Early Stages Of Zebrafish Development Research Articles

Multiparameter analysis of Diuron and its metabolites (DCA and DCPMU) in different stages of zebrafish (Danio rerio) development

IntroductionDiuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a prominent herbicide in Brazilian agriculture, particularly in sugarcane farming. This herbicide persists in the environment and is transformed into potentially toxic metabolites, such as 3,4-dichloroaniline (DCA) and 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU), raising concerns that encompass environmental contamination and One Health, which unites environmental and human health. Zebrafish are an appropriate experimental model to evaluate toxicity: they metabolically and genetically resemble humans and can be applied in ecotoxicology.MethodsIn this study, we have employed multiparameter evaluation to characterize the toxicity of environmentally relevant concentrations (0.5–100 μM) of Diuron, DCA, or DCPMU in Brazil, a developing country. In the early stages of zebrafish development, we performed the fish embryo toxicity test according to the guidelines established in OECD Protocol No. 236, extended to 144 h post fertilization (hpf). We also investigated the oxygen consumption rate in zebrafish embryos at 24 hpf, evaluated the acetylcholinesterase (AChE) enzymatic activity in zebrafish larvae at 96 hpf, and used the Comet Assay to assess DNA fragmentation in zebrafish larvae at 96 hpf. We subjected adult zebrafish to acute exposure for 96 h in accordance with OECD protocol no. 203 for the Acute Toxicity Test in Fish. Following this exposure, we analyzed mutagenicity by using the micronucleus test, which was complemented by quantification of nuclear anomalies (NA) in erythrocytes and further confirmed by histopathological investigations.Results and discussionExposure to any of the tested Diuron concentrations damaged DNA, and NA became significant beyond 10 μM Diuron. At 10 μM, DCA altered AChE activity, suggesting neuromuscular toxicity. Between 0.5 and 5 μM, DCA was potentially genotoxic, and 5–10 μM DCA induced nuclear pleomorphism. At all the tested concentrations, DCPMU damaged DNA, and NA emerged between 1 and 10 μM DCPMU. Adult zebrafish exposed to any of the tested Diuron, DCA, or DCPMU concentrations for 96 h did not exhibit significant histopathological alterations. Erythrocyte morphology analysis did not reveal mutagenicity. Although we observed non-mutagenic genotoxicity, chromosomal instability is of concern. The findings of this study contribute to our understanding of how Diuron and its metabolites affect zebrafish and highlights the need for multiparametric assessment to elucidate how herbicides and their metabolites impact the environment and human health. This endeavor informs regulatory measures for safely using Diuron and similar herbicides and reveals substantial implications for developing nations grappling with escalating water contamination due to pesticide use.

Comparative Study of Condensed and Hydrolysable Tannins during the Early Stages of Zebrafish Development.

In this study, we present data on the effects of condensed tannins (CTs) and hydrolysable tannins (HTs), polyphenols extracted from plants, at different concentrations on zebrafish development to identify the range of concentrations with toxic effects. Zebrafish embryos were exposed to CTs and HTs at two different concentration ranges (5.0-20.0 μgL-1 and 5.0-20.0 mgL-1) for 72 h. The toxicity parameters were observed up to 72 h of treatment. The uptake of CTs and HTs by the zebrafish larvae was assessed via HPLC analysis. A qRT-PCR analysis was performed to evaluate the expressions of genes cd63, zhe1, and klf4, involved in the hatching process of zebrafish. CTs and HTs at 5.0, 10.0, and 20.0 μgL-1 were not toxic. On the contrary, at 5.0, 10.0, and 20.0 mgL-1, HTs induced a delay in hatching starting from 48 h of treatment, while CTs showed a delay in hatching mainly at 48 h. The analysis of gene expression showed a downregulation in the group exposed to HTs, confirming the hatching data. We believe that this study is important for defining the optimal doses of CTs and HTs to be employed in different application fields such as the chemical industry, the animal feed industry, and medical science.

