The zebrafish embryo as a model for chemically-induced steatosis: A case study with three pesticides.

There is an increasing incidence and prevalence of fatty liver disease in the western world, with steatosis as the most prevalent variant. Known causes of steatosis include exposure to food-borne chemicals, and overconsumption of alcohol, carbohydrates and fat, and it is a well-known side effect of certain pharmaceuticals such as tetracycline, amiodarone and tamoxifen (drug-induced hepatic steatosis). Mechanistic knowledge on chemical-induced steatosis has greatly evolved and has been organized into adverse outcome pathways (AOPs) describing the chain of events from first molecular interaction of a substance with a biological system to the adverse outcome, intrahepatic lipid accumulation. In this study, three known steatosis-inducing pesticides (imazalil, clothianidin, and thiacloprid) were tested for their ability to induce hepatic triglyceride accumulation in the zebrafish (Danio rerio) embryo (ZFE) at 5 days post fertilization, both as single compounds and equipotent binary mixtures. The results indicate that the ZFE is very well applicable as a higher tier testing model to confirm effects in downstream key events in AOPs, that is, chemically-induced triglyceride accumulation in the whole organism and production of visible steatosis. Moreover, dose addition could be concluded for binary mixtures of substances with similar and with dissimilar modes of action.

Robotic hiatal hernia repair without mesh.

Background: Newer minimally invasive techniques have supplanted laparotomy and thoracotomy for management of hiatal hernias. Limited data exists on outcomes after robotic hiatal hernia repair without mesh despite the increasing popularity of this approach. We report our high-volume experience with durable robotic hiatal hernia repair with gastric fundoplication without mesh. Methods: A retrospective review was conducted on patients with type I-IV hiatal hernias who underwent an elective robotic-assisted repair from 2016 to 2019 using a novel technique of approximating the hiatus with running barbed absorbable (V-locTM) suture and securing it with interrupted silk sutures. Main outcomes included length of stay, readmission rate, and recurrence rate. Results: A total of 144 patients were reviewed. The average age of the patient was 61 years. Most of the patients were female [95 females (66%) to 49 males], and the average body mass index (BMI) was 29.96 kg/m2. The average operating time was 173 minutes (standard deviation 62 minutes). The average length of stay in the hospital was 2 days, and 89% of patients went home within the first 3 days. Ten patients (6.9%) were readmitted within 30 days, there were no mortalities in 30 days, and there were 6 (4.2%) recurrences on follow up requiring reoperation. Conclusions: Elective robotic hiatal hernia repair with fundoplication and primary closure of the hiatus with V-locTM and nonabsorbable suture without mesh is safe and effective. The robotic approach has similar operative times, lengths of stay, and complications compared to nationally published data on laparoscopic hiatal hernia repairs.

CRISPR-mediated genome editing allows for efficient on demand creation of >200 kb deficiencies with precise boundaries.

Deficiency mapping remains a useful tool in the process of identifying causative genetic lesions in C. elegans mutant strains isolated from forward genetic screens, in particular of non-coding mutants. However, there are significant areas across the genome with no deficiency coverage at all, and the boundaries of many deficiencies remain poorly defined. Here, we describe a simple methodology to generate balanced deficiency strains with up to 230 kb molecularly defined deletions (mini-deficiencies) using CRISPR/Cas9, thus providing a simple path for both precise and tailored deficiency mapping.

Ice modulatory effect of the polysaccharide FucoPol in directional freezing.

Directional freezing harnesses crystal growth development to create aligned solid structures or etchable patterns, useful for directed ice growth in cryobiology and cryoprinting for tissue engineering. We have delved into the ice-modulating properties of FucoPol, a fucose-rich, bio-based polysaccharide. Previous research on FucoPol revealed its non-colligative hysteresis in kinetic freezing point, reduced crystal dimensions and cryoprotective effect. Here, FucoPol reshaped developing sharp, anisotropic obloid ice dendrites into linearly-aligned, thin, isotropic spicules or tubules (cooling rate-dependent morphology). The effect was enhanced by increased concentration and decreased cooling rate, but major reshaping was observed with 5 µM and below. These structures boasted remarkable enhancements: uniform alignment (3-fold), tip symmetry (5.9-fold) and reduced thickness (5.3-fold). The ice-modulating capability of FucoPol resembles the Gibbs-Thomson effect of antifreeze proteins, in particular the ice reshaping profiles of type I antifreeze proteins and rattlesnake venom lectins, evidenced by a 52.6 ± 2.2° contact angle (θ) and spicular structure generation. The high viscosity of FucoPol solutions, notably higher than that of sucrose, plays a crucial role. This viscosity dynamically intensifies during directional freezing, leading to a diffusion-limited impediment that influences dendritic formation. Essentially, the ice-modulating prowess of FucoPol not only reinforces its established cryoprotective qualities but also hints at its potential utility in applications that harness advantageous ice growth for intentional structuring. For instance, its potential in cryobioprinting is noteworthy, offering an economical, biodegradable resource, of easy removal, sidestepping the need for toxic reagents. Moreover, FucoPol fine-tunes resulting ice structures, enabling the ice-etching of biologically relevant patterns within biocompatible matrices for advanced tissue engineering endeavors.

