Early Life Stages Of Zebrafish Research Articles

A transcriptomics-based analysis of mechanisms involved in the neurobehavioral effects of 6PPD-quinone on early life stages of zebrafish

As an emerging pollutant frequently detected in aquatic ecosystems, the toxicity of N-(1,3-dimethylbutyl)-N’-phenyl-p-phenylenediamine-quinone (6PPD-quinone) on fish has been confirmed, but insight into the mechanisms underlying those adverse effects is still limited. Thus, we exposed zebrafish embryos to 6PPD-quinone at 0, 0.25, 2.5, and 25 μg/L until 120 h post-fertilization (hpf), and investigated the variations in their development, behavior, monoamine neurotransmitter levels, and transcriptional profile. Exposure to 6PPD-quinone notably elevated the heart rate of zebrafish at 48 hpf (at 2.5 and 25 μg/L) and 72 hpf (at 0.25, 2.5, and 25 μg/L). In the dark-light transition test, the locomotor activity of zebrafish larvae exposed to 6PPD-quinone significantly increased, especially in the dark periods. Exposure to 6PPD-quinone also altered the dopamine level and its turnover in zebrafish, which exhibited significant correlations to their locomotor activity. RNA sequencing identified 394 differentially expressed genes (DEGs), most of which have the molecular function of binding and catalytic activity. Five DEGs were predicted as the key driver genes in the protein-protein interaction networks associated with circadian rhythm (i.e., npas2), protein processing in endoplasmic reticulum (i.e., hsp90b1 and pdia4), and estrogen signaling pathway (i.e., hsp90aa1.1 and hsp90aa1.2). Our findings provide more insights into mechanisms underlying the toxicity of 6PPD-quinone to teleosts and highlight the necessity to assess its potential risks to aquatic ecosystems.

Unveil the toxicity induced on early life stages of zebrafish (Danio rerio) exposed to 3,4-methylenedioxymethamphetamine (MDMA) and its enantiomers

The increased detection of the recreational drug 3,4-methylenedioxymethamphetamine (MDMA) in aquatic ecosystems, has raised concern worldwide about its possible negative impacts on wildlife. MDMA is produced as racemate but its enantioselective effects on non-target organisms are poorly understood. Therefore, this study aimed to provide a comprehensive study of the toxicity of MDMA and its enantiomers in the early life stages of zebrafish (Danio rerio). Zebrafish embryos (≈3 h post fertilization) were exposed to different concentrations (0.02, 0.2, 2, 20, and 200 μg/L) of (R,S)-MDMA and both pure enantiomers.Both enantiomers induced effects on embryonic development, DNA integrity, and behaviour and enantioselective effects were noted. (S)-MDMA exhibits higher toxic effects on embryonic development level with increased mortality and severity of teratogenic effects, and behavioural abnormalities in acoustic startle-habituation response. (R)-MDMA affected general activity and avoidance behaviour, showing greater inhibitory effects on behavioural activity. Additionally, (R,S)-MDMA induced higher genotoxic effects than the two isolated enantiomers. These results are of concern at populational levels since effects on mortality, development, and behaviour were observed even at environmentally relevant concentrations, which can cause a reduction of larval viability and in the number of individuals in each generation, and an increase in the risk of predation of the organisms. Yet, the bioaccumulation studies showed that MDMA is not accumulated in zebrafish.Therefore, this work demonstrated for the first time the occurrence of MDMA enantiotoxicity in the early life stages of zebrafish, which should be considered in further environmental risk assessments involving fish species or other non-target aquatic organisms.

Immunotoxic response of bio-based plastic on early life stage zebrafish (Danio rerio): A safe alternative to petroleum-based plastics?