Effects of penthiopyrad on the hypothalamic-pituitary-thyroid (HPT) axis in zebrafish

Exposure to specific pesticides has been demonstrated to alter normal thyroid function of aquatic vertebrates. This study aimed to investigate the impact of penthiopyrad (PO) on the thyroid function of zebrafish, further elucidating its toxic mechanisms on the early developmental stages of zebrafish. Exposure to sublethal doses of PO (0.3–1.2 mg/L) for 8 days from 2 h after fertilization resulted in a significant reduction in larval swim bladder size and body weight, accompanied by developmental abnormalities such as pigment deposition and abnormal abdominal development. Perturbations in the hypothalamic-pituitary-thyroid (HPT) axis in larvae manifested as a marked upregulation of crh, tg, ttr, and ugt1ab expression, alongside downregulation of trβ expression, culminating in elevated thyroxine (T4) and triiodothyronine (T3) levels. Additionally, molecular docking results suggest that PO and its metabolites may disrupt the binding of thyroid hormones to thyroid hormone receptor beta (TRβ), compromising the normal physiological function of TRβ. These findings highlight the PO-induced adverse effects on the HPT axis of larvae under sublethal doses, eventually leading to abnormal development and growth inhibition.

Early stress exposure on zebrafish development: effects on survival, malformations and molecular alterations

The effects of stress during early vertebrate development can be especially harmful. Avoiding stressors in fish larvae is essential to ensure the health of adult fish and their reproductive performance and overall production. We examined the consequences of direct exposure to successive acute stressors during early development, including their effects on miR-29a and its targets, survival, hatching and malformation rates, larval behaviour and cartilage and eye development. Our aim was to shed light on the pleiotropic effects of early-induced stress in this vertebrate model species. Our results showed that direct exposure to successive acute stressors during early development significantly upregulated miR-29a and downregulated essential collagen transcripts col2a1a, col6a2 and col11a1a, decreased survival and increased malformation rates (swim bladder, otoliths, cardiac oedema and ocular malformations), promoting higher rates of immobility in larvae. Our results revealed that stress in early stages can induce different eye tissular architecture and cranioencephalic cartilage development alterations. Our research contributes to the understanding of the impact of stressful conditions during the early stages of zebrafish development, serving as a valuable model for vertebrate research. This holds paramount significance in the fields of developmental biology and aquaculture and also highlights miR-29a as a potential molecular marker for assessing novel larval rearing programmes in teleost species.

Combined neurotoxicity of aged microplastics and thiamethoxam in the early developmental stages of zebrafish (Daniorerio)

Microplastics (MPs) pollution, together with its consequential effect on aquatic biota, represent a burgeoning environmental concern that has garnered significant scholarly attention. Thiamethoxam (TMX), a prevalently utilized neonicotinoid insecticide, is renowned for its neurotoxic impact and selective action against targeted pests. The aquatic environment serves as a receptacle for numerous pollutants, such as MPs and neonicotinoid insecticides. However, there is currently a lack of comprehensive understanding regarding the toxic effects of co-exposure to aged MPs and neonicotinoid insecticides in aquatic organisms. Therefore, we endeavor to elucidate the deleterious impacts of aged polystyrene (PS) and TMX on zebrafish (Danio rerio) larvae when present at environmentally relevant concentrations, and to reveal the underlying molecular mechanisms driving these effects. Our study showed that exposure to aged PS, TMX, or their combination notably inhibited the heart rate and locomotion of zebrafish larvae, with a pronounced effect observed under combined exposure. Aged PS and TMX were found to diminish the activity of antioxidative enzymes (SOD, CAT, and GST), elevate MDA levels, and disrupt neurotransmitter homeostasis (5-HT, GABA and ACh). Notably, the mixtures exhibited synergistic effects. Moreover, gene expression related to oxidative stress (e.g., gstr1, gpx1a, sod1, cat1, p38a, ho-1, and nrf2b) and neurotransmission (e.g., ache, ChAT, gat1, gabra1, 5ht1b, and 5ht1aa) was significantly altered upon co-exposure to aged PS and TMX in larval zebrafish. In summary, our findings support the harmful effects of aged MPs and the neonicotinoid insecticides they carry on aquatic organisms. Results from this study enhance our understanding of the biological risks of MPs and insecticides, as well as help fill existing knowledge gaps on neonicotinoid insecticides and MPs coexistence toxicity in aquatic environment.