Convertase-dependent regulation of membrane-tethered and secreted ligands tunes dendrite adhesion.

During neural development, cellular adhesion is crucial for interactions among and between neurons and surrounding tissues. This function is mediated by conserved cell adhesion molecules, which are tightly regulated to allow for coordinated neuronal outgrowth. Here, we show that the proprotein convertase KPC-1 (homolog of mammalian furin) regulates the Menorin adhesion complex during development of PVD dendritic arbors in Caenorhabditis elegans. We found a finely regulated antagonistic balance between PVD-expressed KPC-1 and the epidermally expressed putative cell adhesion molecule MNR-1 (Menorin). Genetically, partial loss of mnr-1 suppressed partial loss of kpc-1, and both loss of kpc-1 and transgenic overexpression of mnr-1 resulted in indistinguishable phenotypes in PVD dendrites. This balance regulated cell-surface localization of the DMA-1 leucine-rich transmembrane receptor in PVD neurons. Lastly, kpc-1 mutants showed increased amounts of MNR-1 and decreased amounts of muscle-derived LECT-2 (Chondromodulin II), which is also part of the Menorin adhesion complex. These observations suggest that KPC-1 in PVD neurons directly or indirectly controls the abundance of proteins of the Menorin adhesion complex from adjacent tissues, thereby providing negative feedback from the dendrite to the instructive cues of surrounding tissues.

Anatomical restructuring of a lateralized neural circuit during associative learning by asymmetric insulin signaling.

Studies of neuronal connectivity in model organisms, i.e., of their connectomes, have been instrumental in dissecting the structure-function relationship of nervous systems. However, the limited sample size of these studies has impeded analyses into how variation of connectivity across populations may influence circuit architecture and behavior. Moreover, little is known about how experiences induce changes in circuit architecture. Here, we show that an asymmetric salt-sensing circuit in the nematode Caenorhabditis elegans exhibits variation that predicts the animals' salt preferences and undergoes restructuring during salt associative learning. Naive worms memorize and prefer the salt concentration they experience in the presence of food through a left-biased neural network architecture. However, animals conditioned at elevated salt concentrations change this left-biased network to a right-biased network. This change in circuit architecture occurs through the addition of new synapses in response to asymmetric, paracrine insulin signaling. Therefore, experience-dependent changes in an animal's neural connectome are induced by insulin signaling and are fundamental to learning and behavior.

Rescue coordination common operating picture: Enhancement through satellite technology.

Natural hazards and human-made disasters are increasing in magnitude and frequency due to climate change. The increase in response assets across agencies is complicating synchronization and communication during lifesaving operations. Technological advancements in wireless communication systems are improving resource tasking, tracking, and awareness but are vulnerable to interruption from the destruction inflicted by natural hazards. However, satellite technology offers solutions to safeguard lifesaving coordination in austere environments. Through a focused review of the relevant literature and policy documents, this study analyzes disaster response command and control, emerging communication capabilities, and satellite technology related to developing a common operating picture for all actors. The aim is to provide practicing emergency managers with the means to overcome communication gaps in wireless and satellite technology to maintain this common picture, save lives in the wake of disasters, and protect emergency responders during hazardous mass rescue operations. Results indicate that communication technology does aid emergency managers in the highly pressurized response environment but the systems being relied upon are themselves vulnerable to disasters. The existing literature also establishes that response actors are leveraging satellite technology to fill gaps in communications connectivity during disasters to reduce risk, which has both national and international -implications.

