Dolutegravir and Folic Acid Interaction during Neural System Development in Zebrafish Embryos.

Dolutegravir (DTG) is one of the most prescribed antiretroviral drugs for treating people with HIV infection, including women of child-bearing potential or pregnant. Nonetheless, neuropsychiatric symptoms are frequently reported. Early reports suggested that, probably in relation to folic acid (FA) shortage, DTG may induce neural tube defects in infants born to women taking the drug during pregnancy. Subsequent reports did not definitively confirm these findings. Recent studies in animal models have highlighted the association between DTG exposure in utero and congenital anomalies, and an increased risk of neurologic abnormalities in children exposed during in utero life has been reported. Underlying mechanisms for DTG-related neurologic symptoms and congenital anomalies are not fully understood. We aimed to deepen our knowledge on the neurodevelopmental effects of DTG exposure and further explore the protective role of FA by the use of zebrafish embryos. We treated embryos at 4 and up to 144 h post fertilization (hpf) with a subtherapeutic DTG concentration (1 µM) and observed the disruption of the anterior-posterior axis and several morphological malformations in the developing brain that were both prevented by pre-exposure (2 hpf) and rescued by post-exposure (10 hpf) with FA. By whole-mount in situ hybridization with riboprobes for genes that are crucial during the early phases of neurodevelopment (ntl, pax2a, ngn1, neurod1) and by in vivo visualization of the transgenic Tg(ngn1:EGFP) zebrafish line, we found that DTG induced severe neurodevelopmental defects over time in most regions of the nervous system (notochord, midbrain-hindbrain boundary, eye, forebrain, midbrain, hindbrain, spinal cord) that were mostly but not completely rescued by FA supplementation. Of note, we observed the disruption of ngn1 expression in the dopaminergic regions of the developing forebrain, spinal cord neurons and spinal motor neuron projections, with the depletion of the tyrosine hydroxylase (TH)+ dopaminergic neurons of the dorsal diencephalon and the strong reduction in larvae locomotion. Our study further supports previous evidence that DTG can interfere with FA pathways in the developing brain but also provides new insights regarding the mechanisms involved in the increased risk of DTG-associated fetal neurodevelopmental defects and adverse neurologic outcomes in in utero exposed children, suggesting the impairment of dopaminergic pathways.

Noscapine modulates hypoxia-induced angiogenesis and hemodynamics: Insights from a zebrafish model investigation.

We investigated the angiogenesis-modulating ability of noscapine in vitro using osteosarcoma cell line (MG-63) and in vivo using a zebrafish model. MTT assay and the scratch wound healing assay were performed on the osteosarcoma cell line (MG-63) to analyze the cytotoxic effect and antimigrative ability of noscapine, respectively. We also observed the antiangiogenic ability of noscapine on zebrafish embryos by analyzing the blood vessels namely the dorsal aorta, and intersegmental vessels development at 24, 48, and 72 h postfertilization. Real-time polymerase chain reaction was used to analyze the hypoxia signaling molecules' gene expression in MG-63 cells and zebrafish embryos. The findings from the scratch wound healing demonstrated that noscapine stopped MG-63 cancer cells from migrating under both hypoxia and normoxia. Blood vessel development and the heart rate in zebrafish embryos were significantly reduced by noscapine under both hypoxia and normoxia which showed the hemodynamics impact of noscapine. Noscapine also downregulated the cobalt chloride (CoCl2) induced hypoxic signaling molecules' gene expression in MG-63 cells and zebrafish embryos. Therefore, noscapine may prevent MG-63 cancer cells from proliferating and migrating, as well as decrease the formation of new vessels and the production of growth factors linked to angiogenesis in vivo under both normoxic and hypoxic conditions.

Cocktail effects of tire wear particles leachates on diverse biological models: A multilevel analysis.