Bio-based plastics are marketed as environmentally friendly alternatives to petroleum-based plastics, although they require specific composting conditions for degradation, which leads to their accumulation in the environment and potential risks to aquatic organisms. We hypothesized that the accumulation of bio-based plastics may induce immunotoxic responses in fish. Our research focused on the accumulation and immunotoxicity of 80 nm polylactic acid (PLA) and polystyrene (PS) (0.1–10 mg/L) on early life stage zebrafish (Danio rerio) exposed for 7 days. Compared to PS, there was a higher accumulation of PLA in larvae. Exposure to PLA resulted in a significant increase in neutrophils and macrophages, while immune protein levels such as Complement 3 (C3), Immunoglobulin M (IgM), and C-reactive protein (CRP) were significantly reduced. Furthermore, the mRNA expression of pro-inflammatory cytokines, including tnf-α and il-6, were significantly elevated in PLA treatments. Additionally, PLA-exposed zebrafish were more susceptible to infection by Vibrio parahaemolyticus. Interestingly, at the same concentration, exposures to PS did not induce significant changes in macrophages or immune protein levels, C3 and IgM. This suggests that PLA has a greater immunotoxic response relative to PS. Our research findings contradict the popular belief that bio-based plastics are non-toxic and harmless, which may have potential risk to aquatic organisms.

Nano-alumina induced developmental and neurobehavioral toxicity in the early life stage of zebrafish, associated with mTOR

The study aims to investigate the effects of nano-alumina (AlNPs) on the early development and neurobehavior of zebrafish and the role of mTOR in this process. After embryos and grown-up larvae exposed to AlNPs from 0 to 200 μg/mL, we examined the development, neurobehavior, AlNPs content, and mTOR pathway genes. Moreover, embryos were randomly administered with control, negative control, mTOR knockdown, AlNPs, and mTOR knockdown + AlNPs, then examined for development, neurobehavior, oxidative stress, neurotransmitters, and development genes. As AlNPs increased, swimming speed and distance initially increased and then decreased; thigmotaxis and panic-avoidance reflex substantially decreased in the high-dose AlNPs group; aluminum and nanoparticles considerably accumulated in the 100 μg/mL AlNPs group; AlNPs at high dose decreased mTOR gene and protein levels, stimulating autophagy via increasing ULK1 and ULK2. mTOR knockdown exacerbated the harm to normal development rate, eye and body length, and neurobehavior induced by AlNPs through raising ROS, SOD, and ACH levels but decreasing AchE activity and development genes. Therefore, AlNPs suppress neurobehavior through downregulating mTOR, and mTOR knockdown further aggravates their early development and neurobehavior loss, suggesting mTOR could be a potential target for the toxicity of AlNPs.

Exposure to thimerosal induces behavioral abnormality in the early life stages of zebrafish via altering amino acid homeostasis

Thimerosal (THI) has become a significant source of organic mercury pollutants in aquatic ecosystems, but there is limited information regarding its adverse effects on fish. In this study, zebrafish embryos were exposed to THI at 0 (control), 5.0, and 50 ng/L from 0–5 days post fertilization (dpf), and variations in their survival, development, behavior, free amino acid contents, and the biochemical responses involved in monoaminergic systems were examined. Although THI exposure did not significantly affect the survival, heart rate, or hatching time of zebrafish embryos, it substantially increased swimming velocity (136–154 % of the control) and reduced exploratory behavior (141–142 % of the control) in zebrafish larvae at 5 dpf. Exposure also significantly altered the amino acid contents (51–209 % of the control) and monoamine levels (70–154 % of the control) in zebrafish larvae, some of which displayed significant correlations with behavioral traits. THI significantly elevated dopamine receptor gene expression and monoamine oxidase activity in zebrafish larvae. Adding extra phenylalanine or tryptophan to the E3 medium facilitates the recovery of zebrafish larvae from the abnormal behaviors induced by THI. These findings reveal for the first time that THI exposure at the level of ng/L is sufficient to induce neurobehavioral toxic effects in the early life stages of zebrafish, and disrupting amino acid homeostasis is a critical underlying mechanism. This study provides valuable insights into the toxicity of THI to fish and highlights the importance of assessing its potential risks to aquatic ecosystems.

Perfluorohexanesulfonic Acid (PFHxS) Impairs Lipid Homeostasis in Zebrafish Larvae through Activation of PPARα.

