Applications of Zebrafish Embryo Models to Predict Developmental Toxicity for Agrochemical Product Development.

The development of safe crop protection products is a complex process that traditionally relies on intensive animal use for hazard identification. Methods that capture toxicity in early stages of agrochemical discovery programs enable a more efficient and sustainable product development pipeline. Here, we explored whether the zebrafish model can be leveraged to identify mammalian-relevant toxicity. We used transgenic zebrafish to assess developmental toxicity following exposures to known mammalian teratogens and captured larval morphological malformations, including bone and vascular perturbations. We further applied toxicogenomics to identify common biomarker signatures of teratogen exposure. The results show that the larval malformation assay predicted teratogenicity with 82.35% accuracy, 87.50% specificity, and 77.78% sensitivity. Similar and slightly lower accuracies were obtained with the vascular and bone assays, respectively. A set of 20 biomarkers were identified that efficiently segregated teratogenic chemicals from nonteratogens. In conclusion, zebrafish are valuable, robust, and cost-effective models for toxicity testing in the early stages of product development.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) causes seizure activity in larval zebrafish via antagonism of γ-aminobutyric acid type A receptor α1ß2γ2.

Hexahydro-1,3,5-trinitro-1,3,5-triazine, or Royal Demolition Explosive (RDX), is a major component of plastic explosives such as C-4. Acute exposures from intentional or accidental ingestion are a documented clinical concern, especially among young male U.S. service members in the armed forces. When ingested in large enough quantity, RDX causes tonic-clonic seizures. Previous in silico and in vitro experiments predict that RDX causes seizures by inhibiting α1ß2γ2 γ-aminobutyric acid type A (GABAA) receptor-mediated chloride currents. To determine whether this mechanism translates in vivo, we established a larval zebrafish model of RDX-induced seizures. After a 3 h of exposure to 300 µM RDX, larval zebrafish exhibited a significant increase in motility in comparison to vehicle controls. Researchers blinded to experimental group manually scored a 20-min segment of video starting at 3.5 h post-exposure and found significant seizure behavior that correlated with automated seizure scores. Midazolam (MDZ), an nonselective GABAAR positive allosteric modulator (PAM), and a combination of Zolpidem (α1 selective PAM) and compound 2-261 (ß2/3-selective PAM) were effective in mitigating RDX-triggered behavioral and electrographic seizures. These findings confirm that RDX induces seizure activity via inhibition of the α1ß2γ2 GABAAR and support the use of GABAAR-targeted anti-seizure drugs for the treatment of RDX-induced seizures.

Developmental Neurotoxicity Screen of Psychedelics and Other Drugs of Abuse in Larval Zebrafish (Danio rerio).

In recent years, psychedelics have garnered significant interest as therapeutic agents for treating diverse neuropsychiatric disorders. However, the potential for these compounds to produce developmental neurotoxicity has not been rigorously assessed, and much of the available safety data is based on epidemiological studies with limited experimental testing in laboratory animal models. Moreover, the experimental safety data available thus far have focused on adult organisms, and the few studies conducted using developing organisms have tested a limited number of compounds, precluding direct comparisons between various chemical scaffolds. In the present study, 13 psychoactive compounds of different chemical or pharmacological classes were screened in a larval zebrafish model for teratological and behavioral abnormalities following acute and chronic developmental exposures. We found that the psychedelic tryptamines and ketamine were less neurotoxic to larval zebrafish than LSD and psychostimulants. Our work, which leverages the advantage of using zebrafish for higher throughput toxicity screening, provides a robust reference database for comparing the neurotoxicity profiles of novel psychedelics currently under development for therapeutic applications.

Diisopropylfluorophosphate (DFP) volatizes and cross-contaminates wells in a common 96-well plate format used in zebrafish larvae toxicology studies.

Diisopropylfluorophosphate (DFP) is an organophosphate (OP) that is commonly used as a surrogate of OP nerve agents to study the neurotoxic effects of acute OP intoxication. In preliminary studies, we discovered abnormally high incidence of deaths in DMSO control zebrafish larvae housed in the same 96-well plate as DFP-exposed larvae and hypothesized that DFP volatilizes and cross-contaminates wells when using static waterborne exposures. Survivability and acetylcholinesterase activity assays were indicative of the presence of DFP in the tissues of zebrafish ostensibly exposed to DMSO only. These findings are consistent with DFP cross-contamination, which raises concerns for the experimental design of studies evaluating the toxicity of volatile and semi-volatile substances in zebrafish using medium-to-high throughput approaches.

