Evaluation of skin sensitization induced by four ionic liquids.

Ionic liquids (ILs) are synthetic solvents used as replacements for volatile organic solvents. Human exposure occurs through dermal or oral routes. In rodents, several ILs were reported to induce dermal toxicity, irritation, and sensitization. Due to the potential for occupational exposure, and industrial use as nonvolatile solvents, 1-ethyl-3-methylimidazolium chloride (EMIM, 6.25% to 50% v/v), 1-butyl-3-methylimidazolium chloride (BMIM, 3.12% to 12.5% v/v), 1-butyl-1-methylpyrrolidinium chloride (BMPY, 0.825% to 6.25% v/v), and N-butylpyridinium chloride (NBuPY, 0.825% to 12.5% v/v) were nominated to the National Toxicology Program and evaluated for skin sensitization. The test compound was applied to the ears of female BALB/c mice daily for 3 days in a primary irritancy (IRR)/local lymph node assay (LLNA). Sensitization was assessed in vitro in the direct peptide reactivity assay (DPRA), KeratinoSens™ assay, and human cell line activation test (h-CLAT). In the LLNA, the butylated ILs, BMIM, and BMPY were more potent than NBuPY (butylated) or EMIM (ethylated), which was neither an irritant nor a sensitizer. NBuPY induced skin irritation in vivo at ≥3.12% (p ≤ 0.01), and sensitization in vitro in the KeratinoSens™ assay and h-CLAT, but was negative for sensitization in vivo and in the DPRA. Although SI3 was not achieved, dermal treatment with 12.5% BMIM or 6.25% BMPY increased (p ≤ 0.01) lymph node cell proliferation in the LLNA. In vitro, BMIM was positive for sensitization in the h-CLAT, and BMPY was positive in the h-CLAT and KeratinoSens™ assay; both were negative in the DPRA. Integrated data analyses, weighted toward in vivo data, suggested that BMIM and BMPY may induce weak to mild sensitization.

Whole-body inhalation exposure to 2-ethyltoluene for two weeks produced nasal lesions in rats and mice.

OBJECTIVE: Ethyltoluenes are isolated during crude oil refinement for use in gasoline and commercial products and are ubiquitous in the environment. However, minimal toxicity data are available. Previously, we identified 2-ethyltoluene (2-ET) as the most potent isomer via nose-only inhalation exposure in rodents. Here, we expanded the hazard characterization of 2-ET in two rodent models using whole-body inhalation exposure and evaluated the role of prenatal exposure. METHODS: Time-mated Hsd:Sprague Dawley® SD® rats were exposed to 0, 150, 300, 600, 900, or 1200 ppm 2-ET via inhalation starting on gestation day 6 until parturition. Rat offspring (n = 8/exposure/sex) were exposed to the same concentrations as the respective dams for 2 weeks after weaning. Adult male and female B6C3F1/N mice (n = 5/exposure/sex) were exposed to the same concentrations for 2 weeks. RESULTS AND DISCUSSION: Exposure to ≥600 ppm 2-ET produced acute toxicity in rats and mice characterized by large decreases in survival, body weight, adverse clinical observations, and diffuse nasal olfactory epithelium degeneration (rats) or necrosis (mice). Due to the early removal of groups ≥600 ppm, most endpoint evaluations focused on lower exposure groups. In 150 and 300 ppm exposure groups, reproductive performance and littering were not significantly changed and body weights in exposed rats and mice were 9-18% lower than controls. Atrophy of the olfactory epithelium and nerves was observed in all animals exposed to 150 and 300 ppm. These lesions were more severe in mice than in rats. CONCLUSION: Nasal lesions were observed in all animals after whole-body exposure up to 600 ppm 2-ET for 2 weeks. Future studies should focus on 2-ET metabolism and distribution to better understand species differences and refine hazard characterization of this understudied environmental contaminant.

Tolerability and age-dependent toxicokinetics following perinatal hydroxyurea treatment in Sprague Dawley rats.

