Comparison of cardiotoxicity induced by alectinib, apatinib, lenvatinib and anlotinib in zebrafish embryos.

Four tyrosine kinase inhibitors, alectinib, apatinib, lenvatinib and anlotinib, have been shown to be effective in the treatment of clinical tumors, but their cardiac risks have also raised concerns. In this study, zebrafish embryos at 6 h post fertilization (hpf) were exposed to the four drugs at concentrations of 0.05-0.2 mg/L until 72 hpf, and then the development of these embryos was quantified, including heart rate, body length, yolk sac area, pericardial area, distance between venous sinus and balloon arteriosus (SV-BA), separation of cardiac myocytes and endocardium, gene expression, vascular development and oxidative stress. At the same exposure concentrations, alectinib and apatinib had little effect on the cardiac development of zebrafish embryos, while lenvatinib and anlotinib could induce significant cardiotoxicity and developmental toxicity, including shortened of body length, delayed absorption of yolk sac, pericardial edema, prolonged SV-BA distance, separation of cardiomyocytes and endocardial cells, and downregulation of key genes for heart development. Heart rate decreased in all four drug treatment groups. In terms of vascular development, alectinib and apatinib did not inhibit the growth of embryonic intersegmental vessels (ISVs) and retinal vessels, while lenvatinib and anlotinib caused serious vascular toxicity, and the inhibition of anlotinib in vascular development was more obvious. Besides, the level of reactive oxygen species (ROS) in the lenvatinib and anlotinib treatment groups was significantly increased. Our results provide reference for comparing the cardiotoxicity of the four drugs.

Down-regulation of theta amplitude through neurofeedback improves executive control network efficiency in healthy children.

Despite extensive clinical research on neurofeedback (NF) in attention-deficit/hyperactivity disorder (ADHD), few studies targeted the optimization of attention performance in healthy children. As a crucial component of attention networks, the executive control network, involved in resolving response conflicts and allocating cognitive resources, is closely linked to theta activity. Here, we aimed to answer whether theta down-regulating NF can enhance healthy children's attention performance, especially the executive control network. Sixty children aged 6-12 years were randomly assigned to the NF and waitlist control groups. The NF group received theta down-regulation NF training for five days (a total of 100 mins), and the attention performance of both groups was measured by the attention network test (ANT) in the pre, post-NF, and 7-day follow-up. The electroencephalographic (EEG) results demonstrated a significant decrease in resting-state theta amplitude within sessions. For the behavioral results, the NF group exhibited significant improvements in overall attention performance and the efficiency of the executive control network relative to the control group in the post-NF and follow-up assessment, whereas the alerting and orienting networks remained unchanged. These findings proved the feasibility of theta down-regulating NF and its positive effect on attention in the healthy children population. In particular, the facilitation of the efficiency of the executive control network and the unaltered performance of the other two attention networks in the NF group may support the causality between theta rhythm and the executive control network.

Dimethyl fumarate induces cardiac developmental toxicity in zebrafish via down-regulation of oxidative stress.

Dimethyl fumarate (DMF) is an immunosuppressant commonly used to treat multiple sclerosis and other autoimmune diseases. Despite known side effects such as lymphopenia, the effect of DMF on cardiac development remains unclear. To assess this, we used zebrafish to evaluate the cardiac developmental toxicity of DMF. Our study showed that DMF reduced the survival rate of zebrafish embryos, with those exposed to 1, 1.3, and 1.6 mg/L exhibiting heart rate reduction, shortened body length, delayed yolk sac absorption, pericardial edema, increased distance from sinus venous to bulbus arteriosus, and separation of cardiomyocytes and endocardial cells at 72 hpf. Heart development-related genes showed disorder, apoptosis-related genes were up-regulated, and the oxidative stress response was down-regulated. Treatment with cysteamine ameliorated the heart development defects. Our study demonstrates that DMF induces cardiac developmental toxicity in zebrafish, possibly by down-regulating oxidative stress responses. This study provides a certain research basis for further study of DMF-induced cardiac developmental toxicity, and provides some experimental evidence for future clinical application and study of DMF.

