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.

Cannabis constituents reduce seizure behavior in chemically-induced and scn1a-mutant zebrafish.

Current antiepileptic drugs (AEDs) are undesirable for many reasons including the inability to reduce seizures in certain types of epilepsy, such as Dravet syndrome (DS) where in one-third of patients does not respond to current AEDs, and severe adverse effects that are frequently experienced by patients. Epidiolex, a cannabidiol (CBD)-based drug, was recently approved for treatment of DS. While Epidiolex shows great promise in reducing seizures in patients with DS, it is used in conjunction with other AEDs and can cause liver toxicity. To investigate whether other cannabis-derived compounds could also reduce seizures, the antiepileptic effects of CBD, Δ9-tetrahydrocannabinol (THC), cannabidivarin (CBDV), cannabinol (CBN), and linalool (LN) were compared in both a chemically-induced (pentylenetetrazole, PTZ) and a DS (scn1Lab-/-) seizure models. Zebrafish (Danio rerio) that were either wild-type (Tupfel longfin) or scn1Lab-/- (DS) were exposed to CBD, THC, CBDV, CBN, or LN for 24 h from 5 to 6 days postfertilization. Following exposure, total distance traveled was measured in a ViewPoint Zebrabox to determine if these compounds reduced seizure-like activity. Cannabidiol (0.6 and 1 µM) and THC (1 and 4 µM) significantly reduced PTZ-induced total distance moved. At the highest THC concentration, the significant reduction in PTZ-induced behavior was likely the result of sedation as opposed to antiseizure activity. In the DS model, CBD (0.6 µM), THC (1 µM), CBN (0.6 and 1 µM), and LN (4 µM) significantly reduced total distance traveled. Cannabinol was the most effective at reducing total distance relative to controls. In addition to CBD, other cannabis-derived compounds showed promise in reducing seizure-like activity in zebrafish. Specifically, four of the five compounds were effective in the DS model, whereas in the PTZ model, only CBD and THC were, suggesting a divergence in the mode of action among the cannabis constituents.

Susceptibility of larval zebrafish to the seizurogenic activity of GABA type A receptor antagonists.

Previous studies demonstrated that pentylenetetrazole (PTZ), a GABA type A receptor (GABAAR) antagonist, elicits seizure-like phenotypes in larval zebrafish (Danio rerio). Here, we determined whether the GABAAR antagonists, tetramethylenedisulfotetramine (TETS) and picrotoxin (PTX), both listed as credible chemical threat agents, similarly trigger seizures in zebrafish larvae. Larvae of three, routinely used laboratory zebrafish lines, Tropical 5D, NHGRI and Tupfel long fin, were exposed to varying concentrations of PTZ (used as a positive control), PTX or TETS for 20 min at 5 days post fertilization (dpf). Acute exposure to PTZ, PTX or TETS triggered seizure behavior in the absence of morbidity or mortality. While the concentration-effect relationship for seizure behavior was similar across zebrafish lines for each GABAAR antagonist, significantly less TETS was required to trigger seizures relative to PTX or PTZ. Recordings of extracellular field potentials in the optic tectum of 5 dpf Tropical 5D zebrafish confirmed that all three GABAAR antagonists elicited extracellular spiking patterns consistent with seizure activity, although the pattern varied between chemicals. Post-exposure treatment with the GABAAR positive allosteric modulators (PAMs), diazepam, midazolam or allopregnanolone, attenuated seizure behavior and activity but did not completely normalize electrical field recordings in the optic tectum. These data are consistent with observations of seizure responses in mammalian models exposed to these same GABAAR antagonists and PAMs, further validating larval zebrafish as a higher throughput-screening platform for antiseizure therapeutics, and demonstrating its appropriateness for identifying improved countermeasures for TETS and other convulsant chemical threat agents that trigger seizures via GABAAR antagonism.

Teratological and Behavioral Screening of the National Toxicology Program 91-Compound Library in Zebrafish (Danio rerio).

