MAPK) was also indicated as an upstream regulator of observed gene expression changes. Western blot analysis confirmed activation of p38 MAPK in the form of phosphorylated p38 MAPK in the male zebrafish brain. In addition, we compared transcriptomic changes observed in this study to a previous study with an embryonic exposure of 10µg/L Pb by sex, showing unique sets of genes dependent on Pb concentration. Overall, these results show sex-specific and concentration-dependent disturbances of global gene expression patterns in the brain of adult zebrafish exposed to Pb during embryogenesis.

Thyroid disrupting effects of halogenated and next generation chemicals on the swim bladder development of zebrafish.

Endocrine disrupting chemicals (EDCs) can alter thyroid function and adversely affect growth and development. Halogenated compounds, such as perfluorinated chemicals commonly used in food packaging, and brominated flame retardants used in a broad range of products from clothing to electronics, can act as thyroid disruptors. Due to the adverse effects of these compounds, there is a need for the development of safer next generation chemicals. The objective of this study was to test the thyroid disruption potential of old use and next generation halogenated chemicals. Zebrafish embryos were exposed to three old use compounds, perfluorooctanoic acid (PFOA), tetrabromobisphenol A (TBBPA) and tris (1,3-dichloro-2-propyl) phosphate (TDCPP) and two next generation chemicals, 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxdie (DOPO) and perfluorobutyric acid (PFBA). Sub-chronic (0-6days post fertilization (dpf)) and chronic (0-28dpf) exposures were conducted at 1% of the concentration known to kill 50% (LC50) of the population. Changes in the surface area of the swim bladder as well as in expression levels of genes involved in the thyroid control of swim bladder inflation were measured. At 6dpf, zebrafish exposed to all halogenated chemicals, both old use and next generation, had smaller posterior swim bladder and increased expression in the gene encoding thyroid peroxidase, tpo and the genes encoding two swim bladder surfactant proteins, sp-a and sp-c. These results mirrored the effects of thyroid hormone-exposed positive controls. Fish exposed to a TPO inhibitor (methimazole, MMI) had a decrease in tpo expression levels at 28dpf. Effects on the anterior swim bladder at 28dpf, after exposure to MMI as well as both old and new halogenated chemicals, were the same, i.e., absence of SB in ∼50% of fish, which were also of smaller body size. Overall, our results suggest thyroid disruption by the halogenated compounds tested via the swim bladder surfactant system. However, with the exception of TBBPA and TDCPP, the concentrations tested (∼5-137ppm) are not likely to be found in the environment.

Comparative analytical and toxicological assessment of methylcyclohexanemethanol (MCHM) mixtures associated with the Elk River chemical spill.

On January 9, 2014, a chemical mixture containing crude methylcyclohexanemethanol (MCHM) contaminated the water supply of Charleston, West Virginia. Although the mixture was later identified as a mix of crude MCHM and stripped propylene glycol phenyl ethers, initial risk assessment focused on 4-MCHM, the predominant component of crude MCHM. The mixture's exact composition and the toxicity differences between 4-MCHM, crude MCHM, and the tank mixture were unknown. We analyzed the chemical composition of crude MCHM and the tank mixture via GC/MS and, based on identified spectra, found that crude MCHM and the tank mixture differed in chemical composition. To evaluate acute developmental toxicity, zebrafish embryos were exposed to 0, 1, 6.25, 12.5, 25, 50, or 100 parts per million (ppm; mg/L) of 4-MCHM, crude MCHM, or the tank mixture. The percent mortality and percent hatch, larval morphology alterations, and larval visual motor response test were used to establish toxicity profiles for each of the chemicals or mixtures. The acute toxicity differed between 4-MCHM, crude MCHM and the tank mixture with significant differences in survival, hatching, morphology, and locomotion at levels as low as the short-term screening level of 1 ppm, suggesting a need for further research into human health risks. This study is the first to evaluate the developmental toxicity of the tank mixture and highlights that studies evaluating risk should not assume the effects of 4-MCHM or crude MCHM are representative of the Tank 396 mixture.

Atrazine exposure decreases the activity of DNMTs, global DNA methylation levels, and dnmt expression.