BPA induces hepatotoxicity in zebrafish through oxidative stress and apoptosis pathways.

BPA is so ubiquitous that 27 million tons of BPA-containing plastic, including mineral water bottles and baby bottles, is produced worldwide each year. The potential toxicity of BPA to humans and aquatic organisms has been the subject of intense research. In this study, a zebrafish model system was used to assess BPA-mediated hepatotoxicity. Zebrafish larvae at 72-144 hpf were exposed to BPA at different concentrations (0,1, 3 and 5mg/L). For example, BPA-treated zebrafish larvae showed increased mortality, delayed uptake of nutrients in yolk sac, shortened body length, smaller liver area, abnormal expression of genes related to liver development, and pathological changes in the liver tissue. Mechanistically, BPA exposure induced excessive oxidative stress in the liver of zebrafish and increased the level of hepatocyte apoptosis in zebrafish larvae, and the antioxidant astaxanthin could rescue the BPA-mediated liver toxicity.

Apatinib induces zebrafish hepatotoxicity by inhibiting Wnt signaling and accumulation of oxidative stress.

Apatinib, a small-molecule VEGFR2-tyrosine kinase inhibitor, has shown potent anticancer activity in various clinical cancer treatments, but also different adverse reactions. Therefore, it is necessary to study its potential toxicity and working mechanism. We used zebrafish to investigate the effects of apatinib on the development of embryos. Zebrafish exposed to 2.5, 5, and 10 µM apatinib showed adverse effects such as decreased liver area, pericardial oedema, slow yolk absorption, bladder atrophy, and body length shortening. At the same time, it leads to abnormal liver tissue structure, liver function and related gene expression. Furthermore, after exposure to apatinib, oxidative stress levels were significantly elevated but liver developmental toxicity was effectively ameliorated with oxidative stress inhibitor treatment. Apatinib induces down-regulation of key target genes of Wnt signaling pathway in zebrafish, and it is found that Wnt activator can significantly rescue liver developmental defects. These results suggest that apatinib may induce zebrafish hepatotoxicity by inhibiting the Wnt signaling pathway and up-regulating oxidative stress, helping to strengthen our understanding of rational clinical application of apatinib.

Roxadustat induces hepatotoxicity in zebrafish embryos via inhibiting Notch signaling.

Roxadustat is a novel and effective small-molecule inhibitor of hypoxia-inducible factor prolyl hydroxylase (HIF-PHI). However, little research has been done on its toxicity to vertebrate embryonic development. In this study, we used zebrafish to assess the effects of roxadustat on early embryonic development. Exposure to 14, 28, and 56 µM roxadustat resulted in abnormal embryonic development in zebrafish embryos, such as shortened body length and early liver developmental deficiency. Roxadustat exposure resulted in liver metabolic imbalance and abnormal liver tissue structure in adult zebrafish. In addition, roxadustat could up-regulate oxidative stress, and astaxanthin (AS) could partially rescue liver developmental defects by down-regulation of oxidative stress. After exposure to roxadustat, the Notch signaling is down-regulated, and the use of an activator of Notch signaling can partially rescue hepatotoxicity. Therefore, our research indicates that roxadustat may induce zebrafish hepatotoxicity by down-regulating Notch signaling. This study provides a reference for the clinical use of roxadustat.

Cyclosporine A induces hepatotoxicity in zebrafish larvae via upregulating oxidative stress.

