Behavior In Zebrafish Larvae Research Articles

Comparative evaluation and QSAR modeling of developmental neurotoxicity of novel brominated flame retardants in zebrafish

The novel brominated flame retardants (NBFRs) have received wide concerns due to their ubiquitous occurrence in the environment and their potential risks to ecosystems and human health. However, the toxicity data of NBFRs are still lacking, especially their toxicity comparison data, and toxicity predictions for untested NBFRs are extremely limited. In this study, eight commonly used NBFRs and decabromodiphenyl ether (BDE209) were selected to compare their toxicity at concentrations between 0.03 and 3.69 μM, by exposing zebrafish embryos until 120 h post-fertilization (hpf) and evaluating 18 toxicity indicators including basic development indicators and a series of behavioral indicators. The toxicity potency of the tested compounds ranked by the total number of significantly affected endpoints were pentabromobenzene (PBB) ≈ 2,4,6-tribromophenol (TBP) > BDE209 ≈ bis(2-ethylhexyl) tetrabromophthalate (TBPH) > pentabromotoluene (PBT) ≈ 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EHTBB) > 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE) > hexabromobenzene (HBB) > decabromodiphenyl ethane (DBDPE). Almost all the tested compounds affected the locomotor behavior of zebrafish larvae, suggesting that the refined behavioral indicators were sensitive endpoints. Furthermore, the quantitative structure-activity relationship (QSAR) model we developed suggested that molecular surface area (MSA) might be the critical factor for determining the developmental neurotoxicity of NBFRs to zebrafish larvae, except for congeners with larger molecules (e.g. DBDPE, BTBPE). These findings would contribute to elucidating the toxicity differences among various NBFRs and provide important references for their toxicity prediction.

Effects of diuron and two of its metabolites in biochemical markers and behavior of zebrafish larvae.

Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is an herbicide used in many crops, including sugar cane cultivation. It is commonly found in aquatic ecosystem and is of high concern due to its ability to persist in the environment. Diuron metabolites include DCA (3,4-dichloroaniline) and DCPMU (3-(3,4-dichlorophenyl-1-methylurea). The objective of this study was to evaluate the effects of diuron and two of its metabolites in zebrafish (Danio rerio) developing embryos, from biochemical to individual level. Activities of the enzymes acetylcholinesterase (AChE), catalase (CAT), glutathione-S-transferase (GST), andlactate dehydrogenase (LDH) and the levels of lipid peroxidation (LPO), swimming activity, and body length were investigated after an exposure of 120h, and the heart rate was determined after 48h of exposure. The range of concentrations tested was 0.003-3.000mg/L diuron, 0.020-1.500mg/L DCA, and 0.020-2.100mg/L DCPMU. Results showed that AChE activity was inhibited by diuron (3.000mg/L) and DCPMU (0.326, 0.828mg/L). However, the swimming activity of fish larvae exposed to diuron or its metabolites was not affected. The CAT was induced by DCPMU, and GST was induced by diuron. This suggests that CAT is acting to cope with oxidative stress induced by DCPMU and GST might have a role in the detoxification of diuron. In addition, larvae exposed to DCA (0.633 and 1.500mg/L) had a reduction in their length, and larvae exposed to diuron (0.754 and 3.000mg/L) and DCA (0.267, 0.633, and 1.500mg/L) presented bradycardia, suggesting cardiotoxicity. Overall, results indicate that diuron, DCA, or DCPMU induces adverse effects during the early phases of zebrafish development, such as the impairment of neurotransmission and cardiovascular function and alterations in antioxidant enzymes and growth. Diuron appeared as more toxic than its metabolites since the lowest LOEC (0.012mg/L) and higher integrated biomarker response (IBR) values were obtained with exposure to this herbicide. Furthermore, as it is fast degraded into DCA and DCPMU, which also affected the zebrafish developing embryos at environmentally relevant concentrations, its use might be of concern in ecosystems that receive agriculture runoff due to their potential adverse effects to aquatic biota.