Atrazine, a herbicide used on agricultural crops is widely applied in the Midwestern United States as well as other areas of the globe. Atrazine frequently contaminates potable water supplies and is a suspected endocrine disrupting chemical. Previous studies have reported morphological, hormonal, and molecular alterations due to developmental and adulthood atrazine exposure; however, studies examining epigenetic alterations are limited. In this study, the effects of atrazine exposure on DNA methyltransferase (DNMT) activity and kinetics were evaluated. Global DNA methylation levels and dnmt expression in zebrafish larvae exposed to 0, 3, or 30 parts per billion (ppb) atrazine throughout embryogenesis was then assessed. Results indicate that atrazine significantly decreased the activity of maintenance DNMTs and that the inhibition mechanism can be described using non-competitive Michaelis-Menten kinetics. Furthermore, results show that an embryonic atrazine exposure decreases global methylation levels and the expression of dnmt4 and dnmt5. These findings indicate that atrazine exposure can decrease the expression and activity of DNMTs, leading to decreased DNA methylation levels.

Design and Synthesis of Chlorinated and Fluorinated 7-Azaindenoisoquinolines as Potent Cytotoxic Anticancer Agents That Inhibit Topoisomerase I.

The 7-azaindenoisoquinolines are cytotoxic topoisomerase I (Top1) inhibitors. Previously reported representatives bear a 3-nitro group. The present report documents the replacement of the potentially genotoxic 3-nitro group by 3-chloro and 3-fluoro substituents, resulting in compounds with high Top1 inhibitory activities and potent cytotoxicities in human cancer cell cultures and reduced lethality in an animal model. Some of the new Top1 inhibitors also possess moderate inhibitory activities against tyrosyl-DNA phosphodiesterase 1 (TDP1) and tyrosyl-DNA phosphodiesterase 2 (TDP2), two enzymes that are involved in DNA damage repair resulting from Top1 inhibitors, and they produce significantly more DNA damage in cancer cells than in normal cells. Eighteen of the new compounds had cytotoxicity mean-graph midpoint (MGM) GI50 values in the submicromolar (0.033-0.630 µM) range. Compounds 16b and 17b are the most potent in human cancer cell cultures with MGM GI50 values of 0.063 and 0.033 µM, respectively. Possible binding modes to Top1 and TDP1were investigated by molecular modeling.

Embryonic Atrazine Exposure Elicits Alterations in Genes Associated with Neuroendocrine Function in Adult Male Zebrafish.

The developmental origins of health and disease (DOHaD) hypothesis states that exposure to environmental stressors early in life can elicit genome and epigenome changes resulting in an increased susceptibility of a disease state during adulthood. Atrazine, a common agricultural herbicide used throughout the Midwestern United States, frequently contaminates potable water supplies and is a suspected endocrine disrupting chemical. In our previous studies, zebrafish was exposed to 0, 0.3, 3, or 30 parts per billion (µg/l) atrazine through embryogenesis, rinsed, and allowed to mature to adulthood. A decrease in spawning was observed with morphological alterations in offspring. In addition, adult females displayed an increase in ovarian progesterone and follicular atresia, alterations in levels of a serotonin metabolite and serotonin turnover in brain tissue, and transcriptome changes in brain and ovarian tissue supporting neuroendocrine alterations. As reproductive dysfunction is also influenced by males, this study assessed testes histology, hormone levels, and transcriptomic profiles of testes and brain tissue in the adult males. The embryonic atrazine exposure resulted in no alterations in body or testes weight, gonadosomatic index, testes histology, or levels of 11-ketotestosterone or testosterone. To further investigate potential alterations, transcriptomic profiles of adult male testes and brain tissue was completed. This analysis demonstrated alterations in genes associated with abnormal cell and neuronal growth and morphology; molecular transport, quantity, and production of steroid hormones; and neurotransmission with an emphasis on the hypothalamus-pituitary-adrenal and hypothalamus-pituitary-thyroid axes. Overall, this data indicate future studies should focus on additional neuroendocrine endpoints to determine potential functional impairments.

Zebrafish Get Connected: Investigating Neurotransmission Targets and Alterations in Chemical Toxicity.

Neurotransmission is the basis of neuronal communication and is critical for normal brain development, behavior, learning, and memory. Exposure to drugs and chemicals can alter neurotransmission, often through unknown pathways and mechanisms. The zebrafish (Danio rerio) model system is increasingly being used to study the brain and chemical neurotoxicity. In this review, the major neurotransmitter systems, including glutamate, GABA, dopamine, norepinephrine, serotonin, acetylcholine, histamine, and glutamate are surveyed and pathways of synthesis, transport, metabolism, and action are examined. Differences between human and zebrafish neurochemical pathways are highlighted. We also review techniques for evaluating neurological function, including the measurement of neurotransmitter levels, assessment of gene expression through transcriptomic analysis, and the recording of neurobehavior. Finally examples of chemical toxicity studies evaluating alterations in neurotransmitter systems in the zebrafish model are reviewed.