Tire wear particles (TWP) stand out as a major contributor to microplastic pollution, yet their environmental impact remains inadequately understood. This study delves into the cocktail effects of TWP leachates, employing molecular, cellular, and organismal assessments on diverse biological models. Extracted in artificial seawater and analyzed for metals and organic compounds, TWP leachates revealed the presence of polyaromatic hydrocarbons and 4-tert-octylphenol. Exposure to TWP leachates (1.5 to 1000 mg peq L-1) inhibited algae growth and induced zebrafish embryotoxicity, pigment alterations, and behavioral changes. Cell painting uncovered pro-apoptotic changes, while mechanism-specific gene-reporter assays highlighted endocrine-disrupting potential, particularly antiandrogenic effects. Although heavy metals like zinc have been suggested as major players in TWP leachate toxicity, this study emphasizes water-leachable organic compounds as the primary causative agents of observed acute toxicity. The findings underscore the need to reduce TWP pollution in aquatic systems and enhance regulations governing highly toxic tire additives.

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.

Bifenox induces hepatotoxicity and vascular toxicity in zebrafish embryos via ROS production and alterations in signaling pathways.

Existing evidence shows that currently used pesticides pose toxicological risks to exposed wildlife. Chemically, bifenox belongs to diphenyl ethers, a well-known group of herbicides. Its mechanism of action primarily involves inducing lipid peroxidation and blocking protoporphyrinogen oxidases. Toxicity of diphenyl ether herbicides has been elucidated in animal cells; however, in vivo toxicological evaluations of bifenox are required to determine its unexpected effects. This study aimed to determine the negative effects of bifenox, and its effects on higher eukaryotes. We found that early stages of zebrafish embryo exposed to bifenox demonstrated increased mortality and physiological defects, based on the LC50 value. Bifenox severely inhibited blood vessel growth by reducing key elements of complex connectivity; fluorescently tagged transgenic lines (fli1a:EGFP) showed morphological changes. Additionally, transgenic lines that selectively identified hepatocytes (fabp10a:DsRed) showed reduced fluorescence, indicating that bifenox may inhibit liver development. To evaluate the level of oxidative stress, we used 2',7'-dichlorofluorescein diacetate (DCFH-DA) probes in zebrafish embryos to identify the underlying mechanisms causing developmental damage. Our findings demonstrate that exposure to bifenox causes abnormalities in the hepatic and cardiovascular systems during zebrafish embryogenesis. Therefore, this study provides new information for the evaluation of toxicological risks of bifenox in vertebrates.

The combined effects of preservative chemicals in consumer products: An analysis using embryonic and adult zebrafish.

Benzisothiazolinone (BIT) and propyl paraben (PP) are preservatives in cleaning products; however, their toxicities are not well understood. In this study, zebrafish embryos were exposed to BIT, PP, and mixtures of both for 96 h to investigate the effects on growth hormone (GH), insulin-like growth factor-1 (IGF-1), and the transcription of 19 genes related to the GH/IGFs axis. Concentrations of BIT and PP were measured in the whole body of larvae. Zebrafish pairs were also exposed to BIT, PP, and mixtures for 21 d to evaluate the effects on sex hormones, histology in gonad, and transcription of 22 genes related to the hypothalamus-pituitary-gonad axis and vitellogenin. The mixtures had potentiation effects on development, reproduction, hormones, and gene transcripts than individual exposure. Larvae exposed to 229 µg L-1 BIT, 64.5 µg L-1 PP, and mixtures showed reduced growth. Decreased GH and IGF-1 levels were supported by gene regulation associated with the GH/IGFs axis. In larvae, reactive oxygen species, superoxide dismutase, catalase, and glutathione peroxidase levels were increased under all exposures. The gonadosomatic index in males and number of eggs decreased after mixture exposure. In females exposed to mixtures, the percentage of atretic follicle in ovary was significantly increased. The significant decrease in testosterone in males and significant decrease in 17ß-estradiol in females exposed to mixtures suggest anti-estrogenic and anti-androgenic potential. Thus, preservative mixtures in consumer products may be more toxic than the individual substances, which is important for managing the risks of mixing preservatives.

Multi-biomarker approach to evaluate the toxicity of chlorpyrifos (active ingredient and a commercial formulation) on different stages of Biomphalaria straminea.