Analyzing the neurotoxic effects of anatoxin-a and saxitoxin in zebrafish larvae

Global warming due to climate change, as well as freshwater eutrophication caused by anthropogenic activities are responsible, among other factors, for an increasing occurrence of harmful algal blooms (HABs) in aquatic systems. These can lead to the generation of cyanotoxins, secondary metabolites coming from cyanobacteria, producing adverse effects in living organisms including death. This research aims to study the effects that two neurotoxins, anatoxin-a (ATX-a) and saxitoxin (STX), have on living organisms. Once the stability of both compounds in water was determined for a 24 h period using ultra-high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer (UPLC-MS/MS), zebrafish larvae were exposed to different levels of toxins (1 ng L−1, 10 ng L−1, 100 ng L−1 and 1 μg L−1) during 24 h. Behavioral studies including vibrational startle response (VSR), habituation to vibrational stimuli, basal locomotor activity (BLM) and visual motor response (VMR) were performed using Danio Vision system, and neurotransmitters (NTs) from 15-head pools of control and exposed zebrafish larvae were extracted and analyzed by UPLC-MS/MS. Both compounds induced hypolocomotion in the individuals, while 10 and 100 ng L−1 of ATX-a significantly increased methionine (120 % and 126 %, respectively) and glutamate levels (118 % and 129 %, respectively). Saxitoxin enhanced 3-metoxytyramine (3-MT) levels at 1 ng L−1 by 185 %. The findings of this study show that both studied cyanotoxins influence the behavior of zebrafish larvae as well as their metabolism.

Preparation of PBAT microplastics and their potential toxicity to zebrafish embryos and juveniles

The extensive use of traditional non-biodegradable plastics results in the generation of microplastics (MPs), forming a new pollutant that can pose significant environmental risks. Biodegradable plastics (BP) possess degradation properties and can partially replace conventional plastics, thereby reducing pollution. However, further investigation is needed into the toxicity of biodegradable microplastics (BMPs) on aquatic organisms. This study explores the toxic effects of PBAT microplastics (PBAT-BMPs) and microplastics produced from degradable PBAT/TPS (thermoplastic starch) composite film (PBAT/TPS-BMPs) on zebrafish embryos. Our findings indicate that the presence of microplastics on the embryo's surface increases with higher BMPs concentration. Nonetheless, PBAT-BMPs tend to aggregate and are blocked by the embryonic membrane, thus diminishing their toxic effects on the embryo. Acute toxicity experiments revealed that 30 mg/L of PBAT-BMPs significantly reduced the survival rate of zebrafish embryos, whereas PBAT/TPS-BMPs had a lesser effect on survival. Both types of BMPs influenced the hatching rate of the embryos, leading to prolonged incubation periods. Additionally, both types of BMPs impacted the locomotor behavior of zebrafish larvae, causing an increase in larval locomotor speed. However, these BMPs had little impact on larval body development and heartbeat behavior. Fluorescent microplastic tracer experiments demonstrated that PBAT-BMPs persisted in juvenile fish for at least 144 h and were difficult to metabolize and excrete. Our study aims to gain a better understanding of the potential effects of BMPs on aquatic ecosystems and biological health, as well as to propose effective strategies for reducing environmental pollution and protecting organisms.

Experience-dependent modulation of collective behavior in larval zebrafish.

Complex group behavior can emerge from simple inter-individual interactions. Commonly, these interactions are considered static and hardwired and little is known about how experience and learning affect collective group behavior. Young larvae use well described visuomotor transformations to guide interindividual interactions and collective group structure. Here, we use naturalistic and virtual-reality (VR) experiments to impose persistent changes in population density and measure their effects on future visually evoked turning behavior and the resulting changes in group structure. We find that neighbor distances decrease after exposure to higher population densities, and increase after the experience of lower densities. These adaptations develop slowly and gradually, over tens of minutes and remain stable over many hours. Mechanistically, we find that larvae estimate their current group density by tracking the frequency of neighbor-evoked looming events on the retina and couple the strength of their future interactions to that estimate. A time-varying state-space model that modulates agents' social interactions based on their previous visual-social experiences, accurately describes our behavioral observations and predicts novel aspects of behavior. These findings provide concrete evidence that inter-individual interactions are not static, but rather continuously evolve based on past experience and current environmental demands. The underlying neurobiological mechanisms of experience dependent modulation can now be explored in this small and transparent model organism.