As a powerful immunosuppressant, cyclosporine A (CsA) is widely used clinically. However, it has been found to have many side effects including nephrotoxicity and neurotoxicity. Despite this, some patients cannot avoid using CsA during pregnancy and this can be detrimental to both the patient and the foetus. This study used zebrafish as a model animal to evaluate the hepatotoxic effects of CsA in zebrafish embryos. Zebrafish embryos cultured at 72 post-fertilization (hpf) were exposed to three concentrations of CsA at 2.5 mg/L, 5 mg/L, and 10 mg/L for 72 h. Liver developmental defects, smaller or missing swim bladder, slower heart rate, reduced body length, and delayed yolk sac absorption were observed. The level of oxidative stress (ROS) increased with the increase of CsA concentration. The indicators of related oxidative stress kinase activities including malondialdehyde (MDA), catalase (CAT) and SOD, all appeared to significantly increased. The use of astaxanthin (ATX) to inhibit oxidative stress was found to be useful for rescuing zebrafish hepatic development defects. Therefore, our results suggest that CsA induces zebrafish embryonic hepatic development defects by activating the oxidative stress. The study of CsA-induced hepatic development defects of zebrafish embryos is helpful for clinical evaluation of the safety of CsA and enables the search for new use without side effects.

Propranolol hydrochloride induces neurodevelopmental toxicity and locomotor disorders in zebrafish larvae.

Propranolol hydrochloride is the first-line drug for the clinical treatment of hypertension, arrhythmia, and other diseases. However, with the increasing use of this drug, its safety and environmental health have received more and more attention. In this study, aquatic vertebrate zebrafish were used as a model to study the toxic effects and mechanisms of propranolol hydrochloride. It was revealed that zebrafish larvae exposed to propranolol hydrochloride showed aberrant head nerve development and locomotor disorders. Additionally, exposure to propranolol hydrochloride could induce oxidative stress, alter the activities of AChE and ATPase, and disrupt the expression of genes involved in neurodevelopment and neurotransmitter pathways. More interestingly, the expression of Parkinson's disease-related genes was altered in zebrafish treated with propranolol hydrochloride. We detected the expression of genes related to the Wnt signaling pathway and found that their expression appeared to be down-regulated. The phenotype of nerve developmental defects and locomotor disorders can be effectively rescued by astaxanthin and Wnt activators. Collectively, the results suggest that propranolol hydrochloride may induce neurotoxicity and abnormal movement behavior with PD-like symptoms in zebrafish larvae.

Exposure to pyrazosulfuron-ethyl induces immunotoxicity and behavioral abnormalities in zebrafish embryos.

Pyrazosulfuron-ethyl is one of the most widely used herbicides in agriculture and can be widely detected in aquatic ecosystems. However, its biosafety, including its potential toxic effects on aquatic organisms and its mechanism, is still poorly understood. As an ideal vertebrate model, zebrafish, the effect of pyrazosulfuron-ethyl on early embryonic development and immunotoxicity of zebrafish can be well evaluated. From 10 to 72 h post fertilization (hpf), zebrafish embryos were exposed to 1, 5, and 9 mg/L pyrazosulfuron-ethyl which led in a substantial reduction in survival, total length, and heart rate, as well as a range of behavioral impairments. In zebrafish larvae, the number of neutrophils and macrophages was considerably decreased and oxidative stress levels increased in a dose-dependent way after pyrazosulfuron-ethyl exposure. And the expression of immune-related genes, such as TLR-4, MyD88 and IL-1ß, were downregulated by pyrazosulfuron-ethyl exposure. Moreover, pyrazosulfuron-ethyl exposure also inhibited motor behavior. Notch signaling was upregulated after exposure to pyrazosulfuron-ethyl, while inhibition of Notch signaling pathway could rescue immunotoxicity. Therefore, our findings suggest that pyrazosulfuron-ethyl has the potential to induce immunotoxicity and neurobehavioral changes in zebrafish larvae.

Pamiparib Induces Neurodevelopmental Defects and Cerebral Haemorrhage in Zebrafish Embryos via Inhibiting Notch Signalling.