Biomphalaria straminea is a freshwater gastropod native to South America and used in toxicological assessments. Our aim was to estimate 48 h-LC50 and sub-chronic effects after the exposure to low concentrations of chlorpyrifos as commercial formulation (CF) and active ingredient (AI) on B. straminea adult, embryos and juveniles. Concentrations between 1 and 5000 µg L-1 were chosen for acute exposures and 0.1 and 1 µg L-1 for the sub-chronic one. After 14 days biochemical parameters, viability and sub-populations of hemocytes, reproductive parameters, embryotoxicity and offspring' survival were studied. Egg masses laid between day 12 and 14 were separated to continue the exposure and the embryos were examined daily. Offspring' survival and morphological changes were registered for 14 days after hatching. 48 h-LC50, NOEC and LOEC were similar between CF and AI, however the CF caused more sub-lethal effects. CF but not the AI decreased carboxylesterases, catalase and the proportion of hyalinocytes with respect to the total hemocytes, and increased superoxide dismutase and the % of granulocytes with pseudopods. Also CF caused embryotoxicity probably due to the increase of embryos' membrane permeability. Acetylcholinesterase, superoxide dismutase, hemocytes sub-populations, the time and rate of hatching and juveniles' survival were the most sensitive biomarkers. We emphasize the importance of the assessment of a battery of biomarkers as a useful tool for toxicity studies including reproduction parameters and immunological responses. Also, we highlight the relevance of incorporating the evaluation of formulations in order to not underestimate the effects of pesticides on the environment.

Comparison of developmental toxicity of graphene oxide and graphdiyne to zebrafish larvae.

Graphdiyne (GDY) is a new member of family of carbon-based 2D nanomaterials (NMs), but the environmental toxicity is less investigated compared with other 2D NMs, such as graphene oxide (GO). In this study, we compared with developmental toxicity of GO and GDY to zebrafish larvae. It was shown that exposure of zebrafish embryos from 5 h post fertilization to GO and GDY for up to 5 days decreased hatching rate and induced morphological deformity. Behavioral tests indicated that GO and GDY treatment led to hyperactivity of larvae. However, blood flow velocity was not significantly affected by GO or GDY. RNA-sequencing data revealed that both types of NMs altered gene expression profiles as well as gene ontology terms and KEGG pathways related with metabolism. We further confirmed that the NMs altered the expression of genes related with lipid droplets and autophagy, which may be account for the delayed development of zebrafish larvae. At the same mass concentrations, GO induced comparable or even larger toxic effects compared with GDY, indicating that GDY might be more biocompatible compared with GO. These results may provide novel understanding about the environmental toxicity of GO and GDY in vivo.

Myclobutanil induces neurotoxicity by activating autophagy and apoptosis in zebrafish larvae (Danio rerio).

Myclobutanil (MYC), a typical broad-spectrum triazole fungicide, is often detected in surface water. This study aimed to explore the neurotoxicity of MYC and the underlying mechanisms in zebrafish and in PC12 cells. In this study, zebrafish embryos were exposed to 0, 0.5 and 1 mg/L of MYC from 4 to 96 h post fertilization (hpf) and neurobehavior was evaluated. Our data showed that MYC decreased the survival rate, hatching rate and heart rate, but increased the malformation rate and spontaneous movement. MYC caused abnormal neurobehaviors characterized by decreased swimming distance and movement time. MYC impaired cerebral histopathological morphology and inhibited neurogenesis in HuC:egfp transgenic zebrafish. MYC also reduced the activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and downregulated neurodevelopment related genes (gfap, syn2a, gap43 and mbp) in zebrafish and PC12 cells. Besides, MYC activated autophagy through enhanced expression of the LC3-II protein and suppressed expression of the p62 protein and autophagosome formation, subsequently triggering apoptosis by upregulating apoptotic genes (p53, bax, bcl-2 and caspase 3) and the cleaved caspase-3 protein in zebrafish and PC12 cells. These processes were restored by the autophagy inhibitor 3-methyladenine (3-MA) both in vivo and in vitro, indicating that MYC induces neurotoxicity by activating autophagy and apoptosis. Overall, this study revealed the potential autophagy and apoptosis mechanisms of MYC-induced neurotoxicity and provided novel strategies to counteract its toxicity.