Manganese Overexposure Alters Neurogranin Expression and Causes Behavioral Deficits in Larval Zebrafish.

Manganese (Mn), a cofactor for various enzyme classes, is an essential trace metal for all organisms. However, overexposure to Mn causes neurotoxicity. Here, we evaluated the effects of exposure to Mn chloride (MnCl2) on viability, morphology, synapse function (based on neurogranin expression) and behavior of zebrafish larvae. MnCl2 exposure from 2.5 h post fertilization led to reduced survival (60%) at 5 days post fertilization. Phenotypical changes affected body length, eye and olfactory organ size, and visual background adaptation. This was accompanied by a decrease in both the fluorescence intensity of neurogranin immunostaining and expression levels of the neurogranin-encoding genes nrgna and nrgnb, suggesting the presence of synaptic alterations. Furthermore, overexposure to MnCl2 resulted in larvae exhibiting postural defects, reduction in motor activity and impaired preference for light environments. Following the removal of MnCl2 from the fish water, zebrafish larvae recovered their pigmentation pattern and normalized their locomotor behavior, indicating that some aspects of Mn neurotoxicity are reversible. In summary, our results demonstrate that Mn overexposure leads to pronounced morphological alterations, changes in neurogranin expression and behavioral impairments in zebrafish larvae.

Key HPI axis receptors facilitate light adaptive behavior in larval zebrafish

The vertebrate stress response (SR) is mediated by the hypothalamic–pituitary–adrenal (HPA) axis and contributes to generating context appropriate physiological and behavioral changes. Although the HPA axis plays vital roles both in stressful and basal conditions, research has focused on the response under stress. To understand broader roles of the HPA axis in a changing environment, we characterized an adaptive behavior of larval zebrafish during ambient illumination changes. Genetic abrogation of glucocorticoid receptor (nr3c1) decreased basal locomotor activity in light and darkness. Some key HPI axis receptors (mc2r [ACTH receptor], nr3c1), but not nr3c2 (mineralocorticoid receptor), were required to adapt to light more efficiently but became dispensable when longer illumination was provided. Such light adaptation was more efficient in dimmer light. Our findings show that the HPI axis contributes to the SR, facilitating the phasic response and maintaining an adapted basal state, and that certain adaptations occur without HPI axis activity.

'Environmental standard limit concentration' arsenic exposure is associated with anxiety, depression, and autism-like changes in early-life stage zebrafish

Arsenic is a worldwide environmental pollutant that can impair human health. Previous studies have identified mental disorders induced by arsenic, but the environmental exposure concentrations in the early life stages associated with these disorders are poorly understood. In the present study, early-life stage zebrafish were used to explore the effects on mental disorders under 'environmental standard limit concentrations' arsenic exposures of 5, 10, 50, 150, and 500 μg/L. The results showed that arsenic exposure at these concentrations changed the locomotor behavior in larval zebrafish and was further associated with anxiety, depression, and autism-like behavior in both larval and juvenile zebrafish. Changes were noted at benchmark dose limit (BMDL) concentrations as low as 0.81 μg/L. Transcriptomics showed that immediate early genes (IEGs) fosab, egr1, egr2a, ier2b, egr3, and jund were decreased after arsenic exposure in larval and juvenile zebrafish. Nervous system impairment and anxiety, depression, and autism-like behaviors in early-life stage zebrafish at 'environmental standard limit concentrations' may be attributed to the downregulation of IEGs. These findings in zebrafish provided new experimental support for an arsenic toxicity threshold for mental disorders, and they suggest that low levels of environmental chemicals may be causative developmental factors for mental disorders.

