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In many human diseases, the molecular pathophysiological mechanisms are not understood, which makes the development and testing of new therapeutic approaches difficult. The generation and characterization of animal models such as mice, rats, fruit flies, worms or fish offers the possibility for in detail studies of a disease's development, its course and potential therapies in an organismal context, which considerably minimizes the risk of therapeutic side effects for patients. Nevertheless, due to the high numbers of experimental animals used in research worldwide, attempts to develop alternative test systems will help in reducing their count. In this regard, the cell culture system displays a suitable option due to its potential of delivering nearly unlimited material and the good opportunities for high-throughput studies such as drug testing. Here, we describe a quick and simple method to isolate and cultivate vital fibroblast-like cells from embryos and adults of two popular teleost model organisms, the Japanese rice fish medaka (Oryzias latipes) and the zebrafish (Danio rerio). [ABSTRACT FROM AUTHOR]

Rare earth elements (REEs) are critical metallic materials with a broad application in industry and biomedicine. The exponential increase in REEs utilization might elevate the toxicity to aquatic animals if they are released into the water due to uncareful handling. The specific objective of our study is to explore comprehensively the critical factor of a model Lanthanide complex electronic structures for the acute toxicity of REEs based on utilizing zebrafish as a model animal. Based on the 96 h LC50 test, we found that the majority of light REEs display lower LC50 values (4.19–25.17 ppm) than heavy REEs (10.30–41.83 ppm); indicating that they are atomic number dependent. Later, linear regression analyses further show that the average carbon charge on the aromatic ring (aromatic Cavg charge) can be the most significant electronic structural factor responsible for the Lanthanides' toxicity in zebrafish embryos. Our results confirm a very strong correlation of LC50 to Lanthanide's atomic numbers (r = 0.72), Milliken charge (r = 0.70), and aromatic Cavg charge (r = −0.85). This most significant correlation suggests a possible toxicity mechanism that the Lanthanide cation's capability to stably bind to the aromatic ring on the residue of targeted proteins via a covalent chelating bond. Instead, the increasing ionic bond character can reduce REEs' toxicity. In addition, Lanthanide toxicity was also evaluated by observing the disruption of photo motor response (PMR) activity in zebrafish embryos. Our study provides the first in vivo evidence to demonstrate the correlation between an atomic number of Lanthanide ions and the Lanthanide toxicity to zebrafish embryos. [ABSTRACT FROM AUTHOR]

Pentachloronitrobenzene (PCNB) is an organochlorine protective fungicide mainly used as a soil and seed fungicide. Currently, there are few reports on the toxicity of PCNB to zebrafish embryo. Here, we evaluated the toxicity of PCNB in aquatic vertebrates using a zebrafish model. Exposure of zebrafish embryos to PCNB at concentrations of 0.25 mg/L, 0.5 mg/L, and 0.75 mg/L from 6 hpf to 72 hpf resulted in abnormal embryonic development, including cardiac malformation, pericardial edema, decreased heart rate, decreased blood flow velocity, deposition at yolk sac, shortened body length, and increased distance between venous sinus and arterial bulb (SV-BA). The expression of genes related to cardiac development was disordered. However, due to the unstable embryo status in the 0.75 mg/L exposure concentration group, the effect of PCNB on the expression levels of cardiac-related genes was not concentration-dependent. We found that PCNB increased reactive oxygen species stress levels in zebrafish, increased malondialdehyde (MDA) content and catalase (CAT) activity, and decreased superoxide dismutase (SOD) activity. The increased level of oxidative stress reduced the proliferation ability of zebrafish cardiomyocytes, and the expressions of zebrafish proliferation-related genes such as cdk-2, cdk-6, ccnd1, and ccne1 were significantly down-regulated. Astaxanthin (AST) attenuates PCNB-induced reduction in zebrafish cardiomyocyte proliferation by reducing oxidative stress levels. Our study shows that PCNB can cause severe oxidative stress in zebrafish, thereby reducing the proliferative capacity of cardiomyocytes, resulting in zebrafish cardiotoxicity. [ABSTRACT FROM AUTHOR]

Pharmaceuticals are widely regarded as a menace to the aquatic environment. The constant consumption of biologically active chemicals for human health has been matched by an increase in the leaking of these compounds in natural habitats over the last two decades. This study was aimed to evaluate the molecular pathway underling the developmental toxicity of exposure in the ecological environment. Zebrafish embryos were exposed at doses of dexamethasone sodium phosphate (DEX) 1 μmol/L, tocilizumab 442.1 μmol/L and dexamethasone + tocilizumab (1 μmol/L and 442.1 μmol/L, respectively) from 24 h post-fertilization (hpf) to 96 hpf. This study confirmed that DEX exposure in association with tocilizumab 442.1 μmol/L at 1 μmol/L (non-toxic concentration) affected the survival and hatching rate, morphology score, and body length. Additionally, it significantly disturbed the antioxidant defense system in zebrafish larvae. Furthermore, a DEX 1 μmol/L and tocilizumab 442.1 μmol/L association also increased the production of apoptosis-related proteins (caspase-3, bax, and bcl-2). [ABSTRACT FROM AUTHOR]

Anisodamine is one of the major components of the tropine alkaloid family and is widely used in the treatment of pain, motion sickness, pupil dilatation, and detoxification of organophosphorus poisoning. As a muscarinic receptor antagonist, the low toxicity and moderate drug effect of anisodamine often result in high doses for clinical use, making it important to fully investigate its toxicity. In this study, zebrafish embryos were exposed to 1.3‐, 2.6‐, and 5.2‐mM anisodamine for 7 days to study the toxic effects of drug exposure on pigmentation, mineral density, craniofacial area, and eye development. The results showed that exposure to anisodamine at 1.3 mM resulted in cranial malformations and abnormal pigmentation in zebrafish embryos; 2.6‐ and 5.2‐mM anisodamine resulted in significant eye development defects and reduced bone density in zebrafish embryos. The associated toxicities were correlated with functional development of neural crest cells through gene expression (col1a2, ddb1, dicer1, mab21l1, mab21l2, sox10, tyrp1b, and mitfa) in the dose of 5.2‐mM exposed group. In conclusion, this study provides new evidence of the developmental toxicity of high doses of anisodamine in aqueous solutions to organisms and provides a warning for the safe use of this drug. We studied the early developmental toxicity of anisodamine in zebrafish. The results showed that anisodamine exposure resulted in toxicity such as increased pigmentation, decreased mineral density, smaller craniofacial area, and smaller eyes. Alterations in related genes were associated with the functional development of neural crest cells. [ABSTRACT FROM AUTHOR]

Tricresyl phosphate (TCP) has received extensive attentions due to its potential adverse effects, while the toxicological information of TCP isomers is limited. In this study, 2 h post-fertilization zebrafish embryos were exposed to tri-o-cresyl phosphate (ToCP), tri-m-cresyl phosphate (TmCP) or tri-p-cresyl phosphate (TpCP) at concentrations of 0, 100, 300 and 600 μg/L until 120 hpf, and the cardiotoxicity and mechanism of TCP isomers in zebrafish embryos/larvae were evaluated. The results showed that ToCP or TmCP exposure induced cardiac morphological defects and dysfunction in zebrafish, characterized by increased distance between sinus venosus and bulbus arteriosis, increased atrium and pericardial sac area, trabecular defects, and decreased heart rate and blood flow velocity, while no adverse effects of TpCP on zebrafish heart were found. Transcriptomic results revealed that extracellular matrix (ECM) and motor proteins, as well as PPAR signaling pathways, were included in the cardiac morphological defects and dysfunction induced by ToCP and TmCP. Co-exposure test with D-mannitol indicated that the inhibition of energy metabolism by ToCP and TmCP affected cardiac morphology and function by decreasing osmoregulation. This study is the first to report the cardiotoxicity induced by TCP in zebrafish from an isomer perspective, providing a new insight into the toxicity of TCP isomers and highlighting the importance of evaluating the toxicity of different isomers. [Display omitted] • Accumulation levels of TCP isomers in zebrafish embryos/larvae were isomer-specific. • ToCP and TmCP, but not TpCP, induced cardiotoxicity in zebrafish embryos/larvae. • Extracellular matrix and motor proteins were included in ToCP- and TmCP-induced cardiotoxicity. • ToCP and TmCP exposure reduced cardiac function by inhibiting energy metabolism. • ToCP and TmCP exposure reduced osmoregulation, thereby exacerbating cardiotoxicity. [ABSTRACT FROM AUTHOR]