Genome-wide DNA methylation changes in Oryzias melastigma embryos exposed to the water accommodated fraction of crude oil.

The water accommodated fraction (WAF) of crude oil exerts considerable impacts on marine fish during embryonic stage. Clarifying changes in epigenetic modifications is helpful for understanding the molecular mechanism underlying the toxicity of embryonic WAF exposure. The aim of this study was to explore genome-wide DNA methylation changes in Oryzias melastigma embryos after exposure to the nominal total petroleum hydrocarbon concentration of 500 µg/L in WAF for 7 days. Whole-genome bisulfite sequencing revealed that 8.47 % and 8.46 % of all the genomic C sites were methylated in the control and WAF-exposed groups, respectively. Among the three sequence contexts, methylated CG site had the largest number in both the two groups. The sequence preferences of nearby methylated cytosines were consistent between the two groups. A total of 4798 differentially methylated regions (DMRs) were identified in the promoter region. Furthermore, Gene Ontology analysis revealed that DMR-related genes were enriched mainly for functions related to development and nervous system. Additionally, the Kyoto Encyclopedia of Genes and Genomes pathways enriched in DMR-related genes were related to nervous system and endocrine system. These novel findings provide comprehensive insights into the genome-wide DNA methylation landscape of O. melastigma following embryonic WAF exposure, shedding light on the epigenetic regulatory mechanisms underlying WAF-induced toxicity.

Effect of biodegradable polymers upon grazing activity of the sea urchin Paracentrotus lividus (Lmk) revealed by morphological, histological and molecular analyses.

In the last years biodegradable polymers (BPs) were largely used as real opportunity to solve plastic pollution. Otherwise, their wide use in commercial products, such as packaging sector, is causing a new pollution alarm, mainly because few data reported about their behaviour in the environment and toxicity on marine organisms. Our previous results showed that embryos of the sea urchin Paracentrotus lividus (Lmk) exposed to poly(ε-caprolactone) (PCL), poly(3-hydroxybutyrate) (PHB) and poly(lactic acid) (PLA) showed delay of their development and morphological malformations, also affecting at the molecular levels the expression of several genes involved in different functional responses. In the present work for the first time, we tested the effects of five microplastics (MPs) obtained from BPs such as PBS, poly(butylene succinate), PBSA, poly(butylene succinate-co-butylene adipate), PCL, PHB and PLA, upon grazing activity of the sea urchin revealed by: i. histological analysis seeing at the gonadic tissues; ii. morphological analysis of the deriving embryos; iii. molecular analyses on these embryos to detect variations of the gene expression of eighty-seven genes involved in stress response, detoxification, skeletogenesis, differentiation and development. All these results will help in understanding how MP accumulated inside various organs in the adult sea urchins, and more in general in marine invertebrates, could represent risks for the marine environment.

Roxithromycin exposure induces motoneuron malformation and behavioral deficits of zebrafish by interfering with the differentiation of motor neuron progenitor cells.

Roxithromycin (ROX), a commonly used macrolide antibiotic, is extensively employed in human medicine and livestock industries. Due to its structural stability and resistance to biological degradation, ROX persists as a resilient environmental contaminant, detectable in aquatic ecosystems and food products. However, our understanding of the potential health risks to humans from continuous ROX exposure remains limited. In this study, we used the zebrafish as a vertebrate model to explore the potential developmental toxicity of early ROX exposure, particularly focusing on its effects on locomotor functionality and CaP motoneuron development. Early exposure to ROX induces marked developmental toxicity in zebrafish embryos, significantly reducing hatching rates (n=100), body lengths (n=100), and increased malformation rates (n=100). The zebrafish embryos treated with a corresponding volume of DMSO (0.1%, v/v) served as vehicle controls (veh). Moreover, ROX exposure adversely affected the locomotive capacity of zebrafish embryos, and observations in transgenic zebrafish Tg(hb9:eGFP) revealed axonal loss in motor neurons, evident through reduced or irregular axonal lengths (n=80). Concurrently, abnormal apoptosis in ROX-exposed zebrafish embryos intensified alongside the upregulation of apoptosis-related genes (bax, bcl2, caspase-3a). Single-cell sequencing further disclosed substantial effects of ROX on genes involved in the differentiation of motor neuron progenitor cells (ngn1, olig2), axon development (cd82a, mbpa, plp1b, sema5a), and neuroimmunity (aplnrb, aplnra) in zebrafish larvae (n=30). Furthermore, the CaP motor neuron defects and behavioral deficits induced by ROX can be rescued by administering ngn1 agonist (n=80). In summary, ROX exposure leads to early-life abnormalities in zebrafish motor neurons and locomotor behavior by hindering the differentiation of motor neuron progenitor cells and inducing abnormal apoptosis.