Synthesis and Hypoglycemic Effect of Insulin from the Venom of Sea Anemone Exaiptasia diaphana.

Sea anemone venom, abundant in protein and peptide toxins, serves primarily for predatory defense and competition. This study delves into the insulin-like peptides (ILPs) present in sea anemones, particularly focusing on their role in potentially inducing hypoglycemic shock in prey. We identified five distinct ILPs in Exaiptasia diaphana, exhibiting varied sequences. Among these, ILP-Ap04 was successfully synthesized using solid phase peptide synthesis (SPPS) to evaluate its hypoglycemic activity. When tested in zebrafish, ILP-Ap04 significantly reduced blood glucose levels in a model of diabetes induced by streptozotocin (STZ) and glucose, concurrently affecting the normal locomotor behavior of zebrafish larvae. Furthermore, molecular docking studies revealed ILP-Ap04's unique interaction with the human insulin receptor, characterized by a detailed hydrogen-bonding network, which supports a unique mechanism for its hypoglycemic effects. Our findings suggest that sea anemones have evolved sophisticated strategies to activate insulin receptors in vertebrates, providing innovative insights into the design of novel drugs for the treatment of diabetes.

Effects of 4 Testing Arena Sizes and 11 Types of Embryo Media on Sensorimotor Behaviors in Wild-Type and chd7 Mutant Zebrafish Larvae.

The larval zebrafish is a highly versatile model across research disciplines, and the expanding use of behavioral analysis has contributed to many advances in neuropsychiatric, developmental, and toxicological studies, often through large-scale chemical and genetic screens. In the absence of standardized approaches to larval zebrafish behavior analysis, however, it is critical to understand the impact on behavior of experimental variables such as the size of testing arenas and the choice of embryo medium. Using a custom-built, modular high-throughput testing system, we examined the effects of 4 testing arena sizes and 11 types of embryo media on conserved sensorimotor behaviors in zebrafish larvae. Our data show that testing arena size impacts acoustic startle sensitivity and kinematics, as well as spontaneous locomotion and thigmotaxis, with fish tested in larger arenas displaying reduced startle sensitivity and increased locomotion. We also find that embryo media can dramatically affect startle sensitivity, kinematics, habituation, and prepulse inhibition, as well as spontaneous swimming, turning, and overall activity. Common medium components such as methylene blue and high calcium concentration consistently reduced startle sensitivity and locomotion. To further address how the choice of embryo medium can impact phenotype expression in zebrafish models of disease, we reared chd7 mutant larvae, a model of CHARGE syndrome with previously characterized morphological and behavioral phenotypes, in five different types of media and observed impacts on all phenotypes. By defining the effects of these key extrinsic factors on larval zebrafish behavior, these data can help researchers select the most appropriate conditions for their specific research questions, particularly for genetic and chemical screens.

Early-life perfluorooctanoic acid exposure disrupts the function of dopamine transporter protein with glycosylation changes implicating the links between decreased dopamine levels and disruptive behaviors in larval zebrafish

Exposure to perfluorooctanoic acid (PFOA) during early embryonic development is associated with the increased risk of developmental neurotoxicity and neurobehavioral disorders in children. In our previous study, we demonstrated that exposure to PFOA affected locomotor activity and disrupted dopamine-related gene expression in zebrafish larvae. Consequently, we continue to study the dopaminergic system with a focus on dopamine levels and dopamine's effect on behaviors in relation to PFOA exposure. In the present study, we found a decrease in dopamine levels in larval zebrafish. We studied the dopamine transporter (DAT) protein, which is responsible for regulating dopamine levels through the reuptake of dopamine in neuronal cells. We demonstrated that exposure to PFOA disrupted the glycosylation process of DAT, inhibited its uptake function, and induced endoplasmic reticulum (ER) stress in dopaminergic cells. Besides, we conducted a light-dark preference test on larval zebrafish and observed anxiety/depressive-like behavioral changes following exposure to PFOA. Dopamine is one of the most prominent neurotransmitters that significantly influences human behavior, with low dopamine levels being associated with impairments such as anxiety and depression. The anxiety-like response in zebrafish larvae exposure to PFOA implies the link with the reduced dopamine levels. Taken together, we can deduce that glycosylation changes in DAT lead to dysfunction of DAT to regulate dopamine levels, which in turn alters behavior in larval zebrafish. Therefore, alternation in dopamine levels may play a pivotal role in the development of anxiety/depressive-like behavioral changes induced by PFOA.