Estradiol withdrawal following a hormone simulated pregnancy induces deficits in affective behaviors and increases ∆FosB in D1 and D2 neurons in the nucleus accumbens core in mice.

In placental mammals, estradiol levels are chronically elevated during pregnancy, but quickly drop to prepartum levels following birth. This may produce an "estrogen withdrawal" state that has been linked to changes in affective states in humans and rodents during the postpartum period. The neural mechanisms underlying these affective changes, however, are understudied. We used a hormone-simulated pseudopregnancy (HSP), a model of postpartum estrogen withdrawal, in adult female C57BL/6 mice to test the impact of postpartum estradiol withdrawal on several behavioral measures of anxiety and motivation. We found that estradiol withdrawal following HSP increased anxiety-like behavior in the elevated plus maze, but not in the open field or marble burying tests. Although hormone treatment during HSP consistently increased sucrose consumption, sucrose preference was generally not impacted by hormone treatment or subsequent estradiol withdrawal. In the social motivation test, estradiol withdrawal decreased the amount of time spent in proximity to a social stimulus animal. These behavioral changes were accompanied by changes in the expression of ∆FosB, a transcription factor correlated with stable long-term plasticity, in the nucleus accumbens (NAc). Specifically, estrogen-withdrawn females had higher ∆FosB expression in the nucleus accumbens core, but ∆FosB expression did not vary across hormone conditions in the nucleus accumbens shell. Using transgenic reporter mice, we found that this increase in ∆FosB occurred in both D1- and D2-expressing cells in the NAc core. Together, these results suggest that postpartum estrogen withdrawal impacts anxiety and motivation and increases ∆FosB in the NAc core.

Restructuring of an asymmetric neural circuit during associative learning.

Asymmetric brain function is common across the animal kingdom and involved in language processing, and likely in learning and memory. What regulates asymmetric brain function remains elusive. Here, we show that the nematode Caenorhabditis elegans restructures an asymmetric salt sensing neural circuit during associative learning. Worms memorize and prefer the salt concentration at which they were raised in the presence of food through a left-biased network architecture. When conditioned at elevated salt concentrations, animals change the left-biased to a right-biased network, which explains the changed salt-seeking behavior. The changes in circuit architecture require new synapse formation induced through asymmetric, paracrine insulin-signaling. Therefore, experience-dependent changes in asymmetric network architecture rely on paracrine insulin signaling and are fundamental to learning and behavior.

Suction-based catheter retrieval of right ventricular clot-in-transit.

Right ventricular clot-in-transit (CIT) is a rare finding in venous thromboembolic disease and carries a high mortality rate. Its optimal treatments have yet to be established in the literature. Here we describe the usage of a suction-based catheter, the INARI FlowTriever® system (INARI Medical Inc.) to successfully retrieve a CIT from the right ventricle of a patient with coronavirus disease 2019 acute respiratory distress syndrome on veno-veno extracorporeal membrane oxygenation.

Prioritization of chemicals in food for risk assessment by integrating exposure estimates and new approach methodologies: A next generation risk assessment case study.

Next generation risk assessment is defined as a knowledge-driven system that allows for cost-efficient assessment of human health risk related to chemical exposure, without animal experimentation. One of the key features of next generation risk assessment is to facilitate prioritization of chemical substances that need a more extensive toxicological evaluation, in order to address the need to assess an increasing number of substances. In this case study focusing on chemicals in food, we explored how exposure data combined with the Threshold of Toxicological Concern (TTC) concept could be used to prioritize chemicals, both for existing substances and new substances entering the market. Using a database of existing chemicals relevant for dietary exposure we calculated exposure estimates, followed by application of the TTC concept to identify substances of higher concern. Subsequently, a selected set of these priority substances was screened for toxicological potential using high-throughput screening (HTS) approaches. Remarkably, this approach resulted in alerts for a selection of substances that are already on the market and represent relevant exposure in consumers. Taken together, the case study provides proof-of-principle for the approach taken to identify substances of concern, and this approach can therefore be considered a supportive element to a next generation risk assessment strategy.

Identification of proteome markers for drug-induced liver injury in zebrafish embryos.