Pamiparib is a poly ADP-ribose polymerase (PARP) inhibitor used in clinical studies, which can penetrate the blood-brain barrier efficiently. At present, there are few studies on its effect on vertebrate neurodevelopment. In this study, we exposed zebrafish embryos to 1, 2 and 3 µM of Pamiparib from 6 to 72 h post-fertilisation (hpf). Results showed that pamiparib can specifically induce cerebral haemorrhage, brain atrophy and movement disorders in fish larvae. In addition, pamiparib exposure leads to downregulation of acetylcholinesterase (AChE) and adenosine triphosphate (ATPase) activities, and upregulation of oxidative stress which then leads to apoptosis and disrupts the gene expression involved in the neurodevelopment, neurotransmitter pathways and Parkinson's disease (PD) like symptoms. Meanwhile, astaxanthin can partially rescue neurodevelopmental defects by downregulating oxidative stress. After exposure to pamiparib, the Notch signalling is downregulated, and the use of an activator of Notch signalling can partially rescue neurodevelopmental toxicity. Therefore, our research indicates that pamiparib may induce zebrafish neurotoxicity by downregulating Notch signalling and provides a reference for the potential neurotoxicity of pamiparib during embryonic development.

Alpha/Theta Ratio Neurofeedback Training for Attention Enhancement in Normal Developing Children: A Brief Report.

Attention plays an important role in children's development and learning, and neurofeedback training (NFT) has been proposed as a promising method to improve attention, mainly in population with attention problems such as attention deficit hyperactivity disorder. However, whether this approach has a positive effect on attention in normal developing children has been rarely investigated. This pilot study conducted ten sessions of alpha/theta ratio (ATR) NFT on eight primary students in school environment, with two to three sessions per week. The results showed inter-individual difference in NFT learning efficacy that was assessed by the slope of ATR over training sessions. In addition, the attention performance was significantly improved after NFT. Importantly, the improvement of attention performance was positively correlated with the NFT learning efficacy. It thus highlighted the need for optimizing ATR NFT protocol for the benefits on attention at the individual level. Future work can employ a double-blind placebo-controlled design with larger sample size to validate the benefits of ATR NFT for attention in normal developing children.

Lenvatinib induces cardiac developmental toxicity in zebrafish embryos through regulation of Notch mediated-oxidative stress generation.

Due to an increasing number of abused drugs dumped into the wastewater, more and more drugs are detected in the water environment, which may affect the survival of aquatic organisms. Lenvatinib is a multi-targeted tyrosine kinase inhibitor, and is clinically used to treat differentiated thyroid cancer, renal epithelial cell carcinoma and liver cancer. However, there are few reports on the effects of lenvatinib in embryos development. In this study, zebrafish embryos were used to evaluate the effect of lenvatinib on cardiovascular development. Well-developed zebrafish embryos were selected at 6 h post fertilization (hpf) and exposed to 0.05 mg/L, 0.1 mg/L and 0.2 mg/L lenvatinib up to 72 hpf. The processed embryos demonstrated cardiac edema, decreased heart rate, prolonged SV-BA distance, inhibited angiogenesis, and blocked blood circulation. Lenvatinib caused cardiac defects in the whole stage of cardiac development and increased the apoptosis of cardiomyocyte. Oxidative stress in the processed embryos was accumulated and inhibiting oxidative stress could rescue cardiac defects induced by lenvatinib. Additionally, we found that lenvatinib downregulated Notch signaling, and the activation of Notch signaling could rescue cardiac developmental defects and downregulate oxidative stress level induced by lenvatinib. Our results suggested that lenvatinib might induce cardiac developmental toxicity through inducing Notch mediated-oxidative stress generation, raising concerns about the harm of exposure to lenvatinib in aquatic organisms.

Cyclosporine A Induces Cardiac Developmental Toxicity in Zebrafish by Up-Regulation of Wnt Signaling and Oxidative Stress.

