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.

BACKGROUND The insecticide tefluthrin is widely used in agriculture, resulting in widespread pollution. Tefluthrin is a type I pyrethroid characterized by its high persistence in the environment. Understanding the mechanisms of toxicity of tefluthrin will improve its risk assessment. OBJECTIVES We aimed to decipher the molecular modes of action of tefluthrin. METHODS Phenotypic developmental toxicity was assessed by exposing zebrafish embryos and larvae to increasing concentrations of tefluthrin. Tg(mnx:mGFP) line was used to assess neurotoxicity. Multi-omics approaches including transcriptomics and lipidomics were applied to analyze RNA and lipid contents, respectively. Finally, an in-silico ligand–protein docking computational method was used to study a possible interaction between tefluthrin and a protein target. RESULTS Tefluthrin exposure caused severe morphological malformations in zebrafish larvae, including motor neuron abnormalities. The differentially expressed genes were associated with neurotoxicity and metabolic disruption. Lipidomics analysis revealed a disruption in fatty acid, phospholipid, and lysophospholipid recycling. Protein docking modeling suggested that the LPCAT3 enzyme, which recycles lysophospholipids in the Land’s cycle, directly interacts with tefluthrin. CONCLUSIONS Tefluthrin exposure causes morphological and neuronal malformations in zebrafish larvae at nanomolar concentrations. Multi-omics results revealed a potential molecular initiating event i.e., inhibition of LPCAT3, and key events i.e., an altered lysophospholipid to phospholipid ratio, leading to the adverse outcomes of neurotoxicity and metabolic disruption.

Perfluorooctane sulfonate (PFOS), a class of synthetic chemicals detected in various environmental compartments, has been associated with dysfunctions of the human central nervous system (CNS). However, the underlying neurotoxicology of PFOS exposure is largely understudied due to the lack of relevant human models. Here, we report bioengineered human midbrain organoid microphysiological systems (hMO-MPSs) to recapitulate the response of a fetal human brain to multiple concurrent PFOS exposure conditions. Each hMO-MPS consists of an hMO on a fully 3D printed holder device with a perfusable organoid adhesion layer for enhancing air-liquid interface culturing. Leveraging the unique, simply-fabricated holder devices, hMO-MPSs are scalable, easy to use, and compatible with conventional well-plates, and allow easy transfer onto a multiple-electrode array (MEA) system for plug-and-play measurement of neural activity. Interestingly, the neural activity of hMO-MPSs initially increased and subsequently decreased by exposure to a concentration range of 0, 30, 100, to 300 μM of PFOS. Furthermore, PFOS exposure impaired neural development and promoted neuroinflammation in the engineered hMO-MPSs. Along with PFOS, our platform is broadly applicable for studies toxicology of various other environmental pollutants.

The regulation of appetite is of growing interest due to the significant rise in global obesity rates. Hunger and satiety are controlled by two hormones with functional activity in the brain; leptin, which is produced in adipocytes and suppresses food intake, and ghrelin, which is produced and released mainly by the stomach and functions as an appetite-stimulatory signal. In this study, zebrafish-based in vivo assays were used to examine whether BPA and five of its analogues, BPAF, BPE, BPC, BPC-CL, and BPS affect appetite regulation. The effect of bisphenol exposure on eating behavior was first examined. Four to six days old zebrafish larvae were exposed to a concentration range of the bisphenols and 17β-estradiol, followed by being fed a stained egg yolk powder at day six. After an hour of feeding, the feed in the gut was imaged by microscopy. Quantitative PCR was used to analyze the gene expression of leptin and ghrelin , as well as eleven other genes involved in appetite control. Exposures to BPA, BPAF, BPE, BPC, BPC-Cl and BPS, resulted in increased amounts of feed in the gut of the larvae in a concentration dependent manner. The qPCR results suggested that leptin mRNA expression was downregulated with the increasing concentrations of BPA, BPAF and BPC-Cl, whereas ghrelin mRNA expression was upregulated. The expression of several additional anorexigenic genes were downregulated by BPAF and BPC-Cl exposure, whereas orexigenic genes were upregulated. In conclusion, bisphenol exposures resulted in an increased eating behavior in zebrafish larvae, which correlated to increased mRNA expression of appetite-stimulatory genes and decreased expression of satiety-inducing genes. In addition, the results suggest that zebrafish larvae can be used to efficiently assess obesogenic capacity of environmental pollutants.### Competing Interest StatementThe authors have declared no competing interest.