Developmental Toxicity of PEDOT:PSS in Zebrafish: Effects on Morphology, Cardiac Function, and Intestinal Health.

Poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) is a conductive polymer commonly used in various technological applications. However, its impact on aquatic ecosystems remains largely unexplored. In this study, we investigated the toxicity effects of PEDOT:PSS on zebrafish. We first determined the lethal concentration (LC50) of PEDOT:PSS in zebrafish and then exposed AB-type zebrafish embryos to different concentrations of PEDOT:PSS for 120 h. Our investigation elucidated the toxicity effects of zebrafish development, including morphological assessments, heart rate measurements, behavioral analysis, transcriptome profiling, and histopathological analysis. We discovered that PEDOT:PSS exhibited detrimental effects on the early developmental stages of zebrafish, exacerbating the oxidative stress level, suppressing zebrafish activity, impairing cardiac development, and causing intestinal cell damage. This study adds a new dimension to the developmental toxicity of PEDOT:PSS in zebrafish. Our findings contribute to our understanding of the ecological repercussions of PEDOT:PSS and highlight the importance of responsible development and application of novel materials in our rapidly evolving technological landscape.

Toxic effects of environmental-relevant exposure to polyethylene terephthalate (PET) micro and nanoparticles in zebrafish early development

Polyethylene terephthalate (PET) is a commonly used thermoplastic in industry due to its excellent malleability and thermal stability, making it extensively employed in packaging manufacturing. Inadequate disposal of PET packaging in the environment and natural physical-chemical processes leads to the formation of smaller particles known as PET micro and nanoplastics (MNPs). The reduced dimensions enhance particle bioavailability and, subsequently, their reactivity. This study involved chemical degradation of PET using trifluoroacetic acid to assess the impact of exposure to varying concentrations of PET MNPs (0.5, 1, 5, 10, and 20 mg/L) on morphological, functional, behavioral, and biochemical parameters during the early developmental stages of zebrafish (Danio rerio). Characterization of the degraded PET revealed the generated microplastics (MPs) ranged in size from 1305 to 2032 μm, and that the generated nanoplastics (NPs) ranged from 68.06 to 955 nm. These particles were then used for animal exposure. After a six-day exposure period, our findings indicate that PET MNPs can diminish spontaneous tail coiling (STC), elevate the heart rate, accumulate on the chorion surface, and reduce interocular distance. These results suggest that PET exposure induces primary toxic effects on zebrafish embryo-larval stage of development.

Evaluation of the Toxicological Effects from Bothrops Jararacussu Snake Venom on Zebrafish (Danio Rerio) Embryos and Larvae

Introduction: Approximately 90% of reported and identified cases of snakebites in Brazil are caused by species belonging to the Bothrops genus. These snakes have clinical rele-vance due to their venom composition, which contains substances capable of triggering local and systemic effects, leading to morbidities and/or mortality. Objective: The objective of this study was to evaluate the toxic and toxinological effects of Both-rops Jararacussu snake venom on zebrafish embryos and larvae. Methods: The stability of B. Jararacussu snake venom under the conditions used in the toxicity experiments in zebrafish embryos and larvae was evaluated on citrated human plasma. Zebrafish embryos and/or larvae mortality, morphological alterations, spontaneous tail movements and heartbeat caused by the venom were quantified within 96 hours. Toxicity parameters and activity of enzyme-related toxicity biomarkers were evaluated in zebrafish after 96 hours of semi-static exposure to the venom. Results: The results indicated that the venom causes toxicity in zebrafish embryos and larvae, in-ducing embryonic mortality, alteration in the number of spontaneous tail movements and activity of biomarker enzymes. The results suggested that the toxic effects caused by the venom in the ear-ly stages of zebrafish development are mediated, in part, by neurotoxic action, induction of oxida-tive and metabolic stress caused by low molecular weight components, and proteins present in this venom. Conclusion: Toxinological evaluations using the zebrafish as a model are scarce; however, this study presented promising results that encourage the development of future research in toxinology using this animal as a model organism.