The zebrafish embryo (ZFE) is a promising alternative non-rodent model in toxicology, and initial studies suggested its applicability in detecting hepatic responses related to drug-induced liver injury (DILI). Here, we hypothesize that detailed analysis of underlying mechanisms of hepatotoxicity in ZFE contributes to the improved identification of hepatotoxic properties of compounds and to the reduction of rodents used for hepatotoxicity assessment. ZFEs were exposed to nine reference hepatotoxicants, targeted at induction of steatosis, cholestasis, and necrosis, and effects compared with negative controls. Protein profiles of the individual compounds were generated using LC-MS/MS. We identified differentially expressed proteins and pathways, but as these showed considerable overlap, phenotype-specific responses could not be distinguished. This led us to identify a set of common hepatotoxicity marker proteins. At the pathway level, these were mainly associated with cellular adaptive stress-responses, whereas single proteins could be linked to common hepatotoxicity-associated processes. Applying several stringency criteria to our proteomics data as well as information from other data sources resulted in a set of potential robust protein markers, notably Igf2bp1, Cox5ba, Ahnak, Itih3b.2, Psma6b, Srsf3a, Ces2b, Ces2a, Tdo2b, and Anxa1c, for the detection of adverse responses.

Pre-hospital caloric deficits in surgical patients.

BACKGROUND: The timing, route, and amount of nutrition for surgical patients with substantial caloric deficits remain active areas of study. Current guidelines are based on in-hospital days NPO after admission to the hospital. This historic process neglects the multiple days of caloric deficit patients experience prior to hospital admission. AIM: To determine the impact of pre-hospital caloric deficit (PHCD) for surgical patients on their outcomes. METHODS: 313 patients admitted with a diagnosis of small bowel obstruction, pancreatitis, or diverticulitis were analyzed for their PHCD's. PHCD's were estimated using patient-reported days with significant emesis, and absent oral intake. Patients with PHCD's were compared to patients with no PHCD for length of stay, status on discharge, disposition, and 30-day readmission rate. RESULTS: There were 313 patients and 42% of the patients were male. The median age was 65 years. Median number of days sick prior to hospital admission was 1 (IQR: 1 to 2). Median PHCD was 1882 kcal (IQR: 1355 to 3650). Median number of days NPO while in-hospital was 3 (IQR: 2 to 5). Median in-hospital caloric deficit was 4268 kcal (IQR: 2825 to 6610). No significant association was observed between discharge disposition, complication rate, ambulatory status, 30-day readmission rate and PHCD. In-hospital caloric deficit was associated with complications after surgery (p < 0.0001). CONCLUSION: Small PHCD's in patients with SBO's, pancreatitis, or diverticulitis do not negatively affect their outcomes. Further research of patients with large PHCD's is needed to best treat surgical patients at risk for malnutrition.

Targeting the Pseudomonas aeruginosa Virulence Factor Phospholipase C With Engineered Liposomes.

Engineered liposomes composed of the naturally occurring lipids sphingomyelin (Sm) and cholesterol (Ch) have been demonstrated to efficiently neutralize toxins secreted by Gram-positive bacteria such as Streptococcus pneumoniae and Staphylococcus aureus. Here, we hypothesized that liposomes are capable of neutralizing cytolytic virulence factors secreted by the Gram-negative pathogen Pseudomonas aeruginosa. We used the highly virulent cystic fibrosis P. aeruginosa Liverpool Epidemic Strain LESB58 and showed that sphingomyelin (Sm) and a combination of sphingomyelin with cholesterol (Ch:Sm; 66 mol/% Ch and 34 mol/% Sm) liposomes reduced lysis of human bronchial and red blood cells upon challenge with the Pseudomonas secretome. Mass spectrometry of liposome-sequestered Pseudomonas proteins identified the virulence-promoting hemolytic phospholipase C (PlcH) as having been neutralized. Pseudomonas aeruginosa supernatants incubated with liposomes demonstrated reduced PlcH activity as assessed by the p-nitrophenylphosphorylcholine (NPPC) assay. Testing the in vivo efficacy of the liposomes in a murine cutaneous abscess model revealed that Sm and Ch:Sm, as single dose treatments, attenuated abscesses by >30%, demonstrating a similar effect to that of a mutant lacking plcH in this infection model. Thus, sphingomyelin-containing liposome therapy offers an interesting approach to treat and reduce virulence of complex infections caused by P. aeruginosa and potentially other Gram-negative pathogens expressing PlcH.

Dose Addition in the Induction of Craniofacial Malformations in Zebrafish Embryos Exposed to a Complex Mixture of Food-Relevant Chemicals with Dissimilar Modes of Action.