Polystyrene nanoplastics synergistically exacerbate diclofenac toxicity in embryonic development and the health of adult zebrafish.

In this study, we investigated the possible ecotoxicological effect of co-exposure to polystyrene nanoplastics (PS-NPs) and diclofenac (DCF) in zebrafish (Danio rerio). After six days of exposure, we noticed that the co-exposure to PS-NP (100 µg/L) and DCF (at 50 and 500 µg/L) decreased the hatching rate and increased the mortality rate compared to the control group. Furthermore, we noted that larvae exposed to combined pollutants showed a higher frequency of morphological abnormalities and increased oxidative stress, apoptosis, and lipid peroxidation. In adults, superoxide dismutase and catalase activities were also impaired in the intestine, and the co-exposure groups showed more histopathological alterations. Furthermore, the TNF-α, COX-2, and IL-1ß expressions were significantly upregulated in the adult zebrafish co-exposed to pollutants. Based on these findings, the co-exposure to PS-NPs and DCF has shown an adverse effect on the intestinal region, supporting the notion that PS-NPs synergistically exacerbate DCF toxicity in zebrafish.

Diving into drug-screening: zebrafish embryos as an in vivo platform for antimicrobial drug discovery and assessment.

The rise of multidrug-resistant bacteria underlines the need for innovative treatments, yet the introduction of new drugs has stagnated despite numerous antimicrobial discoveries. A major hurdle is a poor correlation between promising in vitro data and in vivo efficacy in animal models, which is essential for clinical development. Early in vivo testing is hindered by the expense and complexity of existing animal models. Therefore, there is a pressing need for cost-effective, rapid preclinical models with high translational value. To overcome these challenges, zebrafish embryos have emerged as an attractive model for infectious disease studies, offering advantages such as ethical alignment, rapid development, ease of maintenance, and genetic manipulability. The zebrafish embryo infection model, involving microinjection or immersion of pathogens and potential antibiotic hit compounds, provides a promising solution for early-stage drug screening. It offers a cost-effective and rapid means of assessing the efficacy, toxicity and mechanism of action of compounds in a whole-organism context. This review discusses the experimental design of this model, but also its benefits and challenges. Additionally, it highlights recently identified compounds in the zebrafish embryo infection model and discusses the relevance of the model in predicting the compound's clinical potential.

Dihalogenated nitrophenols exposure induces developmental neurotoxicity in zebrafish embryo.

2,6-Dihalogenated nitrophenols (2,6-DHNPs) are emerging halogenated nitroaromatic pollutants that have been detected in various water environments. However, there is currently limited research available regarding their potential impacts on locomotion behavior and neurotoxicity. Therefore, this study utilized zebrafish embryos to investigate the potential neurotoxic effects of 2,6-DHNPs by examining their impact on the nervous system at a concentration defined as 10% of the median lethal concentration. Our findings demonstrated that exposure to 2,6-DHNPs resulted in a significant 30 % decrease in the total swimming distance of zebrafish larvae, accompanied by notable impairments in motor neuron development and central nervous system. These effects were evidenced by a substantial 25% decrease in axonal growth, as well as disruptions in synapse formation and neuronal differentiation. Additionally, neurotransmitter analysis revealed marked decreases of 40%, 35%, and 30% in dopamine, 5-hydroxytryptamine, and acetylcholine levels respectively, highlighting disturbances in their synthesis, transport, and degradation mechanisms. These results emphasize the considerable neurotoxicity of 2,6-DHNPs at concentrations previously considered safe; thus necessitating a re-evaluation of environmental risk assessments and regulatory standards for such emerging contaminants.