Azithromycin induces neurotoxicity in zebrafish by interfering with the VEGF/Notch signaling pathway

Azithromycin (AZM) is a widely used antibiotic in both human and veterinary medicine, and its use has significantly increased during the COVID-19 pandemic. However, potential adverse effects of AZM on aquatic organisms have not been well studied. In this study, we explored the neurotoxicity of AZM in zebrafish and delved into its underlying mechanisms. Our results showed that AZM exposure resulted in a spectrum of detrimental effects in zebrafish, encompassing abnormal behaviors, damaged neuronal development, aberrant lateral line nervous system development, vascular malformations and perturbed expression of genes related to neural development. Moreover, we observed a concentration-dependent exacerbation of these neurotoxic manifestations with increasing AZM concentrations. Notably, AZM induced excessive cell apoptosis and oxidative stress damage. In addition, alterations in the expression levels of the genes involved in the VEGF/Notch signaling pathway were evident in AZM-exposed zebrafish. Consequently, we hypothesize that AZM may induce neurotoxicity by influencing the VEGF/Notch signaling pathway. To validate this hypothesis, we introduced a VEGF signaling inhibitor, axitinib, and a Notch signaling agonist, valproic acid, alongside AZM exposure. Remarkably, the administration of these rescue compounds significantly mitigated the neurotoxic effects induced by AZM. This dual verification provides compelling evidence that AZM indeed induces neurotoxicity during the early developmental stages of zebrafish, primarily through its interference with the VEGF/Notch pathway. Innovatively, our study reveals the molecular mechanism of AZM-induced neurotoxicity from the perspective of the close connection between blood vessels and nervous system. These findings provide new insights into the potential mechanisms underlying the neurotoxic effect of antibiotics and highlight the need for further investigation into the ecotoxicological effects of antibiotics on aquatic organisms and the potential risks to human health.

Multi-walled carbon nanotubes enhance the toxicity effects of dibutyl phthalate on early life stages of zebrafish (Danio rerio): Research in physiological, biochemical and molecular aspects

Phthalate esters (PAEs) are widely used as plasticizers. PAEs are ubiquitous in natural water bodies, with dibutyl phthalate (DBP) being one of the most common PAEs. DBP is prone to leaching or migration into the environment, posing serious health and environmental risks. Carbon nanotubes (CNTs) have been widely used in various fields with the rapid development of nanotechnology. CNTs could alter the environmental behavior and toxicity of co-existing pollutants. CNTs have been shown to rapidly adsorb PEAs. However, current knowledge about the effects of CNTs on DBP toxicity is limited. Here we show that the toxic effects of single and combined exposure to DBP (0.1, 0.5, 1.0 mg/L) and different CNTs (MWCNTs/MWCNTs-COOH, 0.5 mg/L) on the early growth stage of zebrafish. The results suggested that a significant increase in heart rate and heart malformation rate was observed after co-exposure of DBP and MWCNTs/MWCNTs-COOH (p < 0.05). Furthermore, combined exposure increased antioxidant enzyme activity during early developmental stages in zebrafish (p < 0.05). The qRT-PCR results revealed that DBP and MWCNTs/MWCNTs-COOH co-exposure significantly interfered with the expression of genes related to oxidative stress, energy metabolism, development of cardiac function, and apoptosis (p < 0.05). In addition, for oxidative stress and cardiotoxicity, MWCNTs/MWCNTs-COOH aggravated the toxic effects of 0.5 mg/L DBP on embryos/larvae. The metabolomics results showed that co-exposure mitigated the disturbance of amino acid metabolism mediated by single DBP exposure. In general, MWCNTs/MWCNTs-COOH increased the impact of DBP in the early developmental stages of zebrafish. This study provides new insights into the toxicology of early developmental stages of aquatic organisms exposed to co-exist pollutants of DBP and CNTs.