Hydroxyurea (HU) is a valuable therapy for individuals with sickle cell anemia. With increased use of HU in children and throughout their lives, it is important to understand the potential effects of HU therapy on their development and fertility. Thus, studies were conducted to identify appropriate doses to examine long-term effects of prenatal and early postnatal HU exposure and to understand kinetics of HU at various life stages. Pregnant Sprague Dawley dams were administered HU (0-150 mg/kg/day) via oral gavage from gestation days 17 to 21 and during lactation. Pups were dosed with the same dose as their respective dam starting on postnatal day (PND) 10 and up to PND 34. There was minimal maternal toxicity, and no significant effects on littering at any dose of HU. Starting on ~PND 16, offspring displayed skin discoloration and alopecia at doses ≥75 mg/kg/day and lower body weight compared to controls at doses ≥100 mg/kg/day. Gestational transfer of HU was observed, but there was minimal evidence of lactational transfer. Our toxicokinetic studies suggest that the internal dose in offspring may be altered due to age, but not due to sex. The plasma area under the curve, a measure of systemic exposure, at doses tolerated by offspring was threefold to sevenfold lower than the internal therapeutic dose in humans. Therefore, strategies to establish clinically relevant exposures in animal studies are needed. Overall, these data are useful for the design of appropriate nonclinical studies in the future to evaluate the consequences of long-term HU treatment starting in childhood.

Disposition of ß-N-methylamino-l-alanine (L-BMAA), a neurotoxin, in rodents following a single or repeated oral exposure.

ß-N-methylamino-l-alanine (L-BMAA) is produced by cyanobacteria (blue-green algae). Human exposure to L-BMAA occurs via consumption of L-BMAA-contaminated water and food. It is speculated that exposure to L-BMAA, and subsequent brain accumulation, may contribute to an increased incidence of neurodegenerative diseases indicating the need to evaluate risk of L-BMAA exposure to humans. As an initial step in this process, we have evaluated disposition following a single or repeated gavage administration of 1, 10 or 100mg/kg [14C]L-BMAA in rats and mice. L-BMAA was well absorbed following a single gavage administration with minimal dose, species, or sex-related effect. In both species, the main excretion route was as exhaled CO2 (46-61%) with 7-13% and 1.4-8% of the administered dose excreted in the urine and feces, respectively. L-BMAA was distributed to all tissues examined; the total radioactivity in tissues increased with the dose and was significant in both species (8-20%). In male rats, L-BMAA was slowly eliminated from blood and tissues (half-lives ≥48h). Following 1, 5 and 10days of dosing in male rats, levels in tissues increased with the number of doses demonstrating potential for accumulation of BMAA-derived equivalents. There was no greater affinity for accumulation in the brain compared to other organs and tissues. Following repeated exposure in rats, amino acid mass shifts associated with L-BMAA were detected in brain peptides. However, the low frequency of occurrence suggests that the substitution of an amino acid with L-BMAA is not significant relative to substitutions and/or modifications by other L-BMAA-derived equivalents.

Recent Advances in Asymmetric Hydrogenation of Tetrasubstituted Olefins.

The asymmetric hydrogenation of tetrasubstituted olefins provides direct access to very useful biological molecules and intermediates. The development of the technology has been slow, due in part to the synthetic challenges involved in developing chiral catalysts for a successful asymmetric induction. We briefly recount the breakthroughs in functionalized and unfunctionalized tetrasubstituted olefins, from the reports of Zhou and Buchwald for functionalized and unfunctionalized substrates, respectively, to the advent of chiral phosphoramidite ligands. The main emphasis of this Perspective lies in bringing into focus the complexity and challenges of inducing an asymmetric reduction for these substrates, which includes a brief discussion of the mechanism, the latest developed chiral catalysts, and the enormous scientific opportunities that still exist in developing "go to" catalyst systems for the various substrate types.

In vitro cardiotoxicity assessment of environmental chemicals using an organotypic human induced pluripotent stem cell-derived model.