Effects of developmental exposure to individual and combined PFAS on development and behavioral stress responses in larval zebrafish.

Per- and polyfluoroalkyl substances (PFAS) are a class of synthetic chemicals known for their widespread use and persistence in the environment. Laboratory and epidemiological studies investigating these compounds have signaled their neurotoxic and endocrine-disrupting propensities, prompting further research into their effects on behavioral stress responses and their potential role as risk factors for stress-related disorders such as anxiety and depression. This study elucidates the ramifications of early developmental exposures to individual and combined PFAS on the development and behavioral stress responses of larval zebrafish (Danio rerio), an established model in toxicological research. Wild-type zebrafish embryos were enzymatically dechorionated and exposed to PFOS, PFOA, PFHxS, and PFHxA between 6 and 120 h post-fertilization (hpf). We targeted environmentally relevant concentrations stemming from the USEPA 2016 Hazard Advisory Limit (HAL, 0.07 µg/L) and folds higher (0.35, 0.7, 1.75, and 3.5 µg/L). Evaluations at 120 hpf encompassed mortality, overall development, developmental defects, and larval activity both at baseline stress levels and following exposure to acute stressors (acoustic and visual). Larval exposure to PFOA, PFOS, or PFHxS (0.07 µg/L or higher) elicited significant increases in mortality rates, which capped at 23.1%. Exposure to individual chemicals resulted in limited effects on overall development but increased the prevalence of developmental defects in the body axis, swim bladder, pigmentation, and eyes, as well as the prevalence of yolk sac and pericardial edemas. Larval activity at baseline stress levels and following exposure to acute stimuli was significantly altered. Combined exposure to all four chemicals intensified the breadth of developmental and behavioral alterations, suggesting possible additive or synergistic effects. Our findings shed light on the developmental and neurobehavioral disturbances associated with developmental exposure to PFAS at environmentally relevant concentrations, the added risks of combined exposures to these chemicals, and their possible role as environmental risk factors for stress-related disorders.

Early life stage exposure to fenbuconazole causes multigenerational cardiac developmental defects in zebrafish and potential reasons.

With the increasing use of triazole fungicides in agriculture, triazole pesticides have aroused great concern about their toxicity and ecological risk. The current study investigated the impairments of embryonic exposure to fenbuconazole (FBZ) on cardiac transgenerational toxicity and related mechanisms. The fertilized eggs were exposed to 5, 50 and 500 ng/L FBZ for 72 h, and the larvae were then raised to adulthood in clean water. The adult fish were mated with unexposed fish to produce maternal and paternal F1 and F2 embryos, respectively. The results showed that increased arrhythmia were observed in F0, F1 and F2 larvae. Transcriptome sequencing indicated that the pathway of adrenergic signaling in cardiomyocytes was enriched in F0 and F2 larvae. In both F0 and F1 adult zebrafish hearts, ADRB2 protein expression decreased, and transcription of genes related to cardiac development and Ca2+ homeostasis was downregulated. These alterations might cause cardiac developmental defects. Significantly decreased protein levels of H3K9Ac and H3K14Ac might be linked with the downregulation in transcription of cardiac development genes. Protein‒protein interaction analysis exhibited that the pathway affecting the heart was well inherited in the paternal line. These results provide new ideas for the analysis and prevention of congenital heart disease.

Potential adverse outcome pathway of neurodevelopmental toxicity, inflammatory response, and oxidative stress induction mediated by three alkyl organophosphate flame retardants in zebrafish larvae.