To screen the tens of thousands of chemicals for which no toxicity data currently exists, it is necessary to move from in vivo rodent models to alternative models, such as zebrafish. Here, we used dechorionated Tropical 5D wild-type zebrafish embryos to screen a 91-compound library provided by the National Toxicology Program (NTP) for developmental toxicity. This library contained 86 unique chemicals that included negative controls, flame retardants, polycyclic aromatic hydrocarbons (PAHs), drugs, industrial chemicals, and pesticides. Fish were exposed to 5 concentrations of each chemical or an equal amount of vehicle (0.5% DMSO) in embryo medium from 6 h post-fertilization (hpf) to 5 days post-fertilization (dpf). Fish were examined daily for mortality and teratogenic effects and photomotor behavior was assessed at 4 and 5 dpf. Of the 5 negative control compounds in the library, none caused mortality/teratogenesis, but two altered behavior. Chemicals provided in duplicate produced similar outcomes. Overall, 13 compounds caused mortality/teratology but not behavioral abnormalities, 24 only affected behavior, and 18 altered both endpoints, with behavior affected at concentrations that did not cause mortality/teratology (55/86 hits). Of the compounds that affected behavior, 52% caused behavioral abnormalities at either 4 or 5 dpf. Compounds within the same functional group caused different behavioral abnormalities, while similar behavioral patterns were caused by compounds from different groups. Our data suggest that behavior is a sensitive endpoint for developmental toxicity screening that integrates multiple modes of toxic action and is influenced by the age of the larval fish at the time of testing.

Multigenerational consequences of early-life cannabinoid exposure in zebrafish.

While Δ9-tetrahydrocannabinol (THC) has been widely studied in the realm of developmental and reproductive toxicology, few studies have investigated potential toxicities from a second widely used cannabis constituent, cannabidiol (CBD). CBD is popularized for its therapeutic potential for reducing seizure frequencies in epilepsy. This study investigated developmental origins of health and disease (DOHaD) via multigenerational gene expression patterns, behavior phenotypes, and reproductive fitness of a subsequent F1 following an F0 developmental exposure of zebrafish (Danio rerio) to THC (0.024, 0.12, 0.6 mg/L; 0.08, 0.4, 2 µM) or CBD (0.006, 0.03, 0.15 mg/L; 0.02, 0.1, 0.5 µM). Embryonic exposure at these concentrations did not cause notable morphological abnormalities in either F0 or F1 generations. However, during key developmental stages (14, 24, 48, 72, and 96 h post fertilization) THC and CBD caused differential expression of c-fos, brain-derived neurotrophic factor (bdnf), and deleted-in-azoospermia like (dazl), while in F1 larvae only CBD differentially expressed dazl. Larval photomotor behavior was reduced (F0) or increased (F1) by THC exposure, while CBD had no effect on F0 larvae, but decreased activity in the unexposed F1 larvae. These results support our hypothesis of cannabinoid-related developmental neurotoxicity. As adults, F0 fecundity was reduced, but it was not in F1 adults. Conversely, in the adult open field test there were no significant effects in F0 fish, but a significant reduction in the time in periphery was seen in F1 fish from the highest THC exposure group. The results highlight the need to consider long-term ramifications of early-life exposure to cannabinoids.

Developmental exposure to silver nanoparticles at environmentally relevant concentrations alters swimming behavior in zebrafish (Danio rerio).

Silver nanoparticles (Ag-NPs) are ubiquitous in household and medical products because of their antimicrobial activity. A consequence of the high volume of Ag-NP production and usage is increased amounts of Ag-NPs released into the environment. Their small size (1-100 nm) results in unique physiochemical properties that may increase toxicity relative to their bulk counterpart. Therefore, the goal of the present study was to assess the potential toxicity of environmentally relevant concentrations of Ag-NPs in zebrafish (Danio rerio). Wild-type tropical 5D zebrafish embryos were exposed to Ag-NPs from 4 to 120 h postfertilization at 0.03, 0.1, 0.3, 1, and 3 ppm (mg/L). Inductively coupled plasma-mass spectrometry confirmed concentration-dependent uptake of Ag into zebrafish as well as bioaccumulation over time. A morphological assessment revealed no significant hatching impairment, morphological abnormalities, or mortality at any concentration or time point examined. However, assessment of photomotor behavior at 3 d postfertilization (dpf) revealed significant hyperactivity in the 0.3, 1, and 3 ppm Ag-NP treatment groups. At 4 dpf, significant hyperactivity was observed only in the 3 ppm treatment group, whereas 5 dpf larvae exposed to Ag-NPs displayed no significant abnormalities in photomotor behavior. These findings suggest that nonteratogenic concentrations of Ag-NPs are capable of causing transient behavioral changes during development. Environ Toxicol Chem 2018;37:3018-3024. © 2018 SETAC.

Developmental Effects of Cannabidiol and Δ9-Tetrahydrocannabinol in Zebrafish.