Perfluorohexanesulfonic acid (PFHxS), an emerging short-chain per- and polyfluoroalkyl substance, has been frequently detected in aquatic environments. Adverse outcome pathway studies have shown that perfluorinated compounds impair lipid homeostasis through peroxisome proliferator activated receptors (PPARs). However, many of these studies were performed at high concentrations and may thus be a result of overt toxicity. To better characterize the molecular and key events of PFHxS to biota, early life-stage zebrafish (Danio rerio) were exposed to concentrations detected in the environment (0.01, 0.1, 1, and 10 μg/L). Lipidomic and transcriptomic evaluations were integrated to predict potential molecular targets. PFHxS significantly impaired lipid homeostasis by the dysregulation of glycerophospholipids, fatty acyls, glycerolipids, sphingolipids, prenol lipids, and sterol lipids. Informatic analyses of the lipidome and transcriptome indicated alterations of the PPAR signaling pathway, with downstream changes to retinol, linoleic acid, and glycerophospholipid metabolism. To assess the role of PPARs, potential binding of PFHxS to PPARs was predicted and animals were coexposed to a PPAR antagonist (GW6471). Molecular simulation indicated PFHxS had a 27.1% better binding affinity than oleic acid, an endogenous agonist of PPARα. Antagonist coexposures rescued impaired glycerophosphocholine concentrations altered by PFHxS. These data indicate PPARα activation may be an important molecular initiating event for PFHxS.

Comparative Study of the Effects of Drugs Targeting Adrenergic Receptors on the Early Life Stages of Zebrafish.

Owing to the presence of drugs targeting adrenergic receptors in aquatic ecosystems, considerable attention has been directed towards their environmental distribution and fate in recent decades. However, their potential impacts on non-target aquatic organisms, particularly fish, have received relatively limited investigation. In this study, moxisylyte (MOX) and propranolol (PRO) were selected as representatives of α- or β-adrenergic receptor antagonist, respectively, and we assessed their effects on the early life stages of zebrafish, especially on the nervous and cardiovascular systems. Although both compounds exhibited marginal effects on zebrafish survival, hatching and gross abnormality following exposure to concentrations ranging from 1 to 625 μg/L, they adversely affected the development of cardiovascular and nervous systems, but through different mechanisms of action, as evidenced by variations in gene transcriptional responses and enzyme activities. Notably, cardiovascular responses appear promising for use as potential biomarkers for exposure to drugs targeting adrenergic receptors. This study enhances our understanding of the ecotoxicological risks posed by α- and β-blockers in fish. Nonetheless, further investigation is needed to elucidate the precise mechanisms underlying the impacts of drugs targeting adrenergic receptors due to our limited knowledge of the physiological functions of the adrenergic system in fish.

Exposure Effects of Environmentally Relevant Concentrations of the Tricyclic Antidepressant Amitriptyline in Early Life Stage Zebrafish.

Antidepressants are one of the most globally prescribed classes of pharmaceuticals, and drug target conservation across phyla means that nontarget organisms may be at risk from the effects of exposure. Here, we address the knowledge gap for the effects of chronic exposure (28 days) to the tricyclic antidepressant amitriptyline (AMI) on fish, including for concentrations with environmental relevance, using zebrafish (Danio rerio) as our experimental model. AMI was found to bioconcentrate in zebrafish, was readily transformed to its major active metabolite nortriptyline, and induced a pharmacological effect (downregulation of the gene encoding the serotonin transporter; slc6a4a) at environmentally relevant concentrations (0.03 μg/L and above). Exposures to AMI at higher concentrations accelerated the hatch rate and reduced locomotor activity, the latter of which was abolished after a 14 day period of depuration. The lack of any response on the features of physiology and behavior we measured at concentrations found in the environment would indicate that AMI poses a relatively low level of risk to fish populations. The pseudopersistence and likely presence of multiple drugs acting via the same mechanism of action, however, together with a global trend for increased prescription rates, mean that this risk may be underestimated using current ecotoxicological assessment paradigms.

Ferrocenedicarboxylate modified Bi-MOF for water decontamination via different advanced oxidation processes: Multifunction, mechanisms and biotoxicity test