Metabolic syndrome is increasing worldwide, particularly in rural communities, where residents have a higher risk of exposure to pesticides. We investigated whether six commonly used agricultural pesticides on corn and soy fields possess adipogenic and metabolic disruption activity. Exposure to two of these pesticides, the herbicides acetochlor and metolachlor, induced adipogenesis in vitro in mouse 3T3-L1 preadipocytes. The most potent compound, acetochlor, was selected for further studies in zebrafish. Acetochlor exposure induced morphological malformations and lethality in zebrafish larvae with an EC50 of 7.8 µM and LC50 of 12 µM. Acetochlor exposure at 10 nM resulted in lipid accumulation in zebrafish larvae when simultaneously fed a high-cholesterol diet. To decipher the molecular mechanisms behind acetochlor action, we performed transcriptomic and lipidomic analyses of exposed animals. The combined omics results suggested that acetochlor exposure increased Nrf2 activity in response to reactive oxygen species, as well as induced lipid peroxidation and ferroptosis. We further discovered that acetochlor structurally shares a chloroacetamide group with known inhibitors of glutathione peroxidase 4 (GPX4). Computational docking analysis suggested that acetochlor covalently binds to the active site of GPX4. Consistent with this prediction, Gpx activity was efficiently repressed by acetochlor in zebrafish, whereas lipid peroxidation was increased. We propose that acetochlor disrupts lipid homeostasis by inhibiting GPX activity, resulting in the accumulation of lipid peroxidation, 4-hydroxynonenal, and reactive oxygen species, which in turn activate Nrf2. Because metolachlor, among other acetanilide herbicides, also contains the chloroacetamide group, inhibition of GPX activity may represent a novel, common molecular initiating event of metabolic disruption.

The goal of PrecisionTox is to overcome conceptual barriers to replacing traditional mammalian chemical safety testing by accelerating the discovery of evolutionarily conserved toxicity pathways that are shared by descent among humans and more distantly related animals. An international consortium is systematically testing the toxicological effects of a diverse set of chemicals on a suite of five model species comprising fruit flies, nematodes, water fleas, and embryos of clawed frogs and zebrafish along with human cell lines. Multiple forms of omics and comparative toxicology data are integrated to map the evolutionary origins of biomolecular interactions that are predictive of adverse health effects, to major branches of the animal phylogeny. These conserved elements of adverse outcome pathways (AOPs) and their biomarkers are expected to provide mechanistic insight useful for regulating groups of chemicals based on their shared modes of action. PrecisionTox also aims to quantify risk variation within populations by recognizing susceptibility as a heritable trait that varies with genetic diversity. This initiative incorporates legal experts and collaborates with risk managers to address specific needs within European chemicals legislation, including the uptake of new approach methodologies (NAMs) for setting precise regulatory limits on toxic chemicals.

Triazoles are a major group of azole fungicides commonly used in agriculture, and veterinary and human medicine. Maternal exposure to certain triazole antifungal medication causes congenital malformations, including skeletal malformations. We hypothesized that triazoles used as pesticides in agriculture also pose a risk of causing skeletal malformations in developing embryos. In this study, teratogenic effects of three commonly used triazoles, cyproconazole, paclobutrazol, and triadimenol, were investigated in zebrafish, Danio rerio. Exposure to the triazole fungicides caused bone and cartilage malformations in developing zebrafish larvae. Data from whole-embryo transcriptomics with cyproconazole suggested that exposure to this compound induces adipogenesis while repressing skeletal development. Confirming this finding, the expression of selected bone and cartilage marker genes were significantly downregulated with triazoles exposure as determined by quantitative PCR. The expression of selected adipogenic genes was upregulated by the triazoles. Furthermore, exposure to each of the three triazoles induced adipogenesis and lipid droplet formation in vitro in 3T3-L1 pre-adipocyte cells. In vivo in zebrafish larvae, cyproconazole exposure caused lipid accumulation. These results suggest that exposure to triazoles promotes adipogenesis at the expense of skeletal development, and thus they expand the chemical group of bona fide bone to fat switchers.

Immunocyte infiltration and cytotoxicity play critical roles in both inflammation and immunotherapy. However, current cancer immunotherapy screening methods overlook the capacity of the T cells to penetrate the tumor stroma, thereby significantly limiting the development of effective treatments for solid tumors. Here, we present an automated high-throughput microfluidic platform for simultaneous tracking of the dynamics of T cell infiltration and cytotoxicity within the 3D tumor cultures with a tunable stromal makeup. By recourse to a clinical tumor-infiltrating lymphocyte (TIL) score analyzer, which is based on a clinical data-driven deep learning method, our platform can evaluate the efficacy of each treatment based on the scoring of T cell infiltration patterns. By screening a drug library using this technology, we identified an epigenetic drug (lysine-specific histone demethylase 1 inhibitor, LSD1i) that effectively promoted T cell tumor infiltration and enhanced treatment efficacy in combination with an immune checkpoint inhibitor (anti-PD1) in vivo. We demonstrated an automated system and strategy for screening immunocyte-solid tumor interactions, enabling the discovery of immuno- and combination therapies.