An important target area for addressing data gaps through in vitro screening is the detection of potential cardiotoxicants. Despite the fact that current conservative estimates relate at least 23% of all cardiovascular disease cases to environmental exposures, the identities of the causative agents remain largely uncharacterized. Here, we evaluate the feasibility of a combinatorial in vitro/in silico screening approach for functional and mechanistic cardiotoxicity profiling of environmental hazards using a library of 69 representative environmental chemicals and drugs. Human induced pluripotent stem cell-derived cardiomyocytes were exposed in concentration-response for 30min or 24h and effects on cardiomyocyte beating and cellular and mitochondrial toxicity were assessed by kinetic measurements of intracellular Ca2+ flux and high-content imaging using the nuclear dye Hoechst 33342, the cell viability marker Calcein AM, and the mitochondrial depolarization probe JC-10. More than half of the tested chemicals exhibited effects on cardiomyocyte beating after 30min of exposure. In contrast, after 24h, effects on cell beating without concomitant cytotoxicity were observed in about one third of the compounds. Concentration-response data for in vitro bioactivity phenotypes visualized using the Toxicological Prioritization Index (ToxPi) showed chemical class-specific clustering of environmental chemicals, including pesticides, flame retardants, and polycyclic aromatic hydrocarbons. For environmental chemicals with human exposure predictions, the activity-to-exposure ratios between modeled blood concentrations and in vitro bioactivity were between one and five orders of magnitude. These findings not only demonstrate that some ubiquitous environmental pollutants might have the potential at high exposure levels to alter cardiomyocyte function, but also indicate similarities in the mechanism of these effects both within and among chemicals and classes.

Comparative pulmonary toxicity of inhaled metalworking fluids in rats and mice.

Metalworking fluids (MWFs) are complex formulations designed for effective lubricating, cooling, and cleaning tools and parts during machining operations. Adverse health effects such as respiratory symptoms, dermatitis, and cancer have been reported in workers exposed to MWFs. Several constituents of MWFs have been implicated in toxicity and have been removed from the formulations over the years. However, animal studies with newer MWFs demonstrate that they continue to pose a health risk. This investigation examines the hypothesis that unrecognized health hazards exist in currently marketed MWF formulations that are presumed to be safe based on hazard assessments of individual ingredients. In vivo 13-week inhalation studies were designed to characterize and compare the potential toxicity of four MWFs: Trim VX, Cimstar 3800, Trim SC210, and Syntilo 1023. Male and female Wistar Han rats or Fischer 344N/Tac rats and B6C3F1/N mice were exposed to MWFs via whole-body inhalation at concentrations of 0, 25, 50, 100, 200, or 400 mg/m3 for 13 weeks, after which, survival, body and organ weights, hematology and clinical chemistry, histopathology, and genotoxicity were assessed following exposure. Although high concentrations were used, survival was not affected and toxicity was primarily within the respiratory tract of male and female rats and mice. Minor variances in toxicity were attributed to differences among species as well as in the chemical components of each MWF. Pulmonary fibrosis was present only in rats and mice exposed to Trim VX. These data confirm that newer MWFs have the potential to cause respiratory toxicity in workers who are repeatedly exposed via inhalation.

Temporal and spatial increase of reactive nitrogen species in the kainate model of temporal lobe epilepsy.

Steady-state levels of reactive oxygen species (ROS) and oxidative damage to cellular macromolecules are increased in the rodent hippocampus during epileptogenesis. However, the role of reactive nitrogen species (RNS) in epileptogenesis remains to be explored. The goal of this study was to determine the spatial and temporal occurrence of RNS i.e. nitric oxide levels in a rat model of temporal lobe epilepsy (TLE). Rats were injected with a single high dose of kainate and monitored by video for behavioral seizures for 6weeks to determine the onset and severity of chronic seizures. RNS and tissue/mitochondrial redox status (glutathione redox couple and coenzyme A:glutathione redox couple) were measured in the hippocampus at 8h, 24h, 48h, 1wk, 3wk and 6wk following kainate to assess the level of reactive species in subcellular compartments. We observed a biphasic increase in RNS levels with a return to control values at the 48h time point. However, both tissue and mitochondrial redox status showed permanent and significant decreases during the entire time course of epilepsy development. 3 nitrotyrosine (3NT) protein adducts were found to gradually increase throughout epileptogenesis, conceivably as a result of the local environment under oxidative and nitrosative stress. Colocalization of 3NT immunostaining with neuron- or astrocyte-specific markers revealed neuron-specific localization of 3NT in hippocampal principal neurons. Persistent and concurrent glutathione oxidation and nitrosative stress occur during epileptogenesis suggesting a favorable environment for posttranslational modifications.