Peroxymonosulfate (PMS), peroxydisulfate (PDS) and hydrogen peroxide (H2O2)-based advanced oxidation processes are extensively applied for aqueous pollutants degradation. However, few cases reported the comparison the PMS, PDS and H2O2 activation performances of a heterogeneous catalyst. In this work, 1,1′-ferrocenedicarboxylate (Fc) modified Bi-BDC (Bi-BDC-Fcx) with unique electronic structure and strong oxidation ability was synthesized and applied for degrading bisphenols (BPs) via photocatalytic PMS, PDS, and H2O2 activation, respectively. The optimum Bi-BDC-Fc0.1 exhibited the best photocatalytic PMS, PDS and H2O2 activation performance to degrade various organic pollutants, including BPs, in which the bisphenol A degradation efficiencies followed the order of Bi-BDC-Fc0.1/UVL/PMS, Bi-BDC-Fc0.1/UVL/PDS and Bi-BDC-Fc0.1/UVL/H2O2. The reaction mechanisms of photocatalytic PMS, PDS and H2O2 activation over Bi-BDC-Fc0.1 were comprehensively clarified by experimental results along with DFT calculations. Also, the toxicity and ecotoxicological impact of the pristine BPs and their intermediates were evaluated by the phytotoxicity experiments and zebrafish early-life stage toxicity test. This work presented a comprehensive investigation of degradation performance and the ecotoxicology assessment of Bi-BDC-Fcx/UVL/PMS, Bi-BDC-Fcx/UVL/PDS, and Bi-BDC-Fcx/UVL/H2O2 systems, providing valuable insights to design and utilize the newly developed catalysts for water purification.

Developmental toxicity and mechanism of dibutyl phthalate and alternative diisobutyl phthalate in the early life stages of zebrafish (Danio rerio)

Diisobutyl phthalate (DiBP), is widely chemical replacement for Dibutyl phthalate (DBP). Although DBP and DiBP have been detected in surface water worldwide, few studies to date have systematically assessed the risks of DBP and its alternatives to aquatic organisms. The present study compared DBP and DiBP for their individual and joint toxicity as well as thyroid hormone levels in zebrafish embryo. Transcripts of key genes related to the hypothalamic-pituitary-thyroid (HPT) axis were investigated in developing zebrafish larvae by application of real time polymerase chain reaction. The median half-lethal concentrations of DBP and DiBP to zebrafish at 96 h were 0.545 mg L−1 and 1.149 mg L−1, respectively. The joint toxic effect of DBP–DiBP (0.25–0.53 mg L−1) with the same ratio showed a synergistic effect. Thyroid hormones levels increased with exposure to 10 μg L−1 of DBP or 50 μg L−1 of DiBP, and exposure to both compounds significantly increased thyroid gland-specific transcription of thyroglobulin gene (tg), hyronine deiodinase (dio2), and transthyretin (ttr), indicating an adverse effect associated with the HPT axis. Molecular docking results indicated that DBP (−7.10 kcal/M and −7.53 kcal/M) and DiBP (−6.63 kcal/M and −7.42 kcal/M) had the same docking energy with thyroid hormone receptors. Our data facilities an understand of potential harmful effects of DBP and its alternative (DiBP).

Exposure to acifluorfen induces developmental toxicity in the early life stage of zebrafish

Acifluorfen, a selective herbicide from the diphenyl ether family, targets broad leaf weeds. Diphenyl ether inhibits chlorophyll production in green plants by inhibiting protoporphyrinogen oxidase (PPO), causing cellular damage. Despite its known impacts on plants, the influence of acifluorfen on zebrafish embryo development remains unclear. In this study, we explored the LC50 of acifluorfen in early-stage wild-type zebrafish, determining it to be 54.99 mg/L. Subsequent examinations revealed morphological changes in zebrafish, including reduced body length. Using the cmlc2:dsRED transgenic model, we observed heart dysfunction in acifluorfen-exposed zebrafish, marked by an enlarged heart area, edema, and decreased heart rate. In response to dose-dependent acifluorfen exposure, the inhibition of angiogenesis in the brain was observed in transgenic zebrafish models (fli1a:eGFP). Organ malformations, specifically in the liver and pancreas, were noted, in lfabp:dsRED;elastase:eGFP transgenic models, indicating reduced organ size in acifluorfen-exposed zebrafish. Furthermore, acifluorfen heightened the expression of apoptosis-related genes (casp8, casp9, and tp53) in zebrafish embryos. We then determined whether acifluorfen affected the viability of zebrafish liver (ZFL) cells based on its effects on liver development in vivo. The results indicated that the proliferation of ZFL cells decreased significantly in a dose-dependent manner. Additionally, acifluorfen-treated ZFL cells exhibited a slight increase in apoptotic cells stained with annexin V and propidium iodide. In summary, these findings establish a baseline concentration for acifluorfen's effects on aquatic ecosystems and non-target organisms.

Embryotoxicity Induced by Triclopyr in Zebrafish (Danio rerio) Early Life Stage.