Polystyrene nanoplastics (PS) require a comprehensive evaluation of their toxicity and potential risks to humans and the environment. The zebrafish model, a well-established animal model increasingly utilized for nanotoxicity assessments, was employed in this study. Our research aimed to explore the toxic effects of PS with sizes of 30, 100, 200, and 450 nm on zebrafish embryos. Exposure experiments were conducted on embryos at 4 h post-fertilization (hpf) using various concentrations of nanoparticles (20, 40, 60, 80, and 100 mg/L) until 96 hpf. Notably, PS ranging from 100 to 450 nm did not adversely affect zebrafish embryo development. However, PS with a size of 30 nm at a concentration of 100 mg/L resulted in embryo mortality but not embryonic malformations. Furthermore, our investigation confirmed the uptake of these nanoparticles by zebrafish larvae following the opening of their mouths, with the particles being found predominantly in the digestive system of the larvae. Additionally, 30 nm PS were found to significantly modulate the expression levels of genes associated with oxidative stress and apoptosis. These findings highlight the developmental impacts of 30 nm PS on zebrafish embryos, raising concerns about potential similar consequences in humans. Considering our findings, it is essential to encourage further research into the management and regulation of PS to mitigate their potential environmental and health impacts. • Polystyrene nanoplastics (PSs) ranging from 100 to 450 nm did not adversely affect zebrafish embryo development. • PSs with a size of 30 nm at a concentration of 100 mg/L resulted in embryo mortality without causing embryonic malformations. • Zebrafish larvae exhibited uptake of PS nanoparticles, predominantly in their digestive system, following mouth opening. • 30 nm PSs significantly altered the expression levels of genes related to oxidative stress and apoptosis in zebrafish larvae. [ABSTRACT FROM AUTHOR]

Zebrafish embryo assays are used by pharmaceutical and chemical companies as new approach methodologies (NAMs) in developmental toxicity screening. Despite an overall high concordance of zebrafish embryo assays with in vivo mammalian studies, false negative and false positive results have been reported. False negative results in risk assessment models are of particular concern for human safety, as developmental anomalies may be missed. Interestingly, for several chemicals and drugs that were reported to be false negative in zebrafish, skeletal findings were noted in the in vivo studies. As the number of skeletal endpoints assessed in zebrafish is very limited compared to the in vivo mammalian studies, the aim of this study was to investigate whether the sensitivity could be increased by including a skeletal staining method. Three staining methods were tested on zebrafish embryos that were exposed to four teratogens that caused skeletal anomalies in rats and/or rabbits and were false negative in zebrafish embryo assays. These methods included a fixed alizarin red-alcian blue staining, a calcein staining, and a live alizarin red staining. The results showed a high variability in staining intensity of larvae exposed to mammalian skeletal teratogens, as well as variability between control larvae originating from the same clutch of zebrafish. Hence, biological variability in (onset of) bone development in zebrafish hampers the detection of (subtle) treatment-related bone effects that are not picked-up by gross morphology. In conclusion, the used skeletal staining methods did not increase the sensitivity of zebrafish embryo developmental toxicity assays. • High variability in skeletal staining intensity was observed in larvae exposed to mammalian skeletal teratogensand in control larvae. • Biological variability in bone development hampers the detection of (subtle) treatment-related bone effects that are not picked-up by gross morphology. • The used skeletal staining methods could not increase the sensitivity of zebrafish embryo developmental toxicity assays. [ABSTRACT FROM AUTHOR]

The contamination of water by arsenic (As) has emerged as a significant environmental concern due to its well-documented toxicity. Environmentally relevant concentrations of As have been reported to pose a considerable threat to fish. However, previous studies mainly focused on the impacts of As at environmentally relevant concentrations on adult fish, and limited information is available regarding its impacts on fish at early life stage. In this study, zebrafish embryos were employed to evaluate the environmental risks following exposure to different concentrations (0, 25, 50, 75 and 150 μg/L) of pentavalent arsenate (AsV) for 120 hours post fertilization. Our findings indicated that concentrations ≤ 150 μg/L AsV did not exert significant effects on survival or aberration; however, it conspicuously inhibited heart rate of zebrafish larvae. Furthermore, exposure to AsV significantly disrupted mRNA transcription of genes associated with cardiac development, and elongated the distance between the sinus venosus and bulbus arteriosus at 75 μg/L and 150 μg/L treatments. Additionally, AsV exposure enhanced superoxide dismutase (SOD) activity at 50, 75 and 150 μg/L treatments, and increased mRNA transcriptional levels of Cu/ZnSOD and MnSOD at 75 and 150 μg/L treatments. Concurrently, AsV suppressed metallothionein1 (MT1) and MT2 mRNA transcriptions while elevating heat shock protein70 mRNA transcription levels in zebrafish larvae resulting in elevated malondialdehyde (MDA) levels. These findings provide novel insights into the toxic effects exerted by low concentrations of AsV on fish at early life stage, thereby contributing to an exploration into the environmental risks associated with environmentally relevant concentrations. [Display omitted] • ≤ 150 μg/L AsV had no significant effect on survival and aberration rates of zebrafish larvae. • ≥ 25 μg/L AsV inhibited heart rate of zebrafish embryo. • ≥ 75 μg/L AsV delayed cardiac development of zebrafish larvae. • ≥ 25 μg/L AsV caused oxidative stress and oxidative damage. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Exposure of zebrafish embryos to GO and GDY decreased hatching rate and induced morphological deformity. • Behavioral tests indicated hyperactivity after GO and GDY treatment. • RNA-sequencing data revealed that GO and GDY altered gene ontology terms and KEGG pathways related with metabolism. • GO and GDY altered the expression of genes related with lipid droplets and autophagy. • At the same mass concentrations, GO induced larger toxic effects compared with GDY. [ABSTRACT FROM AUTHOR]

Zebrafish larvae exposed to chemoconvulsants show behavioral seizures and electrographic abnormalities similar to the other mammalian models, making it a potential tool in epilepsy research. During the embryonic stage, zebrafish remains transparent which enables real-time developmental detection and in-situ gene/protein expression. However, pigmentation during the larval stage restricts transparency. Phenylthiourea (1-phenyl-2-thiourea; PTU) is a commonly used pigmentation blocker that maintains larval transparency. It is widely used along with chemoconvulsants to study in situ expressions in epileptic larvae, however, its effect on seizures largely remains unknown. Therefore, in the present study, the effect of PTU-mediated depigmentation was studied on pentylenetetrazol (PTZ)-induced seizures in zebrafish larvae. After spawning, the fish embryos were subjected to standard depigmentation protocol using 0.13 mM PTU. At 7- days post fertilization seizures were induced using 8 mM PTZ. PTU exposure significantly reduced PTZ-mediated hyperactive responses indicated by decreased distance travelled and swimming velocity of the larvae. Furthermore, PTU-exposed depigmented larvae also showed an increase in the latency to the onset of PTZ-mediated clonic-like seizures. The results concluded that PTU depigmentation protocol reduces the seizurogenic response of PTZ, hence its usage for imaging zebrafish larvae must be carefully monitored to avoid erroneous results. • PTU-mediated depigmentation reduced the overall severity of the seizurogenic response of PTZ in zebrafish larvae. • PTU exposure in zebrafish larvae significantly decreased PTZ-induced hyperactive responses. • The depigmentation protocol increased the latency to PTZ-mediated clonic-like seizures in the larvae. [ABSTRACT FROM AUTHOR]

• UV-Aging enhanced adsorption capacity of micro-sized PBAT for heavy metals; • Aged PBAT reduced the toxicity of Cu and Pb to zebrafish embryos and larvae; • Combined aged PBAT and heavy metals induced more severe gut microbiota dysbiosis. Compared with the fossil-based plastics, biodegradable plastics are more easily decomposed into small-sized particles (e.g., microplastics). However, the role of aged biodegradable plastics in being vector of co-existed pollutants and potential toxicological effects remain to be elucidated. The present study selected micro-sized biodegradable polymer Poly (butylene adipate-co-terephthalate) (PBAT) as the object, aiming to explore its aging process, environmental behavior with heavy metals (Cu and Pb), and the toxic effects on zebrafish. The results showed that distinct changes such as cracks and severe deformation can be observed on the surface of PBAT after 60 days of UV aging, and the functional groups changed consequently. The maximum adsorption capacity of aged PBAT for Cu and Pb reached 0.967 and 0.939 mg·g−1, which increased by 1.32 and 1.46 times, respectively. The results of 7-day acute toxicology experiments suggested that the adsorption behavior of aged PBAT may alleviate the toxic effects of heavy metals Cu and Pb on zebrafish in short-term exposure, however it could simultaneously cause a serious imbalance of intestinal microorganisms in zebrafish. As demonstrated, the coexistence of aged PBAT and heavy metals (Cu, Pb) can seriously reduce the intestinal microbial diversity and richness of zebrafish, which may induce more serious toxicity and disease in long-term exposure to pollutants. This study could provide fundamental data for better understanding on the adsorption behavior and ecological risk of aged biodegradable plastics with coexisted pollutants. [Display omitted] [ABSTRACT FROM AUTHOR]