The discovery of diazepinone-based 5-HT3 receptor partial agonists.

Serotonin type 3 (5-HT3) receptor partial agonists have been targeted as potential new drugs for the symptomatic relief of irritable bowel syndrome (IBS). Multiple diazepinone-based compounds have been discovered, which exhibit nanomolar binding affinity for the h5-HT3A receptor and display a range of intrinsic activities (IA=7-87% of 5-HT Emax) in HEK cells heterologously expressing the h5-HT3A receptor. Favorable physicochemical properties and in vitro ADME profile coupled with oral activity in the murine von Bezold-Jarisch reflex model demonstrates the series has promise for producing low to moderate IA partial agonists suitable for an IBS indication.

Disposition of fragrance ingredient [14C]1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)ethanone in male Fisher rats following oral administration and dermal application.

1. Disposition of 1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)ethanone (ß-OTNE), a fragrance ingredient in variety of consumer products, was investigated following a single oral (20 mg/kg) or a dermal (55 or 550 mg/kg) dose of [(14)C]ß-OTNE to male Fisher rats. 2. Following oral administration, 28% and 39% of the dose was recovered in urine and feces, respectively, 48 h following administration. About 73% of a 20 mg/kg dose was excreted in bile within 48 h post-administration supporting significant oral absorption of [(14)C]ß-OTNE. 3. Following dermal application to a covered site, absorption of [(14)C]ß-OTNE 96 h following application was low (ca. 14%) and dose-independent. When the dose site was uncovered, the absorption increased to ca. 33% (55 mg/kg) and ca. 72% (550 mg/kg). 4. [(14)C]ß-OTNE was distributed to tissues following both routes of exposure with the highest radioactive equivalents found in bladder, liver, kidney, adipose and pancreas. 5. Elimination of [(14)C]ß-OTNE equivalents in blood and tissues was slow following both oral and dermal application suggesting potential for accumulation following multiple exposure.

Interlaboratory Study on Zebrafish in Toxicology: Systematic Evaluation of the Application of Zebrafish in Toxicology's (SEAZIT's) Evaluation of Developmental Toxicity.

Embryonic zebrafish represent a useful test system to screen substances for their ability to perturb development. The exposure scenarios, endpoints captured, and data analysis vary among the laboratories who conduct screening. A lack of harmonization impedes the comparison of the substance potency and toxicity outcomes across laboratories and may hinder the broader adoption of this model for regulatory use. The Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative was developed to investigate the sources of variability in toxicity testing. This initiative involved an interlaboratory study to determine whether experimental parameters altered the developmental toxicity of a set of 42 substances (3 tested in duplicate) in three diverse laboratories. An initial dose-range-finding study using in-house protocols was followed by a definitive study using four experimental conditions: chorion-on and chorion-off using both static and static renewal exposures. We observed reasonable agreement across the three laboratories as 33 of 42 test substances (78.6%) had the same activity call. However, the differences in potency seen using variable in-house protocols emphasizes the importance of harmonization of the exposure variables under evaluation in the second phase of this study. The outcome of the Def will facilitate future practical discussions on harmonization within the zebrafish research community.

Post-translational oxidative modification and inactivation of mitochondrial complex I in epileptogenesis.