Triclopyr, an auxin-like herbicide that is widely employed for managing weeds in food crops and pastures, has been identified in various environmental settings, particularly aquatic ecosystems. Limited understanding of the environmental fate of this herbicide, its potential repercussions for both the environment and human health, and its insufficient monitoring in diverse environmental compartments has caused it to be recognized as an emerging contaminant of concern. In this study, we have investigated how triclopyr affects zebrafish, considering a new alternative methodology. We focused on the endpoints of developmental toxicity, neurotoxicity, and behavior of zebrafish embryos and larvae. We determined that triclopyr has a 96 h median lethal concentration of 87.46 mg/L (341.01 µM). When we exposed zebrafish embryos to sublethal triclopyr concentrations (0.5, 1, 5, 10, and 50 μM) for up to 144 h, we found that 50 µM triclopyr delayed zebrafish egg hatchability. Yolk sac malabsorption was significant at 0.5, 1, 5, and 10 µM triclopyr. In zebrafish larvae, uninflated swim bladder was significant only at 50 µM triclopyr. Furthermore, zebrafish larvae had altered swimming activity after exposure to 10 µM triclopyr for 144 h. In summary, these comprehensive results indicate that even low triclopyr concentrations can elicit adverse effects during early zebrafish development.

Multilevel assessment of carbamazepine effects: An integrative approach using zebrafish early-life stages

Carbamazepine (CBZ) is the most commonly used drug in epilepsy treatment, and its metabolites are commonly detected among persistent pharmaceuticals in the aquatic environment. This study aimed to investigate CBZ effects on early-life-stage zebrafish (Danio rerio) (from 2 to 168 hpf) by employing of an integrative approach linking endpoints from molecular to individual level: (i) development; (ii) locomotor activity; (iii) biochemical markers (lactate dehydrogenase, glutathione-S-transferase, acetylcholinesterase and catalase) and (iv) transcriptome analysis using microarray. A 168 h - LC50 of 73.4 mg L−1 and a 72 h - EC50 of 66.8 mg L−1 for hatching were calculated while developmental effects (oedemas and tail deformities) were observed at CBZ concentrations above 37.3 mg L−1. At the biochemical level, AChE activity proved to be the most sensitive parameter, as evidenced by its decrease across all concentrations tested (∼25% maximum reduction, LOEC (lowest observed effect concentration) < 0.6 μg L−1). Locomotor behaviour seemed to be depressed by CBZ although this effect was only evident at the highest concentration tested (50 mg L−1). Molecular analysis revealed a dose-dependent effect of CBZ on gene expression. Although only 25 genes were deregulated in organisms exposed to CBZ when compared to controls, both 0.6 and 2812 μg L−1 treatments impaired gene expression related to development (e.g. crygmxl1, org, klf2a, otos, stx16 and tob2) and the nervous system (e.g. Rtn3, Gdf10, Rtn3), while activated genes were associated with behavioural response (e.g. prlbr and taar). Altogether, our results indicate that environmentally relevant CBZ concentrations might affect biochemical and genetic traits of fish. Thus, the environmental risk of CBZ cannot be neglected, especially in a realistic scenario of constant input of domestic effluents into aquatic systems.

'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.

An Integrated Metabolomics-Based Model, and Identification of Potential Biomarkers, of Perfluorooctane Sulfonic Acid Toxicity in Zebrafish Embryos.

Known for their high stability and surfactant properties, per- and polyfluoroalkyl substances (PFAS) have been widely used in a range of manufactured products. Despite being largely phased out due to concerns regarding their persistence, bioaccumulation, and toxicity, legacy PFAS such as perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid continue to persist at high levels in the environment, posing risks to aquatic organisms. We used high-resolution magic angle spinning nuclear magnetic resonance spectroscopy in intact zebrafish (Danio rerio) embryos to investigate the metabolic pathways altered by PFOS both before and after hatching (i.e., 24 and 72 h post fertilization [hpf], respectively). Assessment of embryotoxicity found embryo lethality in the parts-per-million range with no significant difference in mortality between the 24- and 72-hpf exposure groups. Metabolic profiling revealed mostly consistent changes between the two exposure groups, with altered metabolites generally associated with oxidative stress, lipid metabolism, energy production, and mitochondrial function, as well as specific targeting of the liver and central nervous system as key systems. These metabolic changes were further supported by analyses of tissue-specific production of reactive oxygen species, as well as nontargeted mass spectrometric lipid profiling. Our findings suggest that PFOS-induced metabolic changes in zebrafish embryos may be mediated through previously described interactions with regulatory and transcription factors leading to disruption of mitochondrial function and energy metabolism. The present study proposes a systems-level model of PFOS toxicity in early life stages of zebrafish, and also identifies potential biomarkers of effect and exposure for improved environmental biomonitoring. Environ Toxicol Chem 2024;43:896-914. © 2024 SETAC.