Due to the widely application of Cyclosporine A (CsA) as an immunosuppressant in clinic, it is necessary to study its potential toxicity. Therefore, we used zebrafish as a model animal to evaluate the toxicity of CsA on embryonic development. Exposure of zebrafish embryos to CsA at concentrations of 5 mg/L, 10 mg/L, and 15 mg/L from 12 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, and the apoptotic genes were up-regulated. Oxidative stress level was up-regulated and accumulated in pericardium in a dose-dependent manner. Astaxanthin (ATX) treatment could significantly alleviate zebrafish heart defects. CsA induced up-regulation of Wnt signaling in zebrafish, and IWR-1, an inhibitor of Wnt signaling pathway, could effectively rescue the heart defects induced by CsA. Together, our study indicated that CsA induced cardiac developmental toxicity in zebrafish larvae through up-regulating oxidative stress and Wnt signaling, contributing to a more comprehensive evaluation of the safety of the drug.

Lenvatinib exposure induces hepatotoxicity in zebrafish via inhibiting Wnt signaling.

Lenvatinib is a multi-kinase inhibitor for widely treating thyroid cancer. However, little studies have been done about it or its toxicity on embryonic development of vertebrate. In this study, we used zebrafish to assess the effect of lenvatinib on early embryonic development. Exposure of zebrafish embryos to 58, 117, 176 nM lenvatinib induced abnormal embryonic development, such as decreased heart rate, pericardial edema, delayed yolk absorption, and bladder atrophy. Lenvatinib exposure reduced liver area and down-regulated liver developmental related genes. The proliferation of hepatocytes and the expression of apoptosis-related genes were significantly reduced.by Lenvatinib. Furthermore, the imbalance of liver metabolism and abnormal liver tissue structure were observed in adult zebrafish after Lenvatinib exposure. Oxidative stress was up-regulated by lenvatinib and astaxanthin partially rescued hepatic developmental defects via downregulating oxidative stress. After lenvatinib exposure, Wnt signaling was down-regulated, and activation of Wnt signaling partially rescued hepatic developmental defects. Therefore, these results suggested that lenvatinib might induce zebrafish hepatotoxicity by down-regulating Wnt signaling related genes and inducing oxidative stress. This study provides a reference for the potential hepatotoxicity of lenvatinib during embryonic development and raises health concern about the potential harm of exposure to lenvatinib for foetuses.

A dual-signal sensor for the analysis of parathion-methyl using silver nanoparticles modified with graphitic carbon nitride.

A highly sensitive and selective method was developed for both UV-vis spectrophotometric and fluorimetric determination of organophosphorus pesticides (OPs). This method used silver nanoparticles (AgNPs) modified with graphitic carbon nitride (g-C3N4). The AgNPs reduced the fluorescence intensity of g-C3N4. Acetylthiocholine (ATCh) could be catalytically hydrolyzed by acetylcholinesterase (AChE) to form thiocholine, which induces aggregation of the AgNPs. This aggregation led to the recovery of the blue fluorescence of g-C3N4, with excitation/emission peaks at 310/460 nm. This fluorescence intensity could be reduced again in the presence of OPs because of the inhibitory effect of OPs on the activity of AChE. The degree of reduction was found to be proportional to the concentration of OPs, and the limit of fluorometric detection was 0.0324 µg/L (S/N = 3). In addition, the absorption of the g-C3N4/AgNPs at 390 nm decreased because of the aggregation of the AgNPs, but was recovered in presence of OPs because of the inhibition of enzyme activity by OPs. This method was successfully applied to the analysis of parathion-methyl in real samples.

Three-dimensional cell-culture platform based on hydrogel with tunable microenvironmental properties to improve insulin-secreting function of MIN6 cells.