Because exposure to bisphenol A (BPA) has been linked to health problems in humans and wildlife, BPA analogues have been synthesized to be considered as replacement molecules. We here have examined estrogenic activity of BPA and five of its analogues, BPAF, BPE, BPC, BPC-Cl, and BPS by a combination of zebrafish-based in vivo and in vitro assays. We used transgenic estrogen reporter (5xERE:GFP) fish to study agonistic effects of bisphenols. Exposures to BPA, BPAF, BPE, and BPC, induced GFP expression in estrogen reporter fish at low exposure concentrations in the heart valves and at higher concentrations in the liver, whereas BPC-Cl activated GFP expression mainly in the liver, and BPS faintly in the heart only. The in vivo response was compared to in vitro estrogenicity of bisphenol exposure using reporter cells that express the zebrafish estrogen receptors driving expression of an estrogen response element (ERE)-luciferase reporter. In these cells, BPA, BPAF, BPC, BPE and BPS preferentially activated Esr1, whereas BPC-Cl preferentially activated Esr2a. By quantitative PCR we found that exposure to BPAF induced expression of the classical estrogen target genes vtg1, esr1, and cyp19a1b in a concentration response manner, but the most responsive target gene was f13a1a. Exposure to BPC-Cl resulted in a different expression pattern of vtg1 and f13a1a with an activation at low concentrations, followed by a declining expression at higher concentrations. Because expression of f13a1a was strongly activated by all compounds tested, we suggest including this mRNA as a biomarker for estrogenicity in larval fish. We further showed that exposure to BPAF and BPC-Cl increased E2 levels in zebrafish larvae, indicating that bisphenol exposures result in a feed-forward response that can further augment the estrogenic activity of these compounds.

In toxicology, standard sigmoidal concentration-response curves are used to predict effects concentrations and set chemical regulations. However, current literature also establishes the existence of complex, bimodal concentration-response curves, as is the case for arsenic toxicity. This bimodal response has been observed at the molecular level, but not characterized at the whole organism level. This study investigated the effect of arsenic (sodium arsenite) on post-gastrulated zebrafish embryos and elucidated effects of bimodal concentration responses on different phenotypic perturbations. Six hour post fertilized (hpf) zebrafish embryos were exposed to arsenic to 96 hpf. Hatching success, mortality, and morphometric endpoints were evaluated both in embryos with chorions and dechorionated embryos. Zebrafish embryos exhibited a bimodal response to arsenic exposure. Concentration-response curves for exposed embryos with intact chorions had an initial peak in mortality (88%) at 1.33 mM arsenic, followed by a decrease in toxicity (similar to 20% mortality) at 1.75 mM, and subsequently peaked to 100% mortality at higher concentrations. To account for the bimodal response, two distinct concentration-response curves were generated with estimated LC10 values (and 95% CI) of 0.462 (0.415, 0.508) mM and 1.69 (1.58, 1.78) mM for the 'low concentration' and 'high concentration' peaks, respectively. Other phenotypic analyses, including embryo length, yolk and pericardial edema all produced similar concentration-response patterns. Tests with dechorionated embryos also resulted in a bimodal toxicity response but with lower LC10 values of 0.170 (0.120, 0.220) mM and 0.800 (0.60, 0842) mM, respectively. Similarities in bimodal concentration-responses between with-chorion and dechorionated embryos indicate that the observed effect was not caused by the chorion limiting arsenic availability, thus lending support to other studies such as those that hypothesized a conserved bimodal mechanism of arsenic interference with nuclear receptor activation.