Elucidating the effects of pure glyphosate and a commercial formulation on early life stages of zebrafish using a complete biomarker approach: All-or-nothing!

The search for new methods in the toxicology field has increased the use of early life stages of zebrafish (Danio rerio) as a versatile organism model. Here, we use early stages of zebrafish to evaluate glyphosate as pure active ingredient and within a commercial formulation in terms of oxidative stress. Biomarkers involved in the oxidative status were evaluated along with other markers of neurotoxicity, genotoxicity, cytotoxicity, energy balance and motor performance, and the selected tools were evaluated by its sensitivity in determining early-warning events. Zebrafish embryos exposed to glyphosate active ingredient and glyphosate-based formulation were under oxidative stress, but only the commercial formulation delayed the embryogenesis, affected the cholinergic neurotransmission and induced DNA damage. Both altered the motor performance of larvae at very low concentrations, becoming larvae hypoactive. The energy balance was also impaired, as embryos under oxidative stress had lower lipids reserves. Although data suggest that glyphosate-based formulation has higher toxicity than the active ingredient itself, the most sensitive biomarkers detected early-warning effects at very low concentrations of the active ingredient. Biochemical biomarkers of defense system and oxidative damage were the most sensitive tools, detecting pro-oxidant responses at very low concentrations, along with markers of motor performance that showed high sensitivity and high throughput, suitable for detecting early effects linked to neurotoxicity. Alterations on morphology during embryogenesis showed the lowest sensitivity, thus morphological alterations appeared after several alterations at biochemical levels. Tools evaluating DNA damage and cell proliferation showed mid-sensitivity, but low throughput, thus they could be used as complementary markers.

Cortisol Quantification for Assessing Stress-Induced Changes in Zebrafish Larvae.

The stress response, mainly mediated by cortisol, plays a critical role in the regulation of physiological and behavioral homeostasis through a variety of mechanisms. Different aquatic animal models have been widely employed to understand the pathobiology of stress and stress-related brain disorders. The early life stress can affect the hypothalamic-pituitary-interrenal (HPI) axis and induce cellular and molecular impairments that impact the brain functioning later in life. However, these alterations have been poorly explored mainly due to the lack of suitable models. In this chapter, the vortex flow stimulation, an acute stress that causes a forced swimming and activates the HPI axis, is described and its correlations with behavioral outputs reported. To this end, the early life stages of zebrafish are used as animal models for modeling stress disorders experimentally. The behavioral despair model can be employed as an initial screening tool for assessing neural circuit activation and motor alterations. Taken together, the implementation of this strategy in this animal model allows the analysis of stress responses in a simple manner and its correlation with neural circuitries and motor alterations.

Effects of exposure to 3,6-DBCZ on neurotoxicity and AhR pathway during early life stages of zebrafish (Danio rerio)