Mitochondrial oxidative stress and damage have been implicated in the etiology of temporal lobe epilepsy, but whether or not they have a functional impact on mitochondrial processes during epilepsy development (epileptogenesis) is unknown. One consequence of increased steady-state mitochondrial reactive oxygen species levels is protein post-translational modification (PTM). We hypothesize that complex I (CI), a protein complex of the mitochondrial electron transport chain, is a target for oxidant-induced PTMs, such as carbonylation, leading to impaired function during epileptogenesis. The goal of this study was to determine whether oxidative modifications occur and what impact they have on CI enzymatic activity in the rat hippocampus in response to kainate (KA)-induced epileptogenesis. Rats were injected with a single high dose of KA or vehicle and evidence for CI modifications was measured during the acute, latent, and chronic stages of epilepsy. Mitochondrial-specific carbonylation was increased acutely (48 h) and chronically (6 week), coincident with decreased CI activity. Mass spectrometry analysis of immunocaptured CI identified specific metal catalyzed carbonylation to Arg76 within the 75 kDa subunit concomitant with inhibition of CI activity during epileptogenesis. Computational-based molecular modeling studies revealed that Arg76 is in close proximity to the active site of CI and carbonylation of the residue is predicted to induce substantial structural alterations to the protein complex. These data provide evidence for the occurrence of a specific and irreversible oxidative modification of an important mitochondrial enzyme complex critical for cellular bioenergetics during the process of epileptogenesis.

Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT): Developing a Data Analysis Pipeline for the Assessment of Developmental Toxicity with an Interlaboratory Study.

The embryonic zebrafish is a useful vertebrate model for assessing the effects of substances on growth and development. However, cross-laboratory developmental toxicity outcomes can vary and reported developmental defects in zebrafish may not be directly comparable between laboratories. To address these limitations for gaining broader adoption of the zebrafish model for toxicological screening, we established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) program to investigate how experimental protocol differences can influence chemical-mediated effects on developmental toxicity (i.e., mortality and the incidence of altered phenotypes). As part of SEAZIT, three laboratories were provided a common and blinded dataset (42 substances) to evaluate substance-mediated effects on developmental toxicity in the embryonic zebrafish model. To facilitate cross-laboratory comparisons, all the raw experimental data were collected, stored in a relational database, and analyzed with a uniform data analysis pipeline. Due to variances in laboratory-specific terminology for altered phenotypes, we utilized ontology terms available from the Ontology Lookup Service (OLS) for Zebrafish Phenotype to enable additional cross-laboratory comparisons. In this manuscript, we utilized data from the first phase of screening (dose range finding, DRF) to highlight the methodology associated with the development of the database and data analysis pipeline, as well as zebrafish phenotype ontology mapping.

Short-term toxicity studies of thallium (I) sulfate administered in drinking water to Sprague Dawley rats and B6C3F1/N mice.

Thallium is a heavy metal that is known to induce a broad spectrum of adverse health effects in humans including alopecia, neurotoxicity, and mortality following high dose acute poisoning events. Widespread human exposure to thallium may occur via consumption of contaminated drinking water; limited toxicity data are available to evaluate the corresponding public health risk. To address this data gap, the Division of Translational Toxicology conducted short-term toxicity studies of a monovalent thallium salt, thallium (I) sulfate. Thallium (I) sulfate was administered via dosed drinking water to time-mated Sprague Dawley (Hsd:Sprague Dawley® SD®) rats (F0 dams) and their offspring (F1) from gestation day (GD) 6 until up to postnatal day (PND) 28 at concentrations of 0, 3.13, 6.25, 12.5, 25, or 50 mg/L, and adult male and female B6C3F1/N mice for up to 2 weeks at concentrations of 0, 6.25, 12.5, 25, 50, or 100 mg/L. Rat dams in the 50 mg/L exposure group were removed during gestation, and dams and offspring in the 25 mg/L exposure group were removed on or before PND 0 due to overt toxicity. Exposure to thallium (I) sulfate at concentrations ≤ 12.5 mg/L did not impact F0 dam body weights, maintenance of pregnancy, littering parameters, or F1 survival (PND 4-28). However, in F1 pups, exposure to 12.5 mg/L thallium (I) sulfate resulted in decreased body weight gains relative to control rats and onset of whole-body alopecia. Measurement of thallium concentrations in dam plasma, amniotic fluid, fetuses (GD 18), and pup plasma (PND 4) indicated marked maternal transfer of thallium to offspring during gestation and lactation. Mice exposed to 100 mg/L thallium (I) sulfate were removed early due to overt toxicity, and mice exposed to ≥ 25 mg/L exhibited exposure concentration-related decreases in body weight. Lowest-observed-effect levels of 12.5 mg/L (rats) and 25 mg/L (mice) were determined based on the increased incidence of clinical signs of alopecia in F1 rat pups and significantly decreased body weights for both rats and mice.