Exposure to permethrin or chlorpyrifos causes differential dose- and time-dependent behavioral effects at early larval stages of an endangered teleost species.

Pyrethroid and organophosphate pesticides are two of the most commonly used classes of insecticide worldwide. At sublethal concentrations, permethrin (a pyrethroid) and chlorpyrifos (an organophosphate) impact behavior in model fish species. We investigated behavioral effects of environmentally relevant concentrations of permethrin or chlorpyrifos on early larval delta smelt Hypomesus transpacificus, a Critically Endangered teleost species endemic to the San Francisco Bay Delta, California, USA. Using a photomotor behavioral assay of oscillating light and dark periods, we measured distance moved, turn angle, meander, angular velocity, rotations, thigmotaxis (time spent in the border versus center), and swim speed duration and frequency. The lowest concentrations of permethrin used in the tests (0.05 and 0.5 µg l-1) caused significant increases in distance moved at 72 and 96 h, respectively. At 48, 72, and 96 h of exposure, 5 µg l-1 of permethrin caused a hyperactive state in which the larvae significantly decreased thigmotaxis, quickly turning in short bouts of activity, characterized by significant increases in rotations and freezing events. Larvae exposed to 0.05 µg l-1 chlorpyrifos significantly increased thigmotaxis at 72 and 96 h. In response to 5 µg l-1 chlorpyrifos, larvae significantly increased velocity at 72 h exposure, and significantly increased freezing events at 96 h. Behavioral data on larval delta smelt exposed to contaminants present in their limited habitat have the potential to aid evaluations of the suitability of spawning and rearing habitats for this endangered species, thus improving conservation management strategies focused on this sensitive life stage.

The efficacy of γ-aminobutyric acid type A receptor (GABA AR) subtype-selective positive allosteric modulators in blocking tetramethylenedisulfotetramine (TETS)-induced seizure-like behavior in larval zebrafish with minimal sedation.

The chemical threat agent tetramethylenedisulfotetramine (TETS) is a γ-aminobutyric acid type A receptor (GABA AR) antagonist that causes life threatening seizures. Currently, there is no specific antidote for TETS intoxication. TETS-induced seizures are typically treated with benzodiazepines, which function as nonselective positive allosteric modulators (PAMs) of synaptic GABAARs. The major target of TETS was recently identified as the GABAAR α2ß3γ2 subtype in electrophysiological studies using recombinantly expressed receptor combinations. Here, we tested whether these in vitro findings translate in vivo by comparing the efficacy of GABAAR subunit-selective PAMs in reducing TETS-induced seizure behavior in larval zebrafish. We tested PAMs targeting α1, α2, α2/3/5, α6, ß2/3, ß1/2/3, and δ subunits and compared their efficacy to the benzodiazepine midazolam (MDZ). The data demonstrate that α2- and α6-selective PAMs (SL-651,498 and SB-205384, respectively) were effective at mitigating TETS-induced seizure-like behavior. Combinations of SB-205384 and MDZ or SL-651,498 and 2-261 (ß2/3-selective) mitigated TETS-induced seizure-like behavior at concentrations that did not elicit sedating effects in a photomotor behavioral assay, whereas MDZ alone caused sedation at the concentration required to stop seizure behavior. Isobologram analyses suggested that SB-205384 and MDZ interacted in an antagonistic fashion, while the effects of SL-651,498 and 2-261 were additive. These results further elucidate the molecular mechanism by which TETS induces seizures and provide mechanistic insight regarding specific countermeasures against this chemical convulsant.

Bifenthrin exposure causes hyperactivity in early larval stages of an endangered fish species at concentrations that occur during their hatching season.