Polyhalogenated carbazoles (PHCZs) are emerging environmental pollutants, yet limited information is available on their embryotoxicity and neurotoxicity. Therefore, the current work was performed to investigate the adverse effects of 3,6-dibromocarbazole (3,6-DBCZ), a typical PHCZs homolog, on the early life stages of zebrafish larvae. It revealed that the 96-hour post-fertilization (hpf) median lethal concentration (LC50) value of 3,6-DBCZ in zebrafish larvae was determined to be 0.7988 mg/L. Besides, 3,6-DBCZ reduced survival rates at concentrations ≥ 1 mg/L and decreased hatching rates at ≥ 0.25 mg/L at 48 hpf. In behavior tests, it inhibited locomotor activities and reduced the frequency of recorded acceleration states in response to optesthesia (a sudden bright light stimulus) at concentrations ≥ 160 μg/L. Meanwhile, 3,6-DBCZ exposure decreased the frequency of recorded acceleration states in the startle response (tapping mode) at concentrations ≥ 6.4 μg/L. Pathologically, with the transgenic zebrafish model (hb9-eGFP), we observed a strikingly decreased axon length and number in motor neurons after 3,6-DBCZ treatment, which may be ascribed to the activation of the AhR signaling pathway, as evidenced by the molecular docking analysis and Microscale thermophoresis (MST) assay suggested that 3,6-DBCZ binding to AhR-ARNT2 compound proteins. Through interaction with AhR-ARNT, a striking reduction of the anti-oxidative stress (sod1/2, nqo1, nrf2) and neurodevelopment-related genes (elavl3, gfap, mbp, syn2a) were observed after 3,6-DBCZ challenge, accompanied by a marked increased inflammatory genes (TNFβ, IL1β, IL6). Collectively, our findings reveal a previously unrecognized adverse effect of 3,6-DBCZ on zebrafish neurodevelopment and locomotor behaviors, potentially mediated through the activation of the AhR pathway. Furthermore, it provides direct evidence for the toxic concentrations of 3,6-DBCZ and the potential target signaling in zebrafish larvae, which may be beneficial for the risk assessment of the aquatic ecosystems.

Combined inhibitory effects of microcystin-LR and microcystin-RR on growth and development in zebrafish larvae

Microcystins (MCs) are the most widespread, frequently found, and seriously toxic cyanobacterial toxins in aquatic environments. Microcystin-leucine-arginine (MCLR) and microcystin-arginine-arginine (MCRR) are the most studied MCs. Normally, their levels are low and they coexist in the environment; however, they may also interact with each other. The developmental toxicity of MCLR in the presence of MCRR in the early life stage of zebrafish (from 2 to 120 h post fertilization) was investigated for the first time in this study. Our findings revealed that MCRR treatment marginally elevated thyroxine (T4) and 3,5,3′-triiodothyronine (T3) levels, whereas MCLR treatment alone resulted in a significant increase in T3 and T4 levels, indicating a cooperative effect. Furthermore, clear changes in the expression levels of genes involved in growth and development, accompanied by growth inhibition, were observed after co-treatment with MCRR and MCLR. In addition, zebrafish larvae subjected to MCRR and/or MCLR treatment showed increased levels of superoxide dismutase, glutathione, and malondialdehyde, and decreased levels of catalase in the MCRR + MCLR group, indicating oxidative stress and lipid peroxidation. Thus, we investigated the synergistic developmental toxicity of MCRR and MCLR during the early life stages of zebrafish development.

Analysis of vascular disruption in zebrafish embryos as an endpoint to predict developmental toxicity

Inhibition of angiogenesis is an important mode of action for the teratogenic effect of chemicals and drugs. There is a gap in the availability of simple, experimental screening models for the detection of angiogenesis inhibition. The zebrafish embryo represents an alternative test system which offers the complexity of developmental differentiation of an entire organism while allowing for small-scale and high-throughput screening. Here we present a novel automated imaging-based method to detect the inhibition of angiogenesis in early life stage zebrafish. Video subtraction was used to identify the location and number of functional intersegmental vessels according to the detection of moving blood cells. By exposing embryos to multiple tyrosine kinase inhibitors including SU4312, SU5416, Sorafenib, or PTK787, we confirmed that this method can detect concentration-dependent inhibition of angiogenesis. Parallel assessment of arterial and venal aorta ruled out a potential bias by impaired heart or blood cell development. In contrast, the histone deacetylase inhibitor valproic acid did not affect ISV formation supporting the specificity of the angiogenic effects. The new test method showed higher sensitivity, i.e. lower effect concentrations, relative to a fluorescent reporter gene strain (Tg(KDR:EGFP)) exposed to the same tyrosine kinase inhibitors indicating that functional effects due to altered tubulogenesis or blood transport can be detected before structural changes of the endothelium are visible by fluorescence imaging. Comparison of exposure windows indicated higher specificity for angiogenesis when exposure started at later embryonic stages (24 h post-fertilization). One of the test compounds was showing particularly high specificity for angiogenesis effects (SU4312) and was, therefore, suggested as a model compound for the identification of molecular markers of angiogenic disruption. Our findings establish video imaging in wild-type strains as viable, non-invasive, high-throughput method for the detection of chemical-induced angiogenic disruption in zebrafish embryos.