Astaxanthin activates the Nrf2/Keap1/HO-1 pathway to inhibit oxidative stress and ferroptosis, reducing triphenyl phosphate (TPhP)-induced neurodevelopmental toxicity

Triphenyl phosphate (TPhP) serves as a major organophosphorus flame retardant, and its induced neurodevelopmental toxicity has attracted widespread attention, but the mechanism remains unclear. In this study, we involved zebrafish to explore the new mechanism of TPhP inducing oxidative stress and ferroptosis to promote neurodevelopmental toxicity. The results suggested that TPhP affected the embryonic development, reduced the number of new neurons, and led to abnormal neural behavior in zebrafish larvae. TPhP also induced ROS accumulation, activated the antioxidant defense signal Nrf2 and Keap1, and significantly changed the activities of Acetylcholinesterase (AChE), Adenosine triphosphatase (ATPase) and glutathione S-transferase (GST). In addition, TPhP induced ferroptosis in zebrafish, which was reflected in the increase of Fe2+ content, the abnormal expression of GPX4 protein and genes related to iron metabolism (gpx4a, slc7a11, acsl4b, tfa, slc40a1, fth1b, tfr2, tfr1a, tfr1b and ncoa4). Astaxanthin intervention specifically inhibited ROS levels, and reversed SLC7A11 and GPX4 expression levels and Fe2+ metabolism thus alleviating ferroptosis induced by TPhP. Astaxanthin also partially reversed the activity of AChE, GST and the expression of neurodevelopmental-related genes (gap43, gfap, neurog1 and syn2a), so as to partially rescue the embryonic developmental abnormalities and motor behavior disorders induced by TPhP. More interestingly, the expression of mitochondrial apoptosis-related protein BAX, anti-apoptotic protein BCL-2, Caspase3 and Caspase9 was significantly altered in the TPhP exposed group, which could be also reversed by Astaxanthin intervention. In summary, our results suggested that TPhP exposure can induce oxidative stress and ferroptosis, thereby causing neurodevelopment toxicity to zebrafish, while Astaxanthin can partially reverse oxidative stress and reduce the neurodevelopmental toxicity of zebrafish larvae by activating Nrf2/Keap1/HO-1 signaling pathway.

Modeling Sex-Bias in Anxiety: Pros and Cons of a Larval Zebrafish Model

Anxiety disorders are the most prevalent psychiatric disorders, exhibiting strong female bias. Clinical studies implicate declining estradiol levels in the exacerbation of anxiety symptoms in the premenstrual phase of the menstrual cycle. This study aimed to simulate estradiol fluctuation-linked anxiety behavior in larval zebrafish, using an estradiol treatment withdrawal model. Contrary to model aims, estradiol treatment withdrawal decreased both basal activity and anxiety-like hyperlocomotion (ANOVA main effect of dose, P < 0.0001 and P < 0.01, respectively) in the light/dark transition test. The accuracy of the estradiol washout model was not improved by longer durations of treatment or withdrawal. Basal activity was slightly altered by supraphysiological concentrations of WAY-200070 in the absence of added estradiol. Estrogen receptor (ER) β expression was not upregulated in larvae exposed to physiologically relevant, low concentrations of estradiol. Longer exposure to low concentrations of estradiol increased antioxidant capacity (P < 0.01). In addition, acute exposure to low concentrations of estradiol increased basal activity. Data suggest that in the current models, estradiol-associated altered activity levels were linked to more favorable redox status, rather than reflecting altered anxiety levels. As such, it is recommended that zebrafish larval behavioral analysis be conducted in parallel with mechanistic studies such as redox indicators, for investigations focused on ER signaling.