Strobilurins, among the most used fungicides worldwide, are considered non-toxic to mammals and birds, but there is growing evidence that these compounds are highly toxic to aquatic species. Dimoxystrobin has been included in the 3rd Watch List of the European Commission, and it has been classified as very toxic to aquatic life. However, previous studies focused on acute toxicity and only two reports are available on its impact on fish, and none on its effects during the early life stages. Here, we evaluated for the first time the effects induced on zebrafish embryos and larvae by two dimoxystrobin sublethal concentrations (6.56 and 13.13 μg/L) falling in the range of predicted environmental concentrations. We demonstrated that short-term exposure to dimoxystrobin may exert adverse effects on multiple targets, inducing severe morphological alterations. Moreover, we showed enhanced mRNA levels of genes related to the mitochondrial respiratory chain and ATP production. Impairment of the swim bladder inflation has also been recorded, which may be related to the observed swimming performance alterations. [Display omitted] • Dimoxystrobin induces morphological alterations in zebrafish embryos and larvae • The severity of injuries increases with increasing time and dose of exposure • Dimoxystrobin enhances mRNA level of genes related to the mitochondrial respiration • Swim bladder impairment may be related to swimming performance [ABSTRACT FROM AUTHOR]

• Toxicity of ethinylestradiol and estetrol, in mixture with the progestin drospirenone, was assessed. • Zebrafish behavior at larval stage is sensitive to ethinylestradiol, not estetrol. • Few significant effects on larval proteomic profile were reported with estetrol. • Estetrol presents a more favorable environmental profile than ethinylestradiol. To characterise and compare the toxicity of estetrol (E4) and 17α-ethinylestradiol (EE2), and their respective mixture with the progestin drospirenone (DRSP) in zebrafish (Danio rerio) embryos. Zebrafish embryos were exposed to E4, EE2, DRSP, E4+DRSP, and EE2+DRSP in a fish embryo acute toxicity (FET) test. A second test examined behavioural responses and, using label-free proteomics, identified changes in protein expression in response to hormonal treatments, across a range of concentrations, including those that are considered to be environmentally relevant. In the FET test, no effects were found from E4 at concentrations ≤100 mg/L, while EE2 induced mortality and morphological abnormalities at concentrations of 1-2 mg/L. In the behavioural test, exposure to 30 ng/L EE2 (∼200 × predicted environmental concentration – PEC) resulted in hypoactivity in fish larvae and exposure to 0.3 ng/L EE2 (∼2 × PEC) led to quantitative changes in protein abundance, revealing potential impacts on RNA processing and protein synthesis machinery. Exposure to E4 did not alter behaviour, but several groups of proteins were modulated, mainly at 710 ng/L (∼200 × PEC), including proteins involved in oxidative phosphorylation. When combined with DRSP, EE2 induced reduced effects on behaviour and proteomic responses, suggesting an antagonistic effect of DRSP. E4+DRSP induced no significant effects on behaviour or proteomic profiles at tested concentrations. These findings suggest that E4-based combined oral contraceptives present a more favourable environmental profile than EE2-based contraceptives, particularly during the early developmental stages of fish. [ABSTRACT FROM AUTHOR]

• The DT-VEM accurately divides developmental toxicity innovation into three categories. • Compared with previous research, our model has better performance. In recent years, with the rapid development of society, organic compounds have been released into aquatic environments in various forms, posing a significant threat to the survival of aquatic organisms. The assessment of developmental toxicity is an important part of environmental safety risk systems, helping to identify the potential impacts of organic compounds on the embryonic development of aquatic organisms and enabling early detection and warning of potential ecological risks. Additionally, binary classification models cannot accurately classify organic compounds. Therefore, it is crucial to construct a multiclassification model for predicting the developmental toxicity of organic compounds. In this study, binary and multiclassification models were developed based on the ToxCast™ Phase I chemical library and literature data. The random forest, support vector machine, extreme gradient boosting, adaptive gradient boosting, and C5.0 decision tree algorithms, as well as 8 types of molecular fingerprint were used to establish a multiclassification base model for predicting developmental toxicity through 5-fold cross-validation and external validation. Ultimately, a multiclassification ensemble model was derived through a voting method. The performance of the binary ensemble model, as measured by the balanced accuracy, was 0.918, while that of the multiclassification model was 0.819. The developmental toxicity voting ensemble model (DT-VEM) achieved accuracies of 0.804, 0.834, and 0.855. Furthermore, by utilizing the XGBoost machine learning algorithm to construct separate models for molecular descriptors and substructure molecular fingerprints, we identified several substructures and physical properties related to developmental toxicity. Our research contributes to a more detailed classification of developmental toxicity, providing a new and valuable tool for predicting the developmental toxicity effects of unknown compounds. This supplement addresses the limitations of previous tools, as it offers an enhanced ability to predict potential developmental toxicity in novel compounds. [Display omitted] [ABSTRACT FROM AUTHOR]

The available information regarding the impact of antimony (Sb), a novel environmental pollutant, on the intestinal microbiota and host health is limited. In this study, we conducted physiological characterizations to investigate the response of adult zebrafish to different environmental concentrations (0, 30, 300, and 3000 µg/L) of Sb over a period of 14 days. Biochemical and pathological changes demonstrated that Sb effectively compromised the integrity of the intestinal physical barrier and induced inflammatory responses as well as oxidative stress. Analysis of both intestinal microbial community and metabolome revealed that exposure to 0 and 30 µg/L of Sb resulted in similar microbiota structures; however, exposure to 300 µg/L altered microbial communities' composition (e.g., a decline in genus Cetobacterium and an increase in Vibrio). Furthermore, exposure to 300 µg/L significantly decreased levels of bile acids and glycerophospholipids while triggering intestinal inflammation but activating self-protective mechanisms such as antibiotic presence. Notably, even exposure to 30 µg/L of Sb can trigger dysbiosis of intestinal microbiota and metabolites, potentially impacting fish health through the "microbiota-intestine-brain axis" and contributing to disease initiation. This study provides valuable insights into toxicity-related information concerning environmental impacts of Sb on aquatic organisms with significant implications for developing management strategies. [Display omitted] • Sb caused histopathological damage to the intestine of adult zebrafish. • Sb triggered oxidative stress and inflammatory responses in the intestine. • Sb disrupted intestinal microbial community and metabolism profiles. • Sb can modulate the "microbiota-intestine-brain axis" via intestinal microbiota. [ABSTRACT FROM AUTHOR]

Benzo(a)Pyrene (BaP) is one of the most widespread polycyclic aromatic hydrocarbons (PAHs) with endocrine disrupting properties and carcinogenic effects. In the present study, we tested the effect of BaP on thyroid development and function, using zebrafish as a model system. Zebrafish embryos were treated with 50 nM BaP from 2.5 to 72 h post fertilization (hpf) and compared to 1.2% DMSO controls. The expression profiles of markers of thyroid primordium specification, thyroid hormone (TH) synthesis, hypothalamus-pituitary-thyroid (HPT) axis, TH transport and metabolism, and TH action were analyzed in pools of treated and control embryos at different developmental stages. BaP treatment did not affect early markers of thyroid differentiation but resulted in a significant decrease of markers of TH synthesis (tg and nis) likely secondary to defective expression of the central stimulatory hormones of thyroid axis (trh, tshba) and of TH metabolism (dio2). Consequently, immunofluorescence of BaP treated larvae showed a low number of follicles immunoreactive to T4. In conclusion, our results revealed that the short-term exposure to BaP significantly affects thyroid function in zebrafish, but the primary toxic effects would be exerted at the hypothalamic-pituitary level thus creating a model of central hypothyroidism. [ABSTRACT FROM AUTHOR]