Lmo4a Contributes to Zebrafish Inner Ear and Vestibular Development via Regulation of the Bmp Pathway.

In vertebrates, the development of the inner ear is a delicate process, whereas its relating molecular pathways are still poorly understood. LMO4, an LIM domain-only transcriptional regulator, is drawing an increasing amount of interest for its multiple roles regarding human embryonic development and the modulation of ototoxic side effects of cisplatin including cochlear apoptosis and hearing loss. The aim of the present study is to further explore the role of lmo4a in zebrafish inner ear development and thus explore its functional role. The Spatial Transcript Omics DataBase was referred to in order to evaluate the expression of lmo4a during the first 24 h of zebrafish development. In situ hybridization was applied to validate and extend the expression profile of lmo4a to 3 days post-fertilization. The morpholino (MO) knockdown and CRISPR/Cas9 knockout (KO) of lmo4a was applied. Morphological analyses of otic vesical, hair cells, statoacoustic ganglion and semicircular canals were conducted. The swimming pattern of lmo4a KO and MO zebrafish was tracked. In situ hybridization was further applied to verify the expression of genes of the related pathways. Rescue of the phenotype was attempted by blockage of the bmp pathway via heat shock and injection of Dorsomorphin. lmo4a is constitutively expressed in the otic placode and otic vesicle during the early stages of zebrafish development. Knockdown and knockout of lmo4a both induced smaller otocysts, less hair cells, immature statoacoustic ganglion and malformed semicircular canals. Abnormal swimming patterns could be observed in both lmo4a MO and KO zebrafish. eya1 in preplacodal ectoderm patterning was downregulated. bmp2 and bmp4 expressions were found to be upregulated and extended in lmo4a morphants, and blockage of the Bmp pathway partially rescued the vestibular defects. We concluded that lmo4a holds a regulative effect on the Bmp pathway and is required for the normal development of zebrafish inner ear. Our study pointed out the conservatism of LMO4 in inner ear development between mammals and zebrafish as well as shed more light on the molecular mechanisms behind it. Further research is needed to distinguish the relationships between lmo4 and the Bmp pathway, which may lead to diagnostic and therapeutic approaches towards human inner ear malformation.

Molecular, morphological and behavioral alterations of zebrafish (Danio rerio) embryos/larvae after clorprenaline hydrochloride exposure.

Chlorprenaline hydrochloride (CLOR) is a typical representative of β-adrenergic agonists that may be used illegally as a livestock feed additive and may have adverse impacts on the environment. In the present study, zebrafish embryos were exposed to CLOR to investigate its developmental toxicity and neurotoxicity. The results demonstrated that CLOR exposure led to adverse effects on developing zebrafish, such as morphological changes, a high heart rate, and increased body length, resulting in developmental toxicity. Moreover, the up-regulation of activities of superoxide dismutase (SOD) and catalase (CAT) and the enhancement of malondialdehyde (MDA) content illustrated that CLOR exposure activated oxidative stress in exposed zebrafish embryos. Meanwhile, CLOR exposure also caused alterations in locomotive behavior in zebrafish embryos, including an increase in acetylcholinesterase (AChE) activity. Quantitative polymerase chain reaction (QPCR) results showed that the transcription of genes related to the central nervous system (CNS) development, namely, mbp, syn2a, α1-tubulin, gap43, shha, and elavl3, indicated that CLOR exposure could lead to neurotoxicity in zebrafish embryos. These results showed that CLOR exposure could cause developmental neurotoxicity in the early stages of zebrafish development and that CLOR might induce neurotoxicity by altering the expression of neuro-developmental genes, elevating AChE activity, and activating oxidative stress.