Environmental levels of azoxystrobin disturb male zebrafish behavior: Possible roles of oxidative stress, cholinergic system, and dopaminergic system

A widely applied pesticide of azoxystrobin, is increasingly detected in the water environment. Concern has been raised against its potential detriment to aquatic ecosystems. It has been shown that exposure to azoxystrobin interfere with the locomotor behavior of zebrafish larvae. This study aims to investigate whether exposure to environmental levels of azoxystrobin (2 μg/L, 20 μg/L, and 200 μg/L) changes the behavior of male adult zebrafish. Herein, we evaluated behavioral response (locomotor, anxiety-like, and exploratory behaviors), histopathology, biochemical indicators, and gene expression in male adult zebrafish upon azoxystrobin exposure. The study showed that exposure to azoxystrobin for 42 days remarkably increased the locomotor ability of male zebrafish, resulted in anxiety-like behavior, and inhibited exploratory behavior. After treatment with 200 μg/L azoxystrobin, vasodilatation, and congestion were observed in male zebrafish brains. Exposure to 200 μg/L azoxystrobin notably elevated ROS level, MDA concentration, CAT activity, and AChE activity, while inhibiting SOD activity, GPx activity, ACh concentration, and DA concentration in male zebrafish brains. Moreover, the expression levels of genes related to the antioxidant, cholinergic, and dopaminergic systems were significantly changed. This suggests that azoxystrobin may interfere with the homeostasis of neurotransmitters by causing oxidative stress in male zebrafish brains, thus affecting the behavioral response of male zebrafish.

TMBPF-induced neurotoxicity and oxidative stress in zebrafish larvae: impacts on central nervous system development and dopamine neurons

Bisphenol A (BPA), a common bisphenol molecule, is well known in the environment as an endocrine disruptor. Furthermore, BPs (BPA, BPS, BPF, and BPAF) have been shown in recent years to be neurotoxic to zebrafish. Tetramethyl bisphenol F (TMBPF) has recently been introduced as a substitute for bisphenol A (BPA) in various industries, including plastics and food contact coatings. However, a growing number of studies have demonstrated that the toxicity of some BPA substitutes is similar to or even stronger than BPA, posing potential harm to human health and the environment. In this study, we used zebrafish larvae as a model to investigate the neurodevelopmental effects of TMBPF at different concentrations (0, 0.25, 0.5, 1, 2, 4 and 8 mg/L). Our results showed that exposure to TMBPF at concentrations higher than 4 mg/L for 72 h post-fertilization (hpf) resulted in zebrafish mortality, whereas exposure to 2 mg/L for 144 hpf caused deformities. Furthermore, TMBPF exposure inhibited the development of the central nervous system, motor nerves, and dopamine neurons in zebrafish. Real-time polymerase chain reaction (PCR) analysis revealed that TMBPF exposure significantly down-regulated the expression of oxidative stress-related genes (Cu/Zn-SOD, Mn-SOD, and CAT) and neurodevelopmental genes (mbp, gafp, and syn2a), while up-regulated the expression of dopamine-related genes (th1, th2, and dat). Notably, treatment with the antioxidant N-acetylcysteine (NAC) alleviated TMBPF-induced toxicity. NAC can regulate the expression of genes related to oxidative stress, neurodevelopment and dopamine development, and make the nerve development of zebrafish normal. Overall, our research suggested that TMBPF may disrupt the development of the early central nervous system and dopamine neurons, leading to abnormal motor behavior in zebrafish larvae. These results highlight the potential risks associated with the use of TMBPF in various industries and the importance to evaluate its potential risks to human health and the environment.