Musculoskeletal injuries are common in humans. The cascade of cellular and molecular events following such injuries results either in healing with functional recovery or scar formation. While fibrotic scar tissue serves to bridge between injured planes, it undermines functional integrity. Hence, faithful regeneration is the most desired outcome; however, the potential to regenerate is limited in humans. In contrast, various non-mammalian vertebrates have fascinating capabilities of regenerating even an entire appendage following amputation. Among them, zebrafish is an important and accessible laboratory model organism, sharing striking similarities with mammalian embryonic musculoskeletal development. Moreover, clinically relevant muscle and skeletal injury zebrafish models recapitulate mammalian regeneration. Upon muscle injury, quiescent stem cells – known as satellite cells - become activated, proliferate, differentiate and fuse to form new myofibres, while bone fracture results in a phased response involving hematoma formation, inflammation, fibrocartilaginous callus formation, bony callus formation and remodelling. These models are well suited to testing gene- or pharmaco-therapy for the benefit of conditions like muscle tears and fractures. Insights from further studies on whole body part regeneration, a hallmark of the zebrafish model, have the potential to complement regenerative strategies to achieve faster and desired healing following injuries without any scar formation and, in the longer run, drive progress towards the realisation of large-scale regeneration in mammals. Here, we provide an overview of the basic mechanisms of musculoskeletal regeneration, highlight the key features of zebrafish as a regenerative model and outline the relevant studies that have contributed to the advancement of this field. [ABSTRACT FROM AUTHOR]

Nowadays, increasing interest has recently been given to the exploration of new food preservatives to avoid foodborne outbreaks or food spoilage. Likewise, new compounds that substitute the commonly used synthetic food preservatives are required to restrain the rising problem of microbial resistance. Accordingly, the present study was conducted to examine the chemical composition and the mechanism(s) of action of the Cupressus sempervirens essential oil (CSEO) against Salmonella enterica Typhimuriumand Staphyloccocus aureus. The gas chromatography analysis revealed α-pinene (38.47%) and δ-3-carene (25.14%) are the major components of the CSEO. By using computational methods, such as quantitative structure–activity relationship (QSAR), we revealed that many CSEO components had no toxic effects. Moreover, findings indicated that α-pinene, δ-3-carene and borneol, a minor compound of CSEO, could inhibit the AcrB-TolC and MepR efflux pump activity of S. enterica Typhimurium and S. aureus, respectively. In addition, our molecular docking predictions indicated the high affinity of these three compounds with active sites of bacterial DNA and RNA polymerases, pointing to plausible impairments of the pathogenic bacteria cell replication processes. As well, the safety profile was developed through the zebrafish model. The in vivo toxicological evaluation of (CSEO) exhibited a concentration-dependent manner, with a lethal concentration (LC50) equal to 6.6 µg/mL. [ABSTRACT FROM AUTHOR]

In the trunk of developing zebrafish embryos, adjacent myotome blocks transmit contractile force via myoseptal junctions (MJs), which are dynamic structures that connect the actin cytoskeleton of skeletal muscle cells to extracellular matrix components via transmembrane protein complexes in the sarcolemma. Here, we report that the endolysosomal ion channel, two-pore channel type 1 (TPC1, encoded by tpcn1), generates highly localized non-propagating Ca2+ transients that play a distinct and required role in the capture and attachment of superficial slow skeletal muscle cells at MJs. Use of antisense morpholinos or CRISPR/Cas9 gene editing to disrupt tpcn1 gene expression resulted in abnormal MJ phenotypes, including slow skeletal muscle cells detaching from or crossing the myosepta. We also report that TPC1-decorated endolysosomes are dynamically associated with MJs in a microtubule-dependent manner, and that attenuating tpcn1 expression or TPC1 function disrupted endolysosomal trafficking and resulted in an abnormal distribution of ß-dystroglycan (encoded by dag1; a key transmembrane component of the dystrophin-associated protein complex). Taken together, our data suggest that localized TPC1- generated Ca2+ signals facilitate essential endolysosomal trafficking and membrane contact events, which help form and maintain MJs following the onset of slow skeletal muscle cell contractile activity. [ABSTRACT FROM AUTHOR]

A central element of high throughput screens for chemical effect assessment using zebrafish is the assessment and quantification of phenotypic changes. By application of an automated and more unbiased analysis of these changes using image analysis, patterns of phenotypes may be associated with the mode of action (MoA) of the exposure chemical. The aim of our study was to explore to what extent compounds can be grouped according to their anticipated toxicological or pharmacological mode of action using an automated quantitative multi-endpoint zebrafish test. Chemical-response signatures for 30 endpoints, covering phenotypic and functional features, were generated for 25 chemicals assigned to 8 broad MoA classes. Unsupervised clustering of the profiling data demonstrated that chemicals were partially grouped by their main MoA. Analysis with a supervised clustering technique such as a partial least squares discriminant analysis (PLS-DA) allowed to identify markers with a strong potential to discriminate between MoAs such as mandibular arch malformation observed for compounds interfering with retinoic acid signaling. The capacity for discriminating MoAs was also benchmarked to an available battery of in vitro toxicity data obtained from ToxCast library indicating a partially similar performance. Further, we discussed to which extent the collected dataset indicated indeed differences for compounds with presumably similar MoA or whether other factors such as toxicokinetic differences could have an important impact on the determined response patterns. [ABSTRACT FROM AUTHOR]

Selenium is an essential trace element for humans and other vertebrates, playing an important role in antioxidant defense, neurobiology and reproduction. However, the toxicity of excessive selenium has not been thoroughly evaluated, especially for the visual system of vertebrates. In this study, fertilized zebrafish embryos were treated with 0.5 µM L-selenomethionine to investigate how excessive selenium alters zebrafish eye development. Selenium-stressed zebrafish embryos showed microphthalmia and altered expression of genes required for retinal neurogenesis. Moreover, ectopic proliferation, disrupted mitochondrial morphology, elevated ROS-induced oxidative stress, apoptosis and ferroptosis were observed in selenium-stressed embryos. Two antioxidants—reduced glutathione (GSH) and N-acetylcysteine (NAC)—and the ferroptosis inhibitor ferrostatin (Fer-1) were unable to rescue selenium-induced eye defects, but the ferroptosis and apoptosis activator cisplatin (CDDP) was able to improve microphthalmia and the expression of retina-specific genes in selenium-stressed embryos. In summary, our results reveal that ferroptosis and apoptosis might play a key role in selenium-induced defects of embryonic eye development. The findings not only provide new insights into selenium-induced cellular damage and death, but also important implications for studying the association between excessive selenium and ocular diseases in the future. [ABSTRACT FROM AUTHOR]

With the abundance of chemicals in the environment that could potentially cause neurodevelopmental deficits, there is a need for rapid testing and chemical screening assays. This study evaluated the developmental toxicity and behavioral effects of 61 chemicals in zebrafish (Danio rerio) larvae using a behavioral Light/Dark assay. Larvae (n = 16–24 per concentration) were exposed to each chemical (0.0001–120 μM) during development and locomotor activity was assessed. Approximately half of the chemicals (n = 30) did not show any gross developmental toxicity (i.e., mortality, dysmorphology or non-hatching) at the highest concentration tested. Twelve of the 31 chemicals that did elicit developmental toxicity were toxic at the highest concentration only, and thirteen chemicals were developmentally toxic at concentrations of 10 µM or lower. Eleven chemicals caused behavioral effects; four chemicals (6-aminonicotinamide, cyclophosphamide, paraquat, phenobarbital) altered behavior in the absence of developmental toxicity. In addition to screening a library of chemicals for developmental neurotoxicity, we also compared our findings with previously published results for those chemicals. Our comparison revealed a general lack of standardized reporting of experimental details, and it also helped identify some chemicals that appear to be consistent positives and negatives across multiple laboratories. [ABSTRACT FROM AUTHOR]

(1) Background: Disperse Blue 14, Disperse Red 9, Solvent Red 169 and Solvent Yellow 33 have been used to color smoke; however, they have not been comprehensively assessed for their potential health hazards. (2) Methods: To assess the effects of these dyes, zebrafish embryos were exposed from 6 to 120 h post fertilization (hpf) to 10–55 µM Disperse Red 9, 1–50 µM Solvent Red 169, 7.5–13.5 µM Solvent Yellow 33 or 133–314 µM Disperse Blue 14. Embryos were monitored for adverse effects on gene expression at 48 hpf as well as for mortality, development and behavior at 120 hpf. The dyes were examined for their potential to cross the blood–brain barrier. (3) Results: Solvent Yellow 33 and Disperse Blue 14 impaired development and behavior at all concentrations. Disperse Red 9 impaired behavior at all concentrations and development at all concentrations except for 10 µM. Solvent Red 169 caused no effects. Mortality was only seen in Disperse Blue 14 at 261.5 and 314 µM. Gene expression indicated impacts on neurodevelopment and folate and retinol metabolism as potential mechanisms of toxicity. (4) Conclusions: Smoke dyes have a high potential for causing developmental changes and neurotoxicity and should be examined more closely using comprehensive approaches as used here. [ABSTRACT FROM AUTHOR]