BACKGROUND: Humans are exposed to combinations of chemicals. In cumulative risk assessment (CRA), regulatory bodies such as the European Food Safety Authority consider dose addition as a default and sufficiently conservative approach. The principle of dose addition was confirmed previously for inducing craniofacial malformations in zebrafish embryos in binary mixtures of chemicals with either similar or dissimilar modes of action (MOAs). OBJECTIVES: In this study, we explored a workflow to select and experimentally test multiple compounds as a complex mixture with each of the compounds at or below its no observed adverse effect level (NOAEL), in the same zebrafish embryo model. METHODS: Selection of candidate compounds that potentially induce craniofacial malformations was done using in silico methods-structural similarity, molecular docking, and quantitative structure-activity relationships-applied to a database of chemicals relevant for oral exposure in humans via food (EuroMix inventory, n=1,598). A final subselection was made manually to represent different regulatory fields (e.g., food additives, industrial chemicals, plant protection products), different chemical families, and different MOAs. RESULTS: A final selection of eight compounds was examined in the zebrafish embryo model, and craniofacial malformations were observed in embryos exposed to each of the compounds, thus confirming the developmental toxicity as predicted by the in silico methods. When exposed to a mixture of the eight compounds, each at its NOAEL, substantial craniofacial malformations were observed; according to a dose-response analysis, even embryos exposed to a 7-fold dilution of this mixture still exhibited a slight abnormal phenotype. The cumulative effect of the compounds in the mixture was in accordance with dose addition (added doses of the individual compounds after adjustment for relative potencies), despite different MOAs of the compounds involved. DISCUSSION: This case study of a complex mixture inducing craniofacial malformations in zebrafish embryos shows that dose addition can adequately predicted the cumulative effect of a mixture of multiple substances at low doses, irrespective of the (expected) MOA. The applied workflow may be useful as an approach for CRA in general. https://doi.org/10.1289/EHP9888.

Selection of M2+ -Independent RNA-Cleaving DNAzymes with Side-Chains Mimicking Arginine and Lysine.

Sequence-specific cleavage of RNA by nucleic acid catalysts in the absence of a divalent metal cation (M2+ ) has remained an important goal in biomimicry with potential therapeutic applications. Given the lack of functional group diversity in canonical nucleotides, modified nucleotides with amino acid-like side chains were used to enhance self-cleavage rates at a single embedded ribonucleoside site. Previous works relied on three functional groups: an amine, a guanidine and an imidazole ensconced on three different nucleosides. However, to date, few studies have systematically addressed the necessity of all three modifications, as the value of any single modified nucleoside is contextualized at the outset of selection. Herein, we report on the use of only two modified dNTPs, excluding an imidazole, i. e. 5-(3-guanidinoallyl)-2'-dUTP (dUga TP) and 5-aminoallyl-2'-dCTP (dCaa TP), to select in-vitro self-cleaving DNAzymes that cleave in the absence of M2+ in a pH-independent fashion. Cleavage shows biphasic kinetics with rate constants that are significantly higher than in unmodified DNAzymes and compare favorably to certain DNAzymes involving an imidazole.

Large-scale integration of single-cell transcriptomic data captures transitional progenitor states in mouse skeletal muscle regeneration.

Skeletal muscle repair is driven by the coordinated self-renewal and fusion of myogenic stem and progenitor cells. Single-cell gene expression analyses of myogenesis have been hampered by the poor sampling of rare and transient cell states that are critical for muscle repair, and do not inform the spatial context that is important for myogenic differentiation. Here, we demonstrate how large-scale integration of single-cell and spatial transcriptomic data can overcome these limitations. We created a single-cell transcriptomic dataset of mouse skeletal muscle by integration, consensus annotation, and analysis of 23 newly collected scRNAseq datasets and 88 publicly available single-cell (scRNAseq) and single-nucleus (snRNAseq) RNA-sequencing datasets. The resulting dataset includes more than 365,000 cells and spans a wide range of ages, injury, and repair conditions. Together, these data enabled identification of the predominant cell types in skeletal muscle, and resolved cell subtypes, including endothelial subtypes distinguished by vessel-type of origin, fibro-adipogenic progenitors defined by functional roles, and many distinct immune populations. The representation of different experimental conditions and the depth of transcriptome coverage enabled robust profiling of sparsely expressed genes. We built a densely sampled transcriptomic model of myogenesis, from stem cell quiescence to myofiber maturation, and identified rare, transitional states of progenitor commitment and fusion that are poorly represented in individual datasets. We performed spatial RNA sequencing of mouse muscle at three time points after injury and used the integrated dataset as a reference to achieve a high-resolution, local deconvolution of cell subtypes. We also used the integrated dataset to explore ligand-receptor co-expression patterns and identify dynamic cell-cell interactions in muscle injury response. We provide a public web tool to enable interactive exploration and visualization of the data. Our work supports the utility of large-scale integration of single-cell transcriptomic data as a tool for biological discovery.