Stereoselective toxicity of acetochlor chiral isomers on the nervous system of zebrafish larvae

Acetochlor (ACT) is a widely detected pesticide globally, and the neurotoxic effects of its chiral isomers on humans and environmental organisms remain uncertain. Zebrafish were used to study the neurotoxicity of ACT and its chiral isomers. Our study reveals that the R-ACT, Rac-ACT, and S-ACT induce neurotoxicity in zebrafish larvae by impairing vascular development and disrupting the blood-brain barrier. These detrimental effects lead to apoptosis in brain cells, hindered development of the central nervous system, and manifest as altered swimming behavior and social interactions in the larvae. Importantly, the neurotoxicity caused by the S-ACT exhibits the most pronounced impact and significantly diverges from the effects induced by the R-ACT. The neurotoxicity associated with the Rac-ACT falls intermediate between that of the R-ACT and S-ACT. Fascinatingly, we observed a remarkable recovery in the S-ACT-induced abnormalities in BBB, neurodevelopment, and behavior in zebrafish larvae upon supplementation of the Wnt/β-catenin signaling pathway. This observation strongly suggests that the Wnt/β-catenin signaling pathway serves as a major target of S-ACT-induced neurotoxicity in zebrafish larvae. In conclusion, S-ACT significantly influences zebrafish larval neurodevelopment by inhibiting the Wnt/β-catenin signaling pathway, distinguishing it from R-ACT neurotoxic effects.

Neurotoxicity and the potential molecular mechanisms of mono-2-ethylhexyl phthalic acid (MEHP) in zebrafish

Mono-2-ethylhexyl phthalic acid (MEHP) is the most toxic metabolite of plasticizer di-2-ethylhexyl phthalic acid (DEHP), and there is limited information available on the effects of MEHP on neurotoxicity. This study aims to examine the neurotoxicity of MEHP and preliminarily explore its potential molecular mechanisms. We found that MEHP impeded the growth of zebrafish embryos and the neurodevelopmental-related gene expression at environmentally relevant concentrations. MEHP exposure also induces oxidative stress response and brain cell apoptosis accompanied by a decrease in acetylcholinesterase (AChE) activity in zebrafish larvae. RNA-Seq and bioinformatics analysis showed that MEHP treatment altered the nervous system, neurogenic diseases, and visual perception pathways. The locomotor activity in dark-to-light cycles and phototaxis test confirmed the abnormal neural behavior of zebrafish larvae. Besides, the immune system has produced a large number of differentially expressed genes related to neural regulation. Inflammatory factor IL1β and IL-17 signaling pathways highly respond to MEHP, indicating that inflammation caused by immune system imbalance is a potential mechanism of MEHP-induced neurotoxicity. This study expands the understanding of the toxicity and molecular mechanisms of MEHP, providing a new perspective for in-depth neurotoxicity exploration of similar compounds.

Neurotoxicity of tetramethylammonium ion on larval and juvenile zebrafish: Effects on neurobehaviors and multiple biomarkers

Tetramethylammonium hydroxide (TMAH) is an important compound that utilized and released by the rapidly expanding semiconductor industry, which could hardly be removed by the conventional wastewater treatment techniques. As a cholinergic agonist, the tetramethylammonium ion (TMA+) has been reported to induce toxicity to muscular and respiratory systems of mammals and human, however the toxicity on aquatic biota remains poorly known. We investigated the neurotoxic effects of TMA+ exposure on zebrafish, based on neurobehavior tests and a series of biomarkers. Significant inhibitions on the swimming distance of zebrafish larvae were observed when the exposure level exceeded 50 mg/L, and significant alterations on swimming path angles (straight and deflective movements) occurred even at 10 mg/L. The tested neurobehavioral endpoints of zebrafish larvae were significantly positively correlated with reactive oxygen species (ROS) and malondialdehyde (MDA), significantly negatively related with the activities of antioxidant enzymes, but not significantly correlated with the level of acetylcholinesterase (AChE). Such relationship indicates that the observed neurotoxic effects on swimming behavior of zebrafish larvae is mainly driven by oxidative stress, rather than the alterations of neurotransmitter. At the highest exposure concentration (200 mg/L), TMA+ evoked more severe toxicity on zebrafish juveniles, showing significantly stronger elevation on the MDA activity, and greater inhibitions on the activities of antioxidant enzymes and AChE, suggesting juveniles were more susceptible to TMA+ exposure than larval zebrafish.