Bifenthrin is a pyrethroid insecticide commonly used in agricultural and urban sectors, and is found in watersheds worldwide. As a sodium channel blocker, at sublethal concentrations it causes off-target effects, including disruption of calcium signaling and neuronal growth. At the whole organism level, sublethal concentrations of bifenthrin cause behavioral effects in fish species, raising concerns about the neurotoxic properties of the compound on fish populations. Here we describe the application of a high-throughput behavioral system to evaluate contaminant impacts on the sensitive early-life stages of Delta smelt (Hypomesus transpacificus), a critically endangered teleost species endemic to the San Francisco Bay Delta (SFBD), California, USA. Leveraging the natural behavior of early-larval Delta smelt, whereby they increase movement in bright light and decrease movement in the dark, we developed a test using a cycle of light and dark periods in a closed chamber to test hyper- or hypoactivity for this species. We show that early-larval Delta smelt have a significant preference to move toward light, and utilized the behavioral test to evaluate the impact of exposure to bifenthrin at concentrations found in habitats where Delta smelt reportedly spawn, ranging up to concentrations detected in tributaries to these habitats. All tested concentrations of bifenthrin (nominal 2, 10, or 100 ng/L) caused hyperactivity, over a 96 h exposure, with noted significance determined during the light period of the test. To further understand the impact of bifenthrin exposure, expression of a suite of genes relevant to neurodevelopment, the mechanistic target of rapamycin (mTOR) signaling pathway, and biotransformation in exposed larvae were also measured. Following exposure to picomolar concentrations of bifenthrin, expression of genes in the mTOR signaling and neurogenesis pathways were altered alongside behavior. This study demonstrates how light and dark cycle behavioral tests can be used to assess sensitive alterations in swimming activity in Delta smelt at early developmental stages and how gene expression can complement these assays. This approach can be used to assess the impact of multiple compounds that occur within the restricted habitat of Delta smelt, thus having the potential to greatly inform conservation management strategies for this critically sensitive life stage.

Differential regulation of select osmoregulatory genes and Na+/K+-ATPase paralogs may contribute to population differences in salinity tolerance in a semi-anadromous fish.

The Sacramento splittail (Pogonichthys macrolepidotus) is a species of special concern that is native to the San Francisco Estuary, USA. Two genetically distinct populations exist and differ in maximal salinity tolerances. We examined the expression of 12 genes representative of osmoregulatory functions in the gill over a 14  day time course at two different salinities [11 or 14 PSU (Practical Salinity Units)] and revealed that each population showed distinct patterns of gene expression consistent with population differences in response to osmotic regimes. The relatively more salinity-tolerant San Pablo population significantly upregulated nine out of the 12 transcripts investigated on day 1 of 11 PSU salinity exposure in comparison to the day zero freshwater control. Three transcripts (nka1a, nka1b, and mmp13) were differentially expressed between the populations at 7 and 14 days of salinity exposure, suggesting a reduced ability of the relatively salinity-intolerant Central Valley population to recover. Additionally, a phylogenetic analysis of several Sacramento splittail Na+/K+-ATPase α1 sequences resulted in grouping by proposed paralog rather than species, suggesting that different paralogs of this gene may exist. These findings, together with prior research conducted on the Sacramento splittail, suggest that the San Pablo population may be able to preferentially regulate select osmoregulatory genes, including different Na+/K+-ATPase α1 paralogs, to better cope with salinity challenges.

Developmental Staging and Salinity Tolerance in Embryos of the Delta Smelt, Hypomesus transpacificus.

Delta smelt (Hypomesus transpacificus) is a critically endangered species endemic to the San Francisco Bay Delta (SFBD). Important for the conservation of this species is understanding the physiological and ecological impacts contributing to their population decline, and current studies lack information on embryonic development. Changes in patterns of salinity across the SFBD may be a particularly important environmental stressor contributing to the recruitment and survival of the species. Throughout their ontogeny, delta smelt may exhibit unique requirements and tolerances to environmental conditions including salinity. Here, we describe 22 stages of embryonic development of H. transpacificus that characterize early differentiation from the fertilized egg until hatching, allowing the identification of critical morphological features unique to this species. Additionally, we investigated aspects of physiological tolerance to environmental salinity during development. Embryos survived incubation at salinity treatments between 0.4 and 20 ppt, yet had lower hatch success at higher salinities. Prior to hatching, embryos exposed to higher salinities had increased osmolalities and reduced fractions of yolk implying that the elevated external salinity altered the physiology of the embryo and the environment internal to the chorion. Lastly, egg activation and fertilization appear to also be impacted by salinity. Altogether, we suggest that any potential tolerance to salinity during embryogenesis, a common feature in euryhaline teleost species, impacts life cycle transitions into, and out of, embryonic development. Results from this investigation should improve conservation and management practices of this species and further expand our understanding of the intimate relationship between an embryo and its environment.