Hepatic lipid metabolism disorders and immunotoxicity induced by cysteamine in early developmental stages of zebrafish

Cysteamine, a sulfhydryl compound, is an intermediate in the metabolism of coenzyme A to taurine in living organisms. However, the potential side effects of cysteamine such as hepatotoxicity in pediatric patients have been reported in some studies. To evaluate the impact of cysteamine on infants and children, larval zebrafish (a vertebrate model) were exposed to 0.18, 0.36 and 0.54 mM cysteamine from 72 hpf to 144 hpf. Alterations in general and pathological evaluation, biochemical parameters, cell proliferation, lipid metabolism factors, inflammatory factors and Wnt signaling pathway levels were examined. Increased liver area and lipid accumulation were observed in liver morphology, staining and histopathology in a dose-dependent manner with cysteamine exposure. In addition, the experimental cysteamine group exhibited higher alanine aminotransferase, aspartate aminotransferase, total triglyceride and total cholesterol levels than the control group. Meanwhile, the levels of lipogenesis-related factors ascended whereas lipid transport-related factors descended. Oxidative stress indicators such as reactive oxygen species, MDA and SOD were upregulated after cysteamine exposure. Afterwards, transcription assays revealed that biotinidase and Wnt pathway-related genes were upregulated in the exposed group, and inhibition of Wnt signaling partially rescued the abnormal liver development. The current study found that cysteamine-induced hepatotoxicity in larval zebrafish is due to inflammation and abnormal lipid metabolism, which is mediated by biotinidase (a potential pantetheinase isoenzyme) and Wnt signaling. This provides a perspective on the safety of cysteamine administration in children and identifies potential targets for protection against adverse reactions.

Characterization of N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD)-induced cardiotoxicity in larval zebrafish (Danio rerio)

N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) is a type of p-phenylenediamine (PPD), which is widely used in the manufacture of rubber tires owing to its excellent antiozonant properties. In this study, the developmental cardiotoxicity of 6PPD was evaluated in zebrafish larvae, and the LC50 was approximately 737 μg/L for the larvae at 96 h post fertilization (hpf). In the 6PPD treatment of 100 μg/L, the accumulation concentrations of 6PPD were up to 2658 ng/g in zebrafish larvae, and 6PPD induced significant oxidative stress and cell apoptosis in the early developmental stages of zebrafish. Transcriptome analysis showed that 6PPD exposure could potentially cause cardiotoxicity in larval zebrafish by affecting the transcription of the genes related to the calcium signal pathway and cardiac muscle contraction. The genes related to calcium signaling pathway (slc8a2b, cacna1ab, cacna1da, and pln) were verified by qRT-PCR, which were significantly downregulated in larval zebrafish after exposing to 100 μg/L of 6PPD. Simultaneously, the mRNA levels of the genes related to cardiac functions (myl7, sox9, bmp10, and myh71) also respond accordingly. H&E staining and heart morphology investigation indicated that cardiac malformation occurred in zebrafish larvae exposed to 100 μg/L of 6PPD. Furthermore, the phenotypic observation of transgenic Tg (myl7: EGFP) zebrafish also confirmed that 100 μg/L of 6PPD exposure could change the distance of atria and ventricles of the heart and inhibit some key genes (cacnb3a, ATP2a1l, ryr1b) related to cardiac function in larval zebrafish. These results revealed the toxic effects of 6PPD on the cardiac system of zebrafish larvae.