Exploring Neurobehaviour in Zebrafish Embryos as a Screening Model for Addictiveness of Substances.

Tobacco use is the leading cause of preventable death worldwide and is highly addictive. Nicotine is the main addictive compound in tobacco, but less is known about other components and additives that may contribute to tobacco addiction. The zebrafish embryo (ZFE) has been shown to be a good model to study the toxic effects of chemicals on the neurological system and thus may be a promising model to study behavioral markers of nicotine effects, which may be predictive for addictiveness. We aimed to develop a testing protocol to study nicotine tolerance in ZFE using a locomotion test with light-dark transitions as behavioral trigger. Behavioral experiments were conducted using three exposure paradigms: (1) Acute exposure to determine nicotine's effect and potency. (2) Pre-treatment with nicotine dose range followed by a single dose of nicotine, to determine which pre-treatment dose is sufficient to affect the potency of acute nicotine. (3) Pre-treatment with a single dose combined with acute exposure to a dose range to confirm the hypothesized decreased potency of the acute nicotine exposure. These exposure paradigms showed that (1) acute nicotine exposure decreased ZFE activity in response to dark conditions in a dose-dependent fashion; (2) pre-treatment with increasing concentrations dose-dependently reversed the effect of acute nicotine exposure; and (3) a fixed pre-treatment dose of nicotine induced a decreased potency of the acute nicotine exposure. This effect supported the induction of tolerance to nicotine by the pre-treatment, likely through neuroadaptation. The interpretation of these effects, particularly in view of prediction of dependence and addictiveness, and suitability of the ZFE model to test for such effects of other compounds than nicotine, are discussed.

Developmental Neurotoxicity of Environmentally Relevant Pharmaceuticals and Mixtures Thereof in a Zebrafish Embryo Behavioural Test.

Humans are exposed daily to complex mixtures of chemical substances via food intake, inhalation, and dermal contact. Developmental neurotoxicity is an understudied area and entails one of the most complex areas in toxicology. Animal studies for developmental neurotoxicity (DNT) are hardly performed in the context of regular hazard studies, as they are costly and time consuming and provide only limited information as to human relevance. There is a need for a combination of in vitro and in silico tests for the assessment of chemically induced DNT in humans. The zebrafish (Danio rerio) embryo (ZFE) provides a powerful model to study DNT because it shows fast neurodevelopment with a large resemblance to the higher vertebrate, including the human system. One of the suitable readouts for DNT testing in the zebrafish is neurobehaviour (stimulus-provoked locomotion) since this provides integrated information on the functionality and status of the entire nervous system of the embryo. In the current study, environmentally relevant pharmaceuticals and their mixtures were investigated using the zebrafish light-dark transition test. Zebrafish embryos were exposed to three neuroactive compounds of concern, carbamazepine (CBZ), fluoxetine (FLX), and venlafaxine (VNX), as well as their main metabolites, carbamazepine 10,11-epoxide (CBZ 10,11E), norfluoxetine (norFLX), and desvenlafaxine (desVNX). All the studied compounds, except CBZ 10,11E, dose-dependently inhibited zebrafish locomotor activity, providing a distinct behavioural phenotype. Mixture experiments with these pharmaceuticals identified that dose addition was confirmed for all the studied binary mixtures (CBZ-FLX, CBZ-VNX, and VNX-FLX), thereby supporting the zebrafish embryo as a model for studying the cumulative effect of chemical mixtures in DNT. This study shows that pharmaceuticals and a mixture thereof affect locomotor activity in zebrafish. The test is directly applicable in environmental risk assessment; however, further studies are required to assess the relevance of these findings for developmental neurotoxicity in humans.