Imidacloprid (IMI) is part of the neonicotinoids family, insecticides widely used by humans and also found in wastewater. This class of compounds, if present in the environment, can cause toxicity to different species such as bees and gammarids, although little is known about vertebrates such as fish. In addition, several substances have been reported in the environment that can cause damage to aquatic species, such as potassium perchlorate (KClO4), if exposed to high concentrations or for long periods. Often, the co-presence of different contaminants can cause a synergistic action in terms of toxicity to fish. In the present study, we first analyzed different concentrations of IMI (75, 100 and 150 mg/L) and KClO4 (1, 1.5 and 5 mM) to highlight the morphological effects at 96 hpf and, subsequently, chose two nontoxic concentrations to evaluate their co-exposure and the pathway involved in their co-toxicity. Morphological alteration, mucus production, messenger RNA (mRNA) expression related to intestinal function and oxidative stress were measured. These results suggest that co-exposure to IMI and KClO4 could affect zebrafish embryo development by increasing gut toxicity and the alteration of antioxidative defense mechanisms. [ABSTRACT FROM AUTHOR]

The growing environmental spreading of food synthetic dyes and bio-colors have the potential for altering organisms' redox states. Here, three model species for aquatic pollution trials, Cucumis sativus seeds, Artemia salina cysts, and Danio rerio embryos, were short-term exposed to a fixed concentration of the artificial red E124, and two red bio-colors, cochineal E120, and vegan red (VEGR). In the animal models, we evaluated the total reactive oxygen species (ROS) and the susceptibility to in vitro oxidative stress, and in C. sativus, H2O2 production and antioxidant capacity. We also measured organismal performance indices (routine oxygen consumption in the animal models, dark oxygen consumption, and photosynthetic efficiency in C. sativus). In C. sativus, only E124 increased ROS and affected dark oxygen consumption and photosynthetic efficiency, while all dyes enhanced the antioxidant defenses. In the A. salina nauplii, all dyes increased ROS, while E120 and E124 reduced the susceptibility to oxidative stress. In D. rerio, treatments did not affect ROS content, and reduced oxidative stress susceptibility. Our data show that red food dyes affect the redox state of the developing organisms, in which ROS plays a significant role. We suggest a potentially toxic role for red food dyes with environmentally relevant consequences. [ABSTRACT FROM AUTHOR]

The combined application of nanoparticles and surfactants has attracted tremendous attention in basic research and industry. However, knowledge of their combined toxicity remains scarce. In this study, we exposed zebrafish embryos to cetylpyridinium chloride (CPC, a cationic surfactant, at 0 and 20 μg/L), zeolitic imidazolate framework nanoparticles (ZIF-NPs, at 0, 30, and 60 mg/L), and their mixtures until 120 h post-fertilization (hpf). Within the used concentration range, both single and combined exposures exhibited limited effects on the survival and hatching of zebrafish. However, the combined exposure of ZIF-NPs and CPC caused more severe effects on the heart rate at both 48 and 72 hpf. The combined exposure also induced significant hyperactivity (i.e., increasing the average swimming velocity) and oxidative stress in zebrafish larvae (at 120 hpf), although all single exposure treatments exhibited limited impacts. Furthermore, the level of reactive oxygen species (or malondialdehyde) exhibited a significantly positive correlation with the heart rate (or average swimming velocity) of zebrafish, suggesting that oxidative stress plays a role in mediating the combined toxicity of CPC and ZIF-NPs to zebrafish. Our findings suggest that the interaction of CPC and ZIF-NPs should not be ignored when assessing the potential risks of their mixtures. [ABSTRACT FROM AUTHOR]

Signal transduction initiation by G-protein-coupled receptors (GPCRs) normally begins upon extracellular ligand binding. Some oncogenic GPCR mutants are capable of inducing G-protein signaling without ligand stimulation, thus behaving as constitutively active receptors. Evaluation of disease-causing capacity of constitutively active mutations in animal models requires months of time-consuming experiments, which hampers research progress. Here, using zebrafish embryos transiently expressing with constitutively active mutations via mRNA microinjection, we describe G-protein-subtype-specific phenotypes that can be evaluated over several days. Exogenous expression of the cysteinyl leukotriene receptor type II (CysLT 2 R) with an oncogenic L1293.43Q mutation by mRNA injection into a fertilized embryo induced developmental arrest during epiboly and eventual embryonic lethality, which were suppressed by treatment with the Gq inhibitor, YM-254890. Embryos with a constitutively active Gα q mutant exhibited an analogous phenotype. Interestingly, expression of constitutively active Gα s , Gα i , and Gα 13 mutants induced distinct phenotypes. These phenotypes may thus serve as useful indicators for rapid in vivo evaluation of signaling activity of GPCR and G-protein mutants. • L129Q CysLT 2 R constitutively activates Gq but not other G proteins (Gs, Gi, and G13). • L129Q CysLT 2 R- and Q209L Gα q -expressing embryos die during epiboly. • The other Gα subunits with the activating QL mutation causes distinct phenotypes (failed epiboly initiation, cyclopia and heart edema in Gα 13 , Gα s and Gα i1 , respectively). • Treatment with the Gq inhibitor YM-254890 rescues L129Q CysLT 2 R-induced lethality in zebrafish embryos. [ABSTRACT FROM AUTHOR]

Peptide-based drug development is an emerging and promising approach in cancer therapeutics. The present study focuses on understanding the mechanism of MP12 peptide (MDNHVCIPLCPP) derived from cysteine-rich trypsin inhibitor protein of virulence factor of pathogenic fungus Aphanomyces invadans. MP12 is involved in antiproliferative activity against the human laryngeal epithelial cell (Hep-2), demonstrated in this study. MP12 sequence showed a significant binding score and has multiple hydrogen bond interactions with the proteins that play a vital role in apoptotic pathways such as Bcl-2, caspase-3, caspase-7, and XIAP. Based on the bioinformatics characterization and molecular docking result, further study was focused on MP12 antiproliferative activity. The peptide showed a dose-dependent inhibition against Hep-2 cell line proliferation, analyzed over MTT and neutral red uptake assays. The IC 50 value of the MP12 peptide was calculated based on the antiproliferative property (24.7 ± 0.34 μM). MP12 treated Hep-2 cells showed significant shrinkage in cell morphology compared to untreated cells, inhibiting the cell cycle. The gene expression analysis validated that the MP12 significantly upregulates the caspase-3, caspase-7, and caspase-9 genes. The developmental toxicity study using zebrafish embryos as in vivo model proved that the MP12 is nontoxic. Based on the obtained results, we proposed that the peptide MP12 derived from cysteine-rich trypsin inhibitor protein of virulence molecule of pathogenic fungus have a potential antiproliferative activity. However, further clinical trials need to be focused on the mechanism and therapeutic application against laryngeal cancer. [Display omitted] • An anticancer peptide from the cysteine-rich trypsin inhibitor of fungus virulent molecule is reported. • The peptide possessed to have antiproliferative property against human laryngeal epithelial cells. • Apoptotic pathways influenced by the peptide, which arrested the cancerous growth. • The peptide is no toxic to in vivo zebrafish; however, high dose and long duration influences toxicity. • It is proposed that the peptide is a potential anticancer agent against laryngeal cancer. [ABSTRACT FROM AUTHOR]

Ochratoxin A (OTA) is one of the most widespread mycotoxin contaminants of agricultural crops. Despite being associated with a range of adverse health effects, a comprehensive systems-level mechanistic understanding of the toxicity of OTA remains elusive. In the present study, metabolic profiling by high-resolution magic angle spinning (HRMAS) NMR, coupled to intact zebrafish embryos, was employed to identify metabolic pathways in relation to a systems-level model of OTA toxicity. Embryotoxicity was observed at sub-micromolar exposure concentrations of OTA. Localization of OTA, based on intrinsic fluorescence, as well as a co-localization of increased reactive oxygen species production, was observed in the liver kidney, brain and intestine of embryos. Moreover, HRMAS NMR showed significant alteration of metabolites related to targeting of the liver (i.e., hepatotoxicity), and pathways associated with detoxification and oxidative stress, and mitochondrial energy metabolism. Based on metabolic profiles, and complementary assays, an integrated model of OTA toxicity is, thus, proposed. Our model suggests that OTA hepatotoxicity compromises detoxification and antioxidant pathways, leading to mitochondrial membrane dysfunction manifested by crosstalk between pathways of energy metabolism. Interestingly, our data additionally aligns with a possible role of mitochondrial fusion as a "passive mechanism" to rescue mitochondrial integrity during OTA toxicity. [ABSTRACT FROM AUTHOR]