Effects of 2-ethylhexyl diphenyl phosphate exposure on the glucolipid metabolism and cardiac developmental toxicity in larval zebrafish based on transcriptomic analysis

2-Ethylhexyl diphenyl phosphate (EHDPP) is an organophosphorus type of flame retardant. It is mainly used as a flame-retardant plasticizer in the production of flexible polyvinyl chloride. EHDPP is widely present in environment, particularly in aquatic environment. In this study, we reported that EHDPP exposure significantly affected glucose and lipid metabolism in zebrafish larvae, which was reflected by changes in the transcription of relevant genes and decreased levels of glucose, pyruvate, and triglycerides. In addition, the transcriptomic analysis revealed that the differentially expressed genes could enrich various endpoints in zebrafish larvae. Interestingly, EHDPP exposure could not only change the transcription of genes related to glucolipid metabolism but also cause cardiotoxicity by affecting the transcription of genes related to calcium signaling pathways in zebrafish larvae. To support these findings, we confirmed that these genes involved in cardiac morphology and development were significantly upregulated in zebrafish larvae after EHDPP exposure. More importantly, the distance and overlapping area of the atrium and ventricle were also changed in the EHDPP-exposed zebrafish larvae of transgenic Tg (myl7: EGFP). Overall, our study revealed that EHDPP exposure could affect various endpoints related to glucolipid metabolism and cardiac development in the early developmental stages of zebrafish.

Exposure to the herbicide fluridone induces cardiovascular toxicity in early developmental stages of zebrafish

Fluridone is a systemic herbicide used to control a range of invasive aquatic plants in irrigation systems, lake, and reservoirs. Since aquatic herbicides are more likely to have a hazardous impact on ecosystems than terrestrially applied herbicides, a risk assessment is needed to determine whether to expand or limit their use. The aim of this study was to investigate the developmental toxicity of fluridone using zebrafish. Diverse toxicological results were observed for the sub-lethal endpoints, including lack of hatching, reduced heartbeat and disturbed blood circulation through dysmorphic heart, and edema formation. Abnormal apoptosis was observed in the brain and yolk sac of fluridone-exposed larvae. A computational analysis was used to predict chemical properties in non-target organisms and revealed that fluridone was highly relevant in the cardiovascular system. Double transgenic zebrafish (fli1a:EGFP;cmlc2:dsRed) were used to evaluate the effects of fluridone on the cardiovascular system during embryonic development. Ectopic growth of sub-intestinal vessels and sprouting angiogenesis in the hindbrain region were highly inhibited. Additionally, essential genes involved in the VEGF signaling and heart development were differentially expressed in dose-dependent manner. Collectively, our toxicological findings in fluridone exposure highlight defects in the cardiovascular development causing embryonic lethality that could damage aquatic communities and natural ecosystems.

Morphological Alterations in the Early Developmental Stages of Zebrafish (Danio rerio; Hamilton 1822) Induced by Exposure to Polystyrene Microparticles

Microplastics (MPs) are emerging pollutants of widespread concern in aquatic environments. The aim of this study was to evaluate the negative impact of pristine MPs of polystyrene of 100μm on embryo and larvae of Danio rerio exposed to three environmentally relevant concentrations of polystyrene (3.84 × 10- 6, 3.84 × 10- 7, and 3.84 × 10- 8g/mL). The exposure effect was evaluated through the general morphology score, biometrics, and integrated biomarker response version 2 index. No mortality was observed but the anatomical structure of fishes was affected showing pigmentation deficiency and alterations in the head region as the main affected endpoints. The general morphology score and the integrated biomarker response values were highly sensitive to address the effect of the three concentrations of MPs used here. Our results provide solid evidence of the negative impact of 100μm pristine polystyrene MPs exposure on early stages of zebrafish.

The toxicological effects of Eryngium foetidum extracts on zebrafish embryos and larvae depend on the type of extract, dose, and exposure time.