Pancreatic ß-cells have been reported to be mechanosensitive to cellular microenvironments, and subjecting the cells to more physiologically relevant microenvironments can produce better results than when subjecting them to the conventional two-dimensional (2D) cell-culture conditions. In this work, we propose a novel three-dimensional (3D) strategy for inducing multicellular spheroid formation based on hydrogels with tunable mechanical and interfacial properties. The results indicate that MIN6 cells can sense the substrates and form tightly clustered monolayers or multicellular spheroids on hydrogels with tunable physical properties. Compared to the conventional 2D cell-culture system, the glucose sensitivities of the MIN6 cells cultured in the 3D culture model is enhanced greatly and their insulin content (relative to the amount of protein) is increased 7.3-7.9 folds. Moreover, the relative gene and protein expression levels of some key factors such as Pdx1, NeuroD1, Piezo1, and Rac1 in the MIN6 cells are significantly higher on the 3D platform, compared to the 2D control group. We believe that this 3D cell-culture system developed for the generation of multicellular spheroids will be a promising platform for diabetes treatment in clinical islet transplantation.

CD36 Upregulation in HT-29 Tumor Cells Cultured on 3D Multicellular Spheroids Culturing Platform Based on Anisotropic Magnetic Hydrogel.

Tumor metastasis and invasiveness are research hotspots in tumor therapy. Previous research demonstrated that cells expressing high level of CD36 might initiate metastasis in certain human oral carcinomas cell lines. Multicellular spheroids (MCS) culture technique has been extensively studied both in fundamental and applied research. In our previous research, we invented novel 3D anisotropic magnetic hydrogels (AMHs) which were composed of magnetic nanoparticle assemblies and biocompatible hydrogels. We further applied them as cell culture substrate in vitro. In this study, HT-29 cells were cultured on the 3D AMHs, and cell viability and protein expression were investigated. Real time-PCR and Western-blot were carried out to determine the expression of a series of proteins associated with metastasis. Results revealed that cell growth was significantly decreased in AMHs group compared with controls. Meanwhile, microenvironment greatly affected the protein expression of the cells within. The CD36 expression in the AMHs group was ∼1.5-fold higher than in the control group. Moreover, The Western blot analysis demonstrated that the level of CD36 was higher in the AMHs group than in the 2D group. Besides, it was found that the expressions of N-cadherin and Snail in the HT29 cells were highly improved in the AMHs. As for the expression of ß-catenin and E-cadherin, an obvious decrease was seen in the AMHs than in the 2D group. Our results demonstrated that the elevated expression of CD36 in the HT29 cells, which were cultured on 3D AMHs, was associated with epithelial-mesenchymal transition (EMT) induction. The results further indicated the importance of 3D AMHs cell culture platform in vitro.

Tamoxifen Attenuates Lipopolysaccharide/Galactosamine-induced Acute Liver Failure by Antagonizing Hepatic Inflammation and Apoptosis.

Bacterial lipopolysaccharide (LPS)-induced acute liver failure (ALF) is a common severe clinical syndrome in intensive care unit. No other methods are available for its prevention apart from supportive treatment and liver transplantation. Tamoxifen (TAM) was reported to attenuate ALF induced by excessive acetaminophen, while its effect on LPS-induced ALF remained unknown. For this, in the present study, we comprehensively assessed whether TAM can attenuate ALF induced by LPS/galactosamine (GaIN). Mice were given TAM once a day for three times. Twelve hours after the last treatment, mice were given LPS/GaIN (intraperitoneally [i.p.]). Survival, plasma transaminases, and histopathology were examined. Serum TNF-α and IL-1ß were analyzed by ELISA. Hepatic apoptosis was analyzed by TUNEL and caspase-3 Western blotting, respectively. Compared to the model group, ALF induced by LPS/GaIN was alleviated remarkably following TAM administration, as evidenced by the improvement of survival (87.5% vs. 37.5%), hepatic swell, moderate transaminases, slightly increased serum TNF-α, IL-1ß (P < 0.05), and moderate histopathology. In respect of apoptosis, severe hepatocellular apoptosis was reduced notably by TAM treatment confirmed by less TUNEL-positive hepatocytes and decreased caspase-3 cleavage. The results demonstrated that TAM could attenuate LPS/GaIN-induced ALF effectively, probably due to hepatic inflammation and apoptosis antagonism. Furthermore, it was the first report about the effect of TAM on LPS/GaIN-induced ALF.