Several anthropogenic products in wastewater are considered a threat to the aquatic environment. In addition to common industrial pollutants, levels of pharmaceuticals have been increasingly found in the environment in recent years, which may present a strong risk to the aquatic species that live there. The constant consumption of biologically active chemicals for human health has been matched by an increase in the leaking of these compounds in natural habitats over the last two decades. This study is aimed at evaluating the developmental toxicity of fotemustine in the ecological environment. Zebrafish embryos were exposed to doses of 25, 50 and 100 µg/mL from 4 h post-fertilization to 120 h. This study confirms that fotemustine exposure at 50 and 100 µg/mL affects the survival and hatching rate, morphology score and body length. Additionally, it significantly disturbs the antioxidant defense system and increases ROS in zebrafish larvae. From the molecular point of view, fotemustine exposure strongly induces apoptosis, endoplasmic reticulum stress (ERS) and the Wnt signaling pathway. [ABSTRACT FROM AUTHOR]

Since the middle of the twentieth century, the use of dyes has become more common in every food group as well as in the pharmaceutical, textile and cosmetic industries. Azo dyes, including carmoisine, are the most important of the dye classes with the widest color range. In this study, the effects of carmoisine exposure on the embryonic development of zebrafish at a wide dose scale, including recommended and overexposure doses (from 4 to 2000 ppm), were investigated in detail. For this purpose, many morphological and physiological parameters were examined in zebrafish exposed to carmoisine at determined doses for 96 h, and the mechanisms of action of the changes in these parameters were tried to be clarified with the metabolite levels determined. The no observed effect concentration (NOEC) and median lethal concentration (LC50) were recorded at 5 ppm and 1230.53 ppm dose at 96 hpf, respectively. As a result, it was determined that the applied carmoisine caused serious malformations, reduction in height and eye diameter, increase in the number of free oxygen radicals, in apoptotic cells and in lipid accumulation, decrease in locomotor activity depending on the dose and at the highest dose, decrease in blood flow rate. In the metabolome analysis performed to elucidate the metabolism underlying all these changes, 45 annotated metabolites were detected. [ABSTRACT FROM AUTHOR]

Aflatoxin B1 (AFB1) is a potent mycotoxin and natural carcinogen. The primary producers of AFB1 are Aspergillus flavus and A. parasiticus. Sterigmatocystin (STC), another mycotoxin, shares its biosynthetic pathway with aflatoxins. While there are abundant data on the biological effects of AFB1, STC is not well characterised. According to published data, AFB1 is more harmful to biological systems than STC. It has been suggested that STC is about one-tenth as potent a mutagen as AFB1 as measured by the Ames test. In this research, the biological effects of S9 rat liver homogenate-activated and non-activated STC and AFB1 were compared using two different biomonitoring systems, SOS-Chromotest and a recently developed microinjection zebrafish embryo method. When comparing the treatments, activated STC caused the highest mortality and number of DNA strand breaks across all injected volumes. Based on the E. coli SOS-Chromotest, the two toxins exerted the same genotoxicities. Moreover, according to the newly developed zebrafish microinjection method, STC appeared more toxic than AFB1. The scarce information correlating AFB1 and STC toxicity suggests that AFB1 is a more potent genotoxin than STC. Our findings contradict this assumption and illustrate the need for more complex biomonitoring systems for mycotoxin risk assessment. [ABSTRACT FROM AUTHOR]

Exposure to cyanotoxins has been linked to neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer's, and Parkinson's disease. While the cyanotoxin β-methylamino-L-alanine (BMAA) has received much attention, cyanobacteria produce many cyanotoxic compounds, several of which have been detected in nature alongside BMAA, including 2,4-diaminobutyric acid (2,4-DAB) and N-(2-aminoethyl)glycine (AEG). Thus, the question of whether 2,4-DAB and AEG also cause neurotoxic effects in vivo is of great interest, as is the question of whether they interact to enhance toxicity. Here, we evaluate the toxic and neurotoxic effects of these cyanotoxins alone or in combination by measuring zebrafish larval viability and behavior after exposure. 2,4-DAB was the most potent cyanotoxin as it decreased larval viability by approximately 50% at 6 days post fertilization, while BMAA and AEG decreased viability by just 16% and 8%, respectively. Although we only observed minor neurotoxic effects on spontaneous locomotion, BMAA and AEG enhanced acoustic startle sensitivity, and they interacted in an additive manner to exert their effects. 2,4-DAB; however, only modulated startle kinematics, an indication of motor dysfunction. To investigate the mechanisms of 2,4-DAB's effects, we analyzed the protein profile of larval zebrafish exposed to 500 µM 2,4-DAB at two time points and identified molecular signatures consistent with neurodegeneration, including disruption of metabolic pathways and downregulation of the ALS-associated genes SOD1 and UBQLN4. Together, our data demonstrate that BMAA and its isomers AEG and 2,4-DAB cause neurotoxic effects in vivo, with 2,4-DAB as the most potent of the three in the zebrafish model. [ABSTRACT FROM AUTHOR]

Triclosan (TCS), as a broad‐spectrum antibacterial agent, is widely used in various pharmaceutical and personal care products. However, the details of ecological environmental health risks of TCS are not clear. In this study, zebrafish embryos/larval were exposed to environmentally relevant concentrations of TCS to evaluate the developmental toxicity. Four‐hour post‐fertilization (hpf) zebrafish embryos were exposed to 0, 2, 10, 50, and 250 μg/L TCS until 96 h. The heart beats at 72 hpf were significantly increased in 2 μg/L TCS group, while significantly decreased in 250 μg/L TCS treated group compared with control. The results of acridine orange staining, terminal deoxynucleotide transferase‐mediated UTPnick end labeling assay, and detection of mitochondrial membrane potential showed that 50 and 250 μg/L TCS resulted in apoptosis. Meanwhile, reactive oxygen species (ROS) and DNA damage were induced, but SOD activity was significantly decreased in 250 μg/L TCS treated group. In addition, SOD(Mn) and GPx gene mRNA expressions were significantly down‐regulated in 50 and 250 μg/L TCS treated groups, while Casp3, Casp9, Puma, Casp8, Apaf1, and Bid genes in 250 μg/L TCS and Mdm2 gene in 50 μg/L treated groups were significantly up‐regulated. P53 protein was significantly up‐regulated in 250 μg/L TCS treated group. The overall results showed that TCS can cause oxidative stress and result in apoptosis via the involvement of ROS‐p53‐caspase‐dependent apoptotic pathway in zebrafish embryos. The present findings suggest the potential mechanisms of TCS‐induced developmental toxicity appears to be the generation of ROS and the consequent triggering of apoptosis genes. [ABSTRACT FROM AUTHOR]

The increasing pollution of aquatic habitats with anthropogenic compounds has led to various test strategies to detect hazardous chemicals. However, information on the effects of pollutants on the thyroid system in fish, which is essential for growth, development, and parts of reproduction, is still scarce. Modified early life-stage tests were carried out with zebrafish exposed to the known thyroid inhibitor potassium perchlorate (0.1, 1, 1.5, 2, 2.5, and 5 mM) to identify adverse effects on embryo development. The endogenous antioxidant defense mechanism is one of the key functions of the thyroid gland; in this regard, we examined the co-exposure to potassium perchlorate (KClO4), which could disrupt thyroid function, with cadmium (Cd), a known pro-oxidant compound. Zebrafish embryos were exposed to control KClO4 1 mM and Cd 0.5 μM for 96 h after fertilization (hpf) individually and in combination. The morphological alteration, body length, and messenger RNA (mRNA) expression related to thyroid function and oxidative stress, thyroid hormone levels, and malondialdehyde were measured. Significant down-regulation of mRNAs related to thyroid function (thyroid hormone receptor-alpha (THRα), thyroid hormone receptor-beta (THRβ), haematopoietically expressed homeobox (hhex)) and decreased thyroxin (T4) levels were observed after co-exposure to KClO4 and Cd, but this was not observed in the individually treated groups. These results suggest that co-exposure to KClO4 and Cd could affect antioxidant defense mechanisms and potentially normally increase Cd toxicity on mRNA expression, altering the thyroid functions important in zebrafish embryonic developmental stages. [ABSTRACT FROM AUTHOR]