Exploring the Influence of Experimental Design on Toxicity Outcomes in Zebrafish Embryo Tests.

Compound toxicity data obtained from independent zebrafish laboratories can vary vastly, complicating the use of zebrafish screening for regulatory decisions. Differences in the assay protocol parameters are the primary source of variability. We investigated this issue by utilizing data from the NTP DNT-DIVER database (https://doi.org/10.22427/NTP-DATA-002-00062-0001-0000-1, last accessed June 2, 2022), which consists of data from zebrafish developmental toxicity (devtox) and locomotor response (designated as "neurotox") screens from 3 independent laboratories, using the same set of 87 compounds. The data were analyzed using the benchmark concentration (BMC) modeling approach, which estimates the concentration of interest based on a predetermined response threshold. We compared the BMC results from 3 laboratories (A, B, C) in 3 toxicity outcome categories: mortality, cumulative devtox, and neurotox, in terms of activity calls and potency values. We found that for devtox screening, laboratories with similar/same protocol parameters (B vs C) had an active call concordance as high as 86% with negligible potency difference. For neurotox screening, active call concordances between paired laboratories are lower than devtox screening (highest 68%). When protocols with different protocol parameters were compared, the concordance dropped, and the potency shift was on average about 3.8-fold for the cumulative devtox outcome and 5.8-fold for the neurotox outcome. The potential contributing protocol parameters for potency shift are listed or ranked. This study provides a quantitative assessment of the source of variability in zebrafish screening protocols and sets the groundwork for the ongoing Systematic Evaluation of the Application of Zebrafish in Toxicology effort at the National Toxicology Program.

Plasma concentrations of tris(1-chloro-2-propyl) phosphate and a metabolite bis(2-chloroisopropyl) 1-carboxyethyl phosphate in Sprague-Dawley rats and B6C3F1/N mice from a chronic study of tris(chloropropyl) phosphate via feed.

Tris(chloropropyl) phosphate (TCPP) is an organophosphorus flame retardant and plasticizer used in manufacturing and multiple consumer products. Commercial TCPP is a ubiquitous environmental contaminant and TCPP or its metabolites have been detected in human plasma and urine. In response to the demonstrated widespread human exposure and lack of toxicity data, the Division of the National Toxicology Program is investigating the chronic toxicity of TCPP following perinatal exposure in HSD:Sprague Dawley®SD® (HSD) rats (up to 20,000 ppm) and adult exposure in B6C3F1/N mice (females, up to 10,000 ppm; males up to 5000 ppm) to TCPP via feed. Systemic exposure and bioaccumulation were assessed by measuring plasma concentrations of tris(1-chloro-2-propyl)phosphate (TCIPP), the most abundant TCPP isomer. TCIPP concentrations in TCPP-exposed rats and mice ranged from 3.43 to 1180 ng/mL and increased with exposure concentration at all time points. No sex differences were observed in rats, but male mice had higher TCIPP concentrations than females. TCIPP did not bioaccumulate in rats or mice over the course of the study. Low TCIPP concentrations were seen in some control rats and mice that were attributed to background TCPP present during sample collection, preparation and/or analysis. Bis(2-chloroisopropyl) 1-carboxyethyl phosphate (BCPCP), a TCPP metabolite, was quantified in plasma from control and selected exposed animals. Results showed increases in BCPCP concentration that were proportional to exposure concentration in rats and mice at concentrations much higher than TCIPP, indicating that BCPCP might be a more suitable biomarker of TCPP exposure.

Implementation of Zebrafish Ontologies for Toxicology Screening.