Valbenazine promotes body growth via growth hormone signaling during zebrafish embryonic development

Vesicular monoamine transporter 2 (VMAT-2) functions by uptake of cytoplasmic monoamines into vesicles for storage. Valbenazine (VBZ) is a newly FDA-approved oral VMAT-2 inhibitor used for the treatment of movement disorders such as tardive dyskinesia (TD), and Tourette syndrome (TS). Clinical data shows that VBZ is a relatively safe drug with no cardiotoxicity or hepatotoxicity. However, the effect of VBZ on embryonic development remains unknown. Here, we use zebrafish larvae as an animal model to demonstrate that VBZ exposure causes premature hatching and increased body size and hyperactivity-like behaviors in zebrafish larvae. In addition, VBZ exposure leads to increased dopamine (DA) and Glutamate (Glu) levels. Moreover, an increase of growth hormone (gh) and enriched PI3K/AKT signaling were found in VBZ-exposed zebrafish larvae, which may explain their accelerated development. In summary, VBZ exposure may be developmentally toxic in zebrafish larvae.

Developmental and neurobehavioral toxicity of 2,2′-methylenebis(6-tert-butyl-4-methylphenol) (antioxidant AO2246) during the early life stage of zebrafish

Background2,2′-Methylenebis (4-methyl-6-tert-butylphenol) (AO2246) is a synthetic phenolic antioxidant extensively used in food packaging bags and cosmetics. Recently, AO2246 was detected with unexpectedly high concentrations in plasma and breast milk samples from pregnant and lactating women. Hence, it is essential to conduct a thorough investigation to evaluate the detrimental effects of AO2246 on biota. ObjectiveTo investigate the developmental and behavioral toxicity of AO2246 in zebrafish, as well as the molecular mechanisms underlying these effects. MethodsZebrafish embryos were exposed to AO2246 at concentrations ranging from 0.05 to 10 μM for up to 6 days postfertilization (dpf). Hatching rate, survival rate, heart rate, and body length were measured. Locomotor behavioral and electrophysiologal analyses were performed. Two fluorescence-labeled transgenic zebrafish lines (endothelium-Tg and macrophage/microglia-Tg) were employed. RNA sequencing was carried out. ResultsAO2246 has a 96-hour LC50 value of 3 μM. The exposure of AO2246 resulted in a significant reduction in both hatching rate and heart rate. Analysis of locomotor behavior demonstrated that larvae exposed to AO2246 doses exceeding 2 μM exhibited a significant decrease in both total distance and mean velocity. Electrophysiological recordings demonstrated a noteworthy reduction in spike activity at a concentration of 3 μM, relative to control conditions. The administration of AO2246 at 3 μM elicited morphological reactivity and immune alteration of the midbrain microglia in the macrophage/microglia-transgenic zebrafish line, indicating a potential contribution of neurological disorders to behavioral defects. RNA sequencing analysis revealed altered gene expression profiles at high AO2246 concentrations, particularly the dysregulation of pathways associated with neuronal function. ConclusionsThe present study demonstrates that AO2246 exposure elicits developmental and neurobehavioral toxicity in zebrafish larvae. Specifically, exposure to AO2246 was found to cause disturbances in neuronal electrophysiological activity and neurological disorders, which ultimately led to the impairment of locomotor behavior in zebrafish larvae.