Despite extensive investigation on the toxicity of microplastics (MPs), an emerging global concern, little is known about the combined toxicity of MPs and co-occurring pollutants in aquatic environments. In this study, the combined toxicity of polystyrene MPs and sulfamethoxazole (SMZ) antibiotics was explored in zebrafish embryos in terms of the developmental, physiological, and endocrine toxicities. Exposure to PS and SMZ induced mortality (rate: 25.0 ± 7.5%) and malformation (rate: 20~35%) at multiple regions and stages of zebrafish development. Physiological toxicity was also induced as shown by the significant decrease in fetal movement (by 31.1~37.0%) and swimming frequency (by 26.9~36.8%) and the increase in heartbeat rate (by 19.0~20.9%). Finally, PS and SMZ exposure also induced extensive endocrine toxicities in zebrafish as confirmed by increases in various biomarkers including vitellogenin, 17β-estradiol, testosterone, and triiodothyronine. The combination index showed that antagonistic effects were present between PS and SMZ toxicity, which slightly decreased their combined toxicity. This study aims to further understand the combined toxicity of MPs and co-occurring pollutants in aquatic environments. [ABSTRACT FROM AUTHOR]

Molecular mechanisms controlling the formation, stabilisation and maintenance of blood vessel connections remain poorly defined. Here, we identify blood flow and the large extracellular protein Svep1 as co-modulators of vessel anastomosis during developmental angiogenesis in zebrafish embryos. Both loss of Svep1 and blood flow reduction contribute to defective anastomosis of intersegmental vessels. The reduced formation and lumenisation of the dorsal longitudinal anastomotic vessel (DLAV) is associated with a compensatory increase in Vegfa/Vegfr pERK signalling, concomittant expansion of apelin-positive tip cells, but reduced expression of klf2a. Experimentally, further increasing Vegfa/Vegfr signalling can rescue the DLAV formation and lumenisation defects, whereas its inhibition dramatically exacerbates the loss of connectivity. Mechanistically, our results suggest that flow and Svep1 co-regulate the stabilisation of vascular connections, in part by modulating the Vegfa/Vegfr signalling pathway. [ABSTRACT FROM AUTHOR]

The occurrence of nanoplastics (NPs) and microplastics (MPs) in aquatic ecosystems and their capacity to sorb hydrophobic pollutants is nowadays an issue of great concern. This study aimed to assess the potential bioavailability and acute toxicity of polystyrene (PS) NPs (50 and 500 nm) and of MPs (4.5 µm), alone and with sorbed benzo(a)pyrene (B(a)P), in the embryo/larval stages of brine shrimps and zebrafish. Exposure to pristine plastics up to 50.1 mg PS/L did not cause significant impact on brine shrimp survival, while some treatments of plastics-B(a)P and all concentrations of B(a)P (0.1–10 mg/L) resulted acutely toxic. In zebrafish, only the highest concentrations of MPs-B(a)P and B(a)P caused a significant increase of malformation prevalence. Ingestion of NPs was observed by 24–48 h of exposure in the two organisms (from 0.069 to 6.87 mg PS/L). In brine shrimps, NPs were observed over the body surface and within the digestive tract, associated with feces. In zebrafish, NPs were localized in the eyes, yolk sac, and tail at 72 h, showing their capacity to translocate and spread into the embryo. MP ingestion was only demonstrated for brine shrimps. In zebrafish embryos exposed to plastics-B(a)P, B(a)P appeared in the yolk sac of the embryos. The presence of B(a)P was also noticeable in brine shrimps exposed to 500 nm NPs-B(a)P. In conclusion, NPs entered and spread into the zebrafish embryo and PS NPs, and MPs were successful vectors of B(a)P to brine shrimp and zebrafish embryos. Particle size played a significant role in explaining the toxicity of plastics–B(a)P. Our study provides support for the idea that plastics may pose a risk to aquatic organisms when combined with persistent organic pollutants such as B(a)P. [ABSTRACT FROM AUTHOR]

Oxidative stress and the AKT serine/threonine kinase (AKT) signaling pathway are essential regulators in cellular migration, metastasis, and angiogenesis. More than 300 withanolides were discovered from the plant family Solanaceae, exhibiting diverse functions. Notably, the relationship between oxidative stress, AKT signaling, and angiogenesis in withanolide treatments lacks comprehensive understanding. Here, we summarize connecting evidence related to oxidative stress, AKT signaling, and angiogenesis in the zebrafish model. A convenient vertebrate model monitored the in vivo effects of developmental and tumor xenograft angiogenesis using zebrafish embryos. The oxidative stress and AKT-signaling-modulating abilities of withanolides were highlighted in cancer treatments, which indicated that further assessments of their angiogenesis-modulating potential are necessary in the future. Moreover, targeting AKT for inhibiting AKT and its AKT signaling shows the potential for anti-migration and anti-angiogenesis purposes for future application to withanolides. This particularly holds for investigating the anti-angiogenetic effects mediated by the oxidative stress and AKT signaling pathways in withanolide-based cancer therapy in the future. [ABSTRACT FROM AUTHOR]

Featured Application: The results highlighted the role of mechanical forces in fish embryo osteogenesis. Such a simple in vivo model can be used to elucidate the responsiveness of osteoblasts to environmental mechanical forces and useful in designing new therapeutic approaches in human bone diseases. Skeletal system development and remodelling is regulated by several different factors, including hormones, cytokines, and mechanical forces. It is known that gravity and pressure stimulate mechanosensors on bone cells which transduce mechanical signals to chemical ones. Nevertheless, few data have been provided about the role of mechanical forces on embryo osteogenesis in vivo. Since the zebrafish is an elective model for developmental studies, in particular on bone formation and tissue mineralization, we analyzed in vivo the effects of a static mechanical force generated by a water column on fertilized zebrafish eggs. The results have shown that an increase in the hydrostatic pressure (HP) of up to 5.9% was lethal for 100% of treated embryos at 48 h post fertilization (hpf). A small decrease in length (−2%) and 49% mortality were found in the +4.4% HP embryos compared with the controls. To analyze skeletal development, we evaluated the number of mineralized vertebral bodies in the trunk at five days post fertilization. The embryos grown under +2.4% HP showed a physiological intramembranous mineralization of vertebral bodies whereas the embryos which grew with +3.4% HP showed a significant decrease in mineralization rate (−54%). Morphological analysis of cartilage and bones in embryos at +3.4% HP revealed a delay of both intramembranous and chondrogenic mineralization, respectively, in axial and head bones, whereas the chondrogenesis appeared normal. These data suggested that developing osteoblasts and different mineralization programs are sensitive to mechanical pressure when applied to early embryogenesis. [ABSTRACT FROM AUTHOR]

Animal models are essential to understanding COVID-19 pathophysiology and for preclinical assessment of drugs and other therapeutic or prophylactic interventions. We explored the small, cheap, and transparent zebrafish larva as a potential host for SARS-CoV-2. Bath exposure, as well as microinjection in the coelom, pericardium, brain ventricle, or bloodstream, resulted in a rapid decrease of SARS-CoV-2 RNA in wild-type larvae. However, when the virus was inoculated in the swim bladder, viral RNA stabilized after 24 h. By immunohistochemistry, epithelial cells containing SARS-CoV-2 nucleoprotein were observed in the swim bladder wall. Our data suggest an abortive infection of the swim bladder. In some animals, several variants of concern were also tested with no evidence of increased infectivity in our model. Low infectivity of SARS-CoV-2 in zebrafish larvae was not due to the host type I interferon response, as comparable viral loads were detected in type I interferon-deficient animals. A mosaic overexpression of human ACE2 was not sufficient to increase SARS-CoV-2 infectivity in zebrafish embryos or in fish cells in vitro. In conclusion, wild-type zebrafish larvae appear mostly non-permissive to SARS-CoV-2, except in the swim bladder, an aerial organ sharing similarities with the mammalian lung. [ABSTRACT FROM AUTHOR]

In cardiac muscle cells, heterodimeric integrin transmembrane receptors are known to serve as mechanotransducers, translating mechanical force to biochemical signaling. However, the roles of many individual integrins have still not been delineated. In this report, we demonstrate that Itga3b is localized to the sarcolemma of cardiomyocytes from 24 to 96 hpf. We further show that heterozygous and homozygous itga3b/bdf mutant embryos display a cardiomyopathy phenotype, with decreased cardiac contractility and reduced cardiomyocyte number. Correspondingly, proliferation of ventricular and atrial cardiomyoctyes and ventricular epicardial cells is decreased in itga3b mutant hearts. The contractile dysfunction of itga3b mutants can be attributed to cardiomyocyte sarcomeric disorganization, including thin myofilaments with blurred and shortened Z-discs. Together, our results reveal that Itga3b localizes to the myocardium sarcolemma, and it is required for cardiac contractility and cardiomyocyte proliferation. • Itga3b localizes to the myocardium sarcolemma of zebrafish embryonic heart. • Itga3b is required for proper sarcomere organization of zebrafish embryonic heart. • Zebrafish Itga3b regulates cardiac contractility and cardiomyocyte proliferation. [ABSTRACT FROM AUTHOR]