Following restrictions on polybrominated flame retardants, trimethyl phosphate (TMP), triethyl phosphate (TEP), and tris(2-butoxyethyl) phosphate (TBEP) have been frequently used as plasticizers for fire-resistant plastics. This study investigated the neurodevelopmental effects, inflammatory response, and oxidative stress induction of three alkyl organophosphate flame retardants using a zebrafish embryo/larvae model. After exposure of zebrafish embryos to TMP, TEP, and TBEP (0, 0.02, 0.2, 2, 20, and 200 µg L-1) for 96 h, survival, development, swimming behavior, changes in acetylcholinesterase (AChE) activity, dopamine, tumor necrosis factor-alpha (TNF-α), interleukin (IL), reactive oxygen species (ROS), and antioxidant enzyme activities were observed. Concentrations of TMP, TEP, and TBEP were also measured in the whole body of exposed larvae. Our results showed that exposure to 200 µg L-1 TEP and ≥20 µg L-1 TBEP significantly reduced larval body length; however, TMP had no significant effects on developmental parameters up to 200 µg L-1. After 96 h of exposure to TBEP, total distance moved, mean velocity, AChE, and dopamine concentrations were significantly decreased. Exposure to TEP and TBEP decreased the expression of genes that regulate central nervous system development (e.g. gap43 and mbpa), whereas ROS, antioxidant enzymes, TNF-α, and IL-1ß concentrations were significantly increased. Notably, pretreatment with an antioxidant N-acetylcysteine reduced neurotoxicity and oxidative stress caused by TEP and TBEP. The results of this study demonstrated that exposure to TEP and TBEP causes oxidative stress and has adverse effects on the neurobehavioral and immune system of zebrafish, leading to hypoactivity and ultimately impairing development.

Dissecting the long-term neurobehavioral impact of embryonic benz[a]anthracene exposure on zebrafish: Social dysfunction and molecular pathway activation.

Benz[a]anthracene (BaA), a prevalent environmental contaminant within the polycyclic aromatic hydrocarbon class, poses risks to both human health and aquatic ecosystems. The impact of BaA on neural development and subsequent social behavior patterns remains inadequately explored. In this investigation, we employed the zebrafish as a model to examine the persisting effects of BaA exposure on social behaviors across various developmental stages, from larvae, juveniles to adults, following embryonic exposure. Our findings indicate that BaA exposure during embryogenesis yields lasting neurobehavioral deficits into adulthood. Proteomic analysis highlights that BaA may impair neuro-immune crosstalk in zebrafish larvae. Remarkably, our proteomic data also hint at the activation of the aryl hydrocarbon receptor (AHR) and cytochrome P450 1A (CYP1A) pathway by BaA, leading to the hypothesis that this pathway may be implicated in the disruption of neuro-immune interactions, contributing to observable behavioral disruptions. In summary, our findings suggest that early exposure to BaA disrupts social behaviors, such as social ability and shoaling behaviors, from the larval stage through to maturity in zebrafish, potentially through the detrimental effects on neuro-immune processes mediated by the AHR-CYP1A pathway.

Effects of nanomolar methylmercury on developing human neural stem cells and zebrafish Embryo.

Exposure to mercury and its organic form methylmercury (MeHg), is of great concern for the developing nervous system. Despite available literature on MeHg neurotoxicity, there is still uncertainty about its mechanisms of action and the doses that trigger developmental effects. Our study combines two alternative methodologies, the human neural stem cells (NSC) and the zebrafish (ZF) embryo, to address the neurotoxic effects of early exposure to nanomolar concentrations of MeHg. Our results show linear or nonmonotonic (hormetic) responses depending on studied parameters. In ZF, we observed a hormetic response in locomotion and larval rotation, but a concentration-dependent response for sensory organ size and habituation. We also observed a possible delayed response as MeHg had greater effects on larval activity at 5 days than at 24 h. In NSC cells, some parameters show a clear dose dependence, such as increased apoptosis and differentiation to glial cells or decreased neuronal precursors; while others show a hormetic response: neuronal differentiation or cell proliferation. This study shows that the ZF model was more susceptible than NSC to MeHg neurotoxicity. The combination of different models has improved the understanding of the underlying mechanisms of toxicity and possible compensatory mechanisms at the cellular and organismal level.