Cannabidiol (CBD) has gained much attention in the past several years for its therapeutic potential in the treatment of drug-resistant epilepsy, such as Dravet syndrome. Although CBD has shown anecdotal efficacy in reducing seizure frequency, little is known regarding the potential adverse side effects of CBD on physiology, development, organogenesis, or behavior. The goal of this project was to compare the relative morphological, behavioral, and gene expression phenotypes resulting after a developmental exposure to Δ9-tetrahydrocannabinol (THC) or CBD. Zebrafish were exposed from blastula through larval stage (96 h postfertilization [hpf]) to 0.3, 0.6, 1.25, 2.5, 5 mg/l (1, 2, 4, 8, 16 µM) THC or 0.07, 0.1, 0.3, 0.6, 1.25 mg/l CBD (0.25, 0.5, 1, 2, 4 µM). Despite the similarity in THC and CBD dysmorphologies, ie, edemas, curved axis, eye/snout/jaw/trunk/fin deformities, swim bladder distention, and behavioral abnormalities, the LC50 for CBD (0.53 mg/l) was nearly 7 times lower than THC (3.65 mg/l). At 96 hpf, c-fos, dazl, and vasa were differentially expressed following THC exposure, but only c-fos expression was significantly increased by CBD. Cannabidiol was more bioconcentrated compared with THC despite higher THC water concentrations. This work supports the potential for persistent developmental impacts of cannabinoid exposure, but more studies are needed to assess latent effects and their molecular mechanisms of toxicity.

Developmental ethanol exposure impairs locomotor movement in Japanese medaka (Oryzias latipes) larvae targeting epigenome.

Evidence indicated ethanol exposure during development disrupts brain functions that induces fetal alcohol spectrum disorder (FASD) phenotypes with behavioral abnormalities. We aimed to investigate whether prenatal ethanol exposure has any potential impact on behavior of a FASD fish model. Fertilized Japanese medaka (Oryzias latipes) eggs were exposed to 100-300 mM ethanol or 2 mM 5-azacytidine (5-azaC), 0-2 day post fertilization (dpf), in embryo-rearing medium (ERM). Survived embryos were maintained in clean ERM and used either for gene expression analysis on 2- and 6-dpf or allowed to hatch for behavioral study. Photomotor response of 3-4 day post hatch larvae were tracked for 3 h with light-dark transitions. It was observed that larval swimming was phototactic; enhanced in presence of light, declined in dark. Phototactic response was also observed in larvae prenatally exposed to ethanol or 5-azaC; however, the total distance swum by these larvae compared to controls declined. Further analysis indicated that, in light phases, total swimming activity and average swimming speed were reduced in larvae prenatally exposed to ethanol (300 mM) or 5-azaC. Expression analysis of baz1a and baz2a in embryos indicated developmental regulation. Ethanol (100-300 mM) or 5-azaC (2 mM) were able to modulate downregulation of both baz1a and baz2a mRNAs only in 6 dpf embryos of 300 mM ethanol and 5-azaC (2 mM) groups. These studies indicated that prenatal exposure to ethanol or 5-azaC was able to disrupt movements and thus swimming behavior in FASD phenotypes probably due to delayed remodeling of genome and epigenome.

Levonorgestrel exposure to fathead minnows (Pimephales promelas) alters survival, growth, steroidogenic gene expression and hormone production.

Human pharmaceuticals are commonly detected in the environment. Concern over these compounds in the environment center around the potential for pharmaceuticals to interfere with the endocrine system of aquatic organisms. The main focus of endocrine disruption research has centered on how estrogenic and androgenic compounds interact with the endocrine system to elicit reproductive effects. Other classes of compounds, such as progestins, have been overlooked. Recently, studies have investigated the potential for synthetic progestins to impair reproduction and growth in aquatic organisms. The present study utilizes the OECD 210 Early-life Stage (ELS) study to investigate the impacts levonorgestrel (LNG), a synthetic progestin, on fathead minnow (FHM) survival and growth. After 28 days post-hatch, survival of larval FHM was impacted at 462 ng/L, while growth was significantly reduced at 86.9 ng/L. Further analysis was conducted by measuring specific endocrine related mRNA transcript profiles in FHM larvae following the 28 day ELS exposure to LNG. Transcripts of 3ß-HSD, 20ß-HSD, CYP17, AR, ERα, and FSH were significantly down-regulated following 28d exposure to 16.3 ng/L LNG, while exposure to 86.9 ng/L significantly down-regulated 3ß-HSD, 20ß-HSD, CYP19A, and FSH. At 2,392 ng/L of LNG, a significant down-regulation occurred with CYP19A and ERß transcripts, while mPRα and mPRß profiles were significantly induced. No significant changes occurred in 11ß-HSD, CYP11A, StAR, LHß, and VTG mRNA expression following LNG exposure. An ex vivo steroidogenesis assay was conducted with sexually mature female FHM following a 7 day exposure 100 ng/L LNG with significant reductions observed in pregnenolone, 17α,20ß-dihydroxy-4-pregnen-3-one (17,20-DHP), testosterone, and 11-ketotestosterone. Together these data suggest LNG can negatively impact FHM larval survival and growth, with significant alterations in endocrine related responses.