Toxicological evaluation of chemicals using early-life stage zebrafish (Danio rerio) involves the observation and recording of altered phenotypes. Substantial variability has been observed among researchers in phenotypes reported from similar studies, as well as a lack of consistent data annotation, indicating a need for both terminological and data harmonization. When examined from a data science perspective, many of these apparent differences can be parsed into the same or similar endpoints whose measurements differ only in time, methodology, or nomenclature. Ontological knowledge structures can be leveraged to integrate diverse data sets across terminologies, scales, and modalities. Building on this premise, the National Toxicology Program's Systematic Evaluation of the Application of Zebrafish in Toxicology undertook a collaborative exercise to evaluate how the application of standardized phenotype terminology improved data consistency. To accomplish this, zebrafish researchers were asked to assess images of zebrafish larvae for morphological malformations in two surveys. In the first survey, researchers were asked to annotate observed malformations using their own terminology. In the second survey, researchers were asked to annotate the images from a list of terms and definitions from the Zebrafish Phenotype Ontology. Analysis of the results suggested that the use of ontology terms increased consistency and decreased ambiguity, but a larger study is needed to confirm. We conclude that utilizing a common data standard will not only reduce the heterogeneity of reported terms but increases agreement and repeatability between different laboratories. Thus, we advocate for the development of a zebrafish phenotype atlas to help laboratories create interoperable, computable data.

Novel serotonin type 3 receptor partial agonists for the potential treatment of irritable bowel syndrome.

Serotonin type 3 (5-HT(3)) receptor partial agonists are being targeted as potential new drugs for the treatment of irritable bowel syndrome (IBS). Two new chemical series bearing indazole and indole cores have exhibited nanomolar binding affinity for the h5-HT(3)A receptor. A range of partial agonist activities in HEK cells heterologously expressing the h5-HT(3)A receptor were measured for the indazole series. Excellent 5-HT(3) receptor selectivity, favorable in vitro metabolic stability and CYP inhibition properties, and good oral in vivo potency in the murine von Bezold-Jarisch reflex model is exemplified thereby indicating the series to have potential utility as improved IBS agents.

Profiling the Tox21 Chemical Collection for Acetylcholinesterase Inhibition.

BACKGROUND: Inhibition of acetylcholinesterase (AChE), a biomarker of organophosphorous and carbamate exposure in environmental and occupational human health, has been commonly used to identify potential safety liabilities. So far, many environmental chemicals, including drug candidates, food additives, and industrial chemicals, have not been thoroughly evaluated for their inhibitory effects on AChE activity. AChE inhibitors can have therapeutic applications (e.g., tacrine and donepezil) or neurotoxic consequences (e.g., insecticides and nerve agents). OBJECTIVES: The objective of the current study was to identify environmental chemicals that inhibit AChE activity using in vitro and in silico models. METHODS: To identify AChE inhibitors rapidly and efficiently, we have screened the Toxicology in the 21st Century (Tox21) 10K compound library in a quantitative high-throughput screening (qHTS) platform by using the homogenous cell-based AChE inhibition assay and enzyme-based AChE inhibition assays (with or without microsomes). AChE inhibitors identified from the primary screening were further tested in monolayer or spheroid formed by SH-SY5Y and neural stem cell models. The inhibition and binding modes of these identified compounds were studied with time-dependent enzyme-based AChE inhibition assay and molecular docking, respectively. RESULTS: A group of known AChE inhibitors, such as donepezil, ambenonium dichloride, and tacrine hydrochloride, as well as many previously unreported AChE inhibitors, such as chelerythrine chloride and cilostazol, were identified in this study. Many of these compounds, such as pyrazophos, phosalone, and triazophos, needed metabolic activation. This study identified both reversible (e.g., donepezil and tacrine) and irreversible inhibitors (e.g., chlorpyrifos and bromophos-ethyl). Molecular docking analyses were performed to explain the relative inhibitory potency of selected compounds. CONCLUSIONS: Our tiered qHTS approach allowed us to generate a robust and reliable data set to evaluate large sets of environmental compounds for their AChE inhibitory activity. https://doi.org/10.1289/EHP6993.