Eryngium foetidum is a herbaceous plant found in tropical and subtropical regions. In vivo pharmacological parameters show that leaf extracts of this plant have antioxidant, anti-inflammatory, antidiabetic, and antimicrobial activities due to their bioactive compounds such as flavonoids and phenols. Despite the evidence for several bioactivities of E. foetidum, information on its safety and tolerability is limited. The objective of this study was to assess the effect and concentration of different extracts of E. foetidum on the development of zebrafish (Danio rerio) embryos. To study the impact of aqueous (AE), ethanolic (EE), and methanolic (ME) extracts, the embryos were exposed to 0.625, 1.25, 2.5, 5, and 10mgmL-1 for up to 120-h postfertilization to assess embryonic developmental toxicity and then to 0.039, 0.078, 0.156, 0.312, and 0.625mgmL-1 to assess the antioxidant responses of the enzymes superoxide dismutase catalase, glutathione S-transferase (GST), and cell apoptosis. The results showed that, depending on the extraction solvent, concentration used, and exposure time, E. foetidum extracts caused mortality, altered the hatching time, and promoted changes in enzymatic activities. Delays in development and increased GST activity were found in all treatments. Apoptosis was not observed in any of the treatments. In conclusion, AE, EE, and ME concentrations above 0.625mgmL-1 can cause adverse effects on the early stages of zebrafish development.

Kdm4aa is required for reproduction and development of zebrafish

Lysine-specific demethylase 4A (KDM4A) catalyzes demethylation of histone lysine residues, which regulates chromatin state and transcription. In drosophila and mice, KDM4A plays an important role in multiple biological processes including development, aging, metabolism, and immunity, however the functions of KDM4A in fish are still unclear. There are two copies of the kdm4a gene in zebrafish, namely kdm4aa and kdm4ab, kdm4aa was edited using CRISPR/Cas9 technology in the present study, then homozygous kdm4aa mutants (kdm4aa−/−) were obtained, and loss of kdm4aa was confirmed by sequencing and increased H3K9me3. Whole-mount in situ hybridization showed that kdm4aa is widely expressed during the embryonic development of zebrafish. Compared with WT zebrafish, kdm4aa−/− zebrafish showed no significant difference in gamete formation and fertilization, but the survival rate of kdm4aa−/− embryos dramatically reduced to 21% at 26 hpf. Further observation showed that about 80% of survived kdm4aa−/− zebrafish experienced disruption in stripe formation, and 10% of survived kdm4aa−/− zebrafish underwent vertebral malformation. Alizarin red S staining demonstrated the abnormal spinal development in kdm4aa−/− zebrafish. These results indicated that kdm4aa is required for normal embryonic development of zebrafish, loss of kdm4aa function leads to decreased survival during the early stages of zebrafish development and morphological variation in adult zebrafish.

Comparative toxicity of [C8mim]Br and [C8py]Br in early developmental stages of zebrafish (Danio rerio) with focus on oxidative stress, apoptosis, and neurotoxicity

The increasing production and usage of ionic liquids (ILs) have raised global ecotoxicological concerns regarding their release into the environment. While the effects of side chains on the IL-induced toxicity in various aquatic organisms have been well-recognized, the role of cationic cores in determining their ecotoxicity remains to be elucidated. Herein, the comparative bioavailability and toxicity of two ILs with different cationic cores but the same anion and side chain in zebrafish embryos were determined. 1-octyl-3-methylimidazolium bromide ([C8mim]Br) has higher accumulation in zebrafish, and triggered developmental toxicity by inducing oxidative stress and apoptosis. Meanwhile, 1-octyl-1-methylpyridium bromide ([C8py]Br) enhanced SOD activity and upregulated anti-apoptotic bcl-2 gene expression, contributing to its much lower neurodevelopmental toxicity. Our study demonstrates the vital role of cationic core in determining the developmental toxicity of ILs and highlights the need for further investigations into the toxicity of imidazolium and pyridinium based ILs in aquatic ecosystems.