The 3T3-L1 murine pre-adipocyte line is an established cell culture model for screening Metabolism Disrupting Chemicals (MDCs). Despite a need to accurately identify MDCs for further evaluation, relatively little research has been performed to comprehensively evaluate reproducibility across laboratories, assess factors that might contribute to varying degrees of differentiation between laboratories (media additives, plastics, cell source, etc.), or to standardize protocols. As such, the goals of this study were to assess interlaboratory variability of efficacy and potency outcomes for triglyceride accumulation and pre-adipocyte proliferation using the mouse 3T3-L1 pre-adipocyte cell assay to test chemicals. Ten laboratories from five different countries participated. Each laboratory evaluated one reference chemical (rosiglitazone) and three blinded test chemicals (tributyltin chloride, pyraclostrobin, and bisphenol A) using: 1) their Laboratory-specific 3T3-L1 Cells (LC) and their Laboratory-specific differentiation Protocol (LP), 2) Shared 3T3-L1 Cells (SC) with LP, 3) LC with a Shared differentiation Protocol (SP), and 4) SC with SP. Blinded test chemical responses were analyzed by the coordinating laboratory. The magnitude and range of bioactivities reported varied considerably across laboratories and test conditions, though the presence or absence of activity for each tested chemical was more consistent. Triglyceride accumulation activity determinations for rosiglitazone ranged from 90 to 100% across test conditions, but 30-70 % for pre-adipocyte proliferation; this was 40-80 % for triglyceride accumulation induced by pyraclostrobin, 80-100 % for tributyltin, and 80-100 % for bisphenol A. Consistency was much lower for pre-adipocyte proliferation, with 30-70 % active determinations for pyraclostrobin, 30-50 % for tributyltin, and 20-40 % for bisphenol A. Greater consistency was observed for the SC/SP assessment. As such, working to develop a standardized adipogenic differentiation protocol represents the best strategy for improving consistency of adipogenic responses using the 3T3-L1 model to reproducibly identify MDCs and increase confidence in reported outcomes.

Nuclear receptors are classically defined as ligand-activated transcription factors that regulate key functions in reproduction, development, and physiology. Humans have 48 nuclear receptors, which when dysregulated are often linked to diseases. Because most nuclear receptors can be selectively activated or inactivated by small molecules, they are prominent therapeutic targets. The basic understanding of this family of transcription factors was accelerated in the 1980s upon the cloning of the first hormone receptors. During the next 20 years, a deep understanding of hormone signaling was achieved that has translated to numerous clinical applications, such as the development of standard-of-care endocrine therapies for hormonally driven breast and prostate cancers. A 2004 issue of this journal reviewed progress on elucidating the structures of nuclear receptors and their mechanisms of action. In the current issue, we focus on the broad application of new knowledge in this field for therapy across diverse disease states including cancer, cardiovascular disease, various inflammatory diseases, the aging brain, and COVID-19.

OBJECTIVE:Fetal exposure to the anticonvulsant drug valproic acid (VPA), used to treat certain types of epilepsy, increases the risk for birth defects, including neural tube defects, as well as learning difficulties and behavioral problems. Here, we investigated neurotoxic effects of VPA exposure using zebrafish as a model organism. The capacity of folic acid (FA) supplementation to rescue the VPA-induced neuronal and behavioral perturbations was also examined. METHODS:Zebrafish embryos of different transgenic lines with neuronal green fluorescent protein expression were exposed to increasing concentrations of VPA with or without FA supplementation. Fluorescence microscopy was used to visualize alterations in brain structures and neural progenitor cells, as well as motor neurons and neurite sprouting. A twitching behavioral assay was used to examine the functional consequences of VPA and FA treatment. RESULTS:In zebrafish embryos, VPA exposure caused a decrease in the midbrain size, an increase in the midline gap of the hindbrain, and perturbed neurite sprouting of secondary motor neurons, in a concentration-dependent manner. VPA exposure also decreased the fluorescence intensity of neuronal progenitor cells in early developmental stages, indicating fewer cells. Furthermore, VPA exposure significantly altered embryonic twitching activity, causing hyperactivity in dark and hypoactivity in light. Supplementation of FA rescued the VPA-induced smaller midbrain size and hindbrain midline gap defects. FA treatment also increased the number of neuronal progenitor cells in VPA-treated embryos and salvaged neurite sprouting of the secondary motor neurons. FA rescued the VPA-induced alterations in twitching activity in light but not in dark. SIGNIFICANCE:We conclude that VPA exposure induces specific neurotoxic perturbations in developing zebrafish embryos, and that FA reversed most of the identified defects. The results demonstrate that zebrafish is a promising model to study VPA-induced teratogenesis and to screen for countermeasures.

Breast cancer is a highly complex, heterogeneous, and multifactorial disease that poses challenges for rapid and efficient treatment and development of personalized therapy. Here, we describe a rapid and reliable method to generate three-dimensional (3D) tumor spheroids in vitro that recapitulate an individual patient's tumor for testing treatments. By employing droplet microfluidics and scaffold materials, tumor cells were encapsulated into a large number of Matrigel-in-oil droplets with precise control over cell numbers and components per droplet. After removal of the oil, large numbers of uniform tumor spheroids were formed within a few hours via Matrigel-supported cell self-assembly. Our microfluidic technique produces uniform-sized tumor spheroids in less than 1 day. This method was used to reproducibly and rapidly generate uniform-sized tumor spheroids derived from patients' breast tumor tissues. As a proof-of-concept application, this method was used to quickly evaluate cancer treatments. We demonstrated that our microfluidic patient-derived tumor cultures not only preserve the genetic characteristics of the original tumor tissue but also provide heterogeneous responses to targeted therapies within 2 days. We believe this method will enable a timely and reliable 3D in vitro culture model, which may be applicable to personalized treatment prediction, drug discovery, and toxicity testing.