Neural retina leucine zipper (NRL) is an essential gene for the fate determination and differentiation of the precursor cells into rod photoreceptors in mammals. Mutations in NRL are associated with the autosomal recessive enhanced S-cone syndrome and autosomal dominant retinitis pigmentosa. However, the exact role of Nrl in regulating the development and maintenance of photoreceptors in the zebrafish (Danio rerio), a popular animal model used for retinal degeneration and regeneration studies, has not been fully determined. In this study, we generated an nrl knockout zebrafish model via the CRISPR-Cas9 technology and observed a surprising phenotype characterized by a reduced number, but not the total loss, of rods and over-growth of green cones. We discovered two waves of rod genesis, nrl-dependent and -independent at the embryonic and post-embryonic stages, respectively, in zebrafish by monitoring the rod development. Through bulk and single-cell RNA sequencing, we characterized the gene expression profiles of the whole retina and each retinal cell type from the wild type and nrl knockout zebrafish. The over-growth of green cones and mis-expression of green-cone-specific genes in rods in nrl mutants suggested that there are rod/green-cone bipotent precursors, whose fate choice between rod versus green-cone is controlled by nrl. Besides, we identified the mafba gene as a novel regulator of the nrl-independent rod development, based on the cell-type-specific expression patterns and the retinal phenotype of nrl/mafba double-knockout zebrafish. Gene collinearity analysis revealed the evolutionary origin of mafba and suggested that the function of mafba in rod development is specific to modern fishes. Furthermore, the altered photoreceptor composition and abnormal gene expression in nrl mutants caused progressive retinal degeneration and subsequent regeneration. Accordingly, this study revealed a novel function of the mafba gene in rod development and established a working model for the developmental and regulatory mechanisms regarding the rod and green-cone photoreceptors in zebrafish. Author summary: Vision is mediated by two types of light-sensing cells named rod and cone photoreceptors in animal eyes. Abnormal generation, dysfunction or death of photoreceptor cells all cause irreversible vision problems. NRL is an essential gene for the generation and function of rod cells in mice and humans. Surprisingly, we found that in the zebrafish, a popular animal model for human diseases and therapeutic testing, there are two types of rod cells, and eliminating the function of nrl gene affects the rod cell formation at the embryonic stage but not at the juvenile and adult stages. The rod cell formation at the post-embryonic is driven by the mafba gene, which has not been reported to play a role in rod cells. In addition to the reduced number of rod cells, deletion of nrl also results in the emergence of rod/green-cone hybrid cells and an increased number of green cones. The ensuing cellular and molecular alterations collectively lead to retinal degeneration. These findings expand our understanding of photoreceptor development and maintenance and highlight the underlying conserved and species-specific regulatory mechanisms. [ABSTRACT FROM AUTHOR]

Pseudoxanthoma elasticum (PXE) is a multisystem ectopic mineralization disorder caused by pathogenic variants in the ABCC6 gene. Though complications of the disease can be treated, PXE itself remains currently intractable. A strategy for rapid and cost-effective discovery of therapeutic drugs would be to perform chemical compound screening using zebrafish, but this approach remains to be validated for PXE. In this paper, we validate a stable CRISPR/Cas9 abcc6a knockout zebrafish model–which has spinal column hypermineralization as its primary phenotypic feature–as a model system for compound screening in ectopic mineralization. We evaluated the anti-mineralization potential of five compounds, which had (anecdotal) positive effects reported in Abcc6 knockout mice and/or PXE patients. Abcc6a knockout zebrafish larvae were treated from 3 to 10 days post-fertilization with vitamin K1, sodium thiosulfate, etidronate, alendronate or magnesium citrate and compared to matching controls. Following alizarin red S staining, alterations in notochord sheath mineralization were semiquantified and found to largely congrue with the originally reported outcomes. Our results demonstrate that the use of this abcc6a knockout zebrafish model is a validated and promising strategy for drug discovery against ectopic mineralization. [ABSTRACT FROM AUTHOR]

1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) is a neurotoxin that damages dopaminergic neurons. Zebrafish has been shown to be a suitable model organism to investigate the molecular pathways in the pathogenesis of Parkinson's disease and also for potential therapeutic agent research. Boron has been shown to play an important role in the neural activity of the brain. Boronic acids are used in combinatorial approaches in drug design and discovery. The effect of 3-pyridinylboronic acid which is an important sub-class of heterocyclic boronic acids has not been evaluated in case of MPTP exposure in zebrafish embryos. Accordingly, this study was designed to investigate the effects of 3-pyridinylboronic acid on MPTP exposed zebrafish embryos focusing on the molecular pathways related to neurodegeneration and apoptosis by RT-PCR. Zebrafish embryos were exposed to MPTP (800 μM); MPTP + Low Dose 3-Pyridinylboronic acid (50 μM) (MPTP + LB) and MPTP + High Dose 3-Pyridinylboronic acid (100 μM) (MPTP + HB) in well plates for 72 hours post fertilization. Results of our study showed that MPTP induced a P53 dependent and Bax mediated apoptosis in zebrafish embryos and 3-pyridinylboronic acid restored the locomotor activity and gene expressions related to mitochondrial dysfunction and oxidative stress due to the deleterious effects of MPTP, in a dose-dependent manner. [ABSTRACT FROM AUTHOR]

Chemical UV filters are increasingly used in cosmetics to protect skin from UV radiation. As a consequence, they are released into the aquatic environment via recreational activities and wastewaters. In aquatic ecosystems, fish eggs in contact with sediment can be affected by organic and lipophilic pollutants such as UV filters. The present study aims to evaluate the toxicity of six individual UV filters, diethylhexyl butamido triazone (DBT), diethylamino hydroxybenzoyl hexyl benzoate (DHHB), ethylhexyl triazone (ET), 2-ethylhexyl salicylate (ES), homosalate (HS), and octocrylene (OC), in the embryo-larval stages of zebrafish Danio rerio. Contamination of fish eggs and larvae with UV filters occurred through contact with spiked sediment for 96 h at a concentration of 10 μg g−1. Among the six UV filters tested, OC delayed hatching success, whereas ES significantly increased the heartbeat rate of embryo–larvae after sediment exposure, probably as a stress response. [ABSTRACT FROM AUTHOR]

The amount of pharmaceuticals transferred to the aquatic environment via municipal and hospital waste water is steadily increasing. The progress in medical research has resulted in the manufacture of active substances of increased stability, specificity, and potency, which can trigger adverse effects in aquatic organisms. Moreover, advanced analytical methods allow the detection of pharmaceuticals in environmental matrices at very low concentrations, which increases the number of substances to be assessed. Levonorgestrel is a synthetic gestagen commonly used in medicinal products for contraception. Because progestogenic compounds could have an impact on fish maturation processes, a life cycle test was performed to assess the effects of levonorgestrel exposure of the embryonic to the adult stages of zebrafish (Danio rerio) at mean measured concentrations of 0.06, 0.16, 0.47, 1.64, and 5.45 ng/L. Apical endpoints were survival, growth, reproduction, and sex ratio. Determination of endocrine modulation was completed by measurement of vitellogenin and 11‐keto testosterone in blood plasma, as well as by histopathological analysis of gonads. For all parameters, control values were within the recommended quality range. The most prominent levonorgestrel effect was a shift toward an increased number of male fish at 1.64 and especially 5.45 ng/L, at which point all fish were histologically determined to be males and no spawning occurred; 11‐keto testosterone was significantly decreased. A no‐observed‐effect concentration (NOEC) of 0.47 ng levonorgestrel/L was confirmed by the fertilization capability of adult fish, the male maturation stages, and female gonad histopathology. Whereas hatch and juvenile growth were not affected, posthatch survival was significantly impeded at ≥0.47 ng levonorgestrel/L, although it was not clearly related to the test concentration. For male length and weight, the same NOEC of 0.16 ng/L was obtained at study termination. Environ Toxicol Chem 2022;41:580–591. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. [ABSTRACT FROM AUTHOR]

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