The precise positioning and arrangement of cell spheroids and organoids are critical to reconstructing complex tissue architecture for tissue engineering and regenerative medicine. Here, we present a digital acoustofluidic method to manipulate cell spheroids and organoids with unprecedented dexterity. By introducing localized vibrations via a C-shaped integrated digital transducer (IDT), we can generate a trapping node to immobilize cell spheroids with a diameters ranging from 20μm to 300μm. Moreover, we digitally trapped multiple cell spheroids atop the C-shaped IDTs within a closed or open microfluidic chamber. By programming the trapping nodes within a 3 × 3 C-shaped IDT array, we can precisely position cell spheroids into designed patterns. We also demonstrated that our digital acoustofluidic device can accurately control the interaction of spheroid cells and organoids. Along with a simple fabrication procedure and setup, our digital acoustofluidic method can provide precisely manipulate and position various cell spheroids or organoids in a contactless, label-free, and highly biocompatible manner. We believe this technology can be widely used for tissue engineering, regenerative medicine, and fundamental cell biology research.

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

The high volume production compound bisphenol A (BPA) is of environmental concern largely because of its estrogenic activity. Consequently, BPA analogues have been synthesized to be considered as replacement molecules for BPA. These analogues need to be thoroughly evaluated for their estrogenic activity. Here, we combined mechanism zebrafish-based assays to examine estrogenic and anti-estrogenic activities of BPA and two of its analogues, bisphenol AF (BPAF) and bisphenol C (BPC) in vitro and in vivo. In vitro reporter cell lines were used to investigate agonistic and antagonistic effects of the three bisphenols on the three zebrafish estrogen receptors. The transgenic Tg(5 × ERE:GFP) and Cyp19a1b-GFP zebrafish lines were then used to analyze estrogenic and anti-estrogenic responses of the three bisphenols in vivo. BPA, BPAF and BPC were agonists with different potencies for the three zebrafish estrogen receptors in vitro. The potent zfERα-mediated activity of BPA and BPAF in vitro resulted in vivo by activation of GFP expression in zebrafish larvae in the heart (zfERα-dependent) at lower concentrations, and in the liver (zfERβ-dependent) at higher concentrations. BPC induced zfERβ-mediated luciferase expression in vitro, and the zfERβ agonism led to activation of GFP expression in the liver and the brain in vivo. In addition, BPC acted as a full antagonist on zfERα, and completely inhibited estrogen-induced GFP expression in the heart of the zebrafish larvae. To summarize, applying a combination of zebrafish-based in vitro and in vivo methods to evaluate bisphenol analogues for estrogenic activity will facilitate the prioritization of these chemicals for further analysis in higher vertebrates as well as the risk assessment in humans.

BACKGROUND:Polyphenol catechins from green tea, particularly (-)-epigallocatechin-3-gallate (EGCG), exhibits numerous beneficial health effects, although the mechanisms remain unclear. METHODS:In this study, the mechanism of EGCG-mediated healing in an experimentally injured zebrafish model was examined at the cellular and molecular level using confocal microscopy and gene expression analysis. RESULTS:The mechanisms of action of EGCG were shown to involve: (1) reducing neutrophil response (accumulation, travel speed, and distance) and (2) downregulating the expression of IL-1β, TNFα, and related signaling pathways. As determined by dynamic time-lapse tracking studies, the local accumulation of neutrophils with high migration speeds after wounding (n=33 cells, v=0.020 μm/s, d=37.8 μm), underwent significant reduction following treatment with EGCG doses of 300 μM (n=22 cells, v=0.013 μm/s, d=39.5 μm) and 600 μM (n=18 cells, v=0.008 μm/s, d=9.53 μm). Reverse transcription polymerase chain reaction studies revealed that several signature genes in the IL-1β, TNFα, and related signaling pathways were downregulated after EGCG treatment. CONCLUSION:The convenience, transparency, and simplicity of the zebrafish model facilitate tracking of fluorescent neutrophils in real time, in order to monitor inflammation, and assess the impact of therapeutic agents.