Isavuconazole Induces Neurodevelopment Defects and Motor Behaviour Impairment in Zebrafish Larvae.

Isavuconazole is a broad-spectrum antifungal drug used for the treatment of serious infections caused by invasive aspergillosis and mucormycosis in adults. With the continuous use of this drug, its safety and environmental impact have received increasing attention. However, information on the adverse effects of the drug is very limited. Fish is a particularly important model for assessing environmental risks. In this study, the aquatic vertebrate zebrafish was used as a model to study the toxic effects and mechanisms of isavuconazole. We exposed zebrafish embryos to 0.25, 0.5, and 1 mg/L of isavuconazole 6 h after fertilization. The results showed that at 72 hpf, isavuconazole exposure reduced heart rate, body length, and survival of zebrafish embryos compared to controls. Secondly, when isavuconazole reached a certain dose level (0.25 mg/L), it caused morphological changes in the Tg(elavl3:eGFP) transgenic fish line, with the head shrunk, the body bent, the fluorescence intensity becoming weaker, the abnormal motor behaviour, etc. At the same time, exposure of zebrafish embryos to isavuconazole downregulated acetylcholinesterase (AchE) and adenosine triphosphate (ATPase) activities but upregulated oxidative stress, thereby disrupting neural development and gene expression of neurotransmitter pathways. In addition, astaxanthin partially rescued the neurodevelopmental defects of zebrafish embryos by downregulating oxidative stress. Thus, our study suggests that isavuconazole exposure may induce neurodevelopment defects and behavioural disturbances in larval zebrafish.

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.

Hexafluoropropylene oxide trimer acid, a perfluorooctanoic acid alternative, induces cardiovascular toxicity in zebrafish embryos.

As an increasingly used alternative to perfluorooctanoic acid (PFOA), hexafluoropropylene oxide trimer acid (HFPO-TA) has been widely detected in global water environments. However, little is known regarding its toxic effects on cardiovascular development. Here, zebrafish embryos were treated with egg water containing 0, 60, 120, or 240 mg/L HFPO-TA. Results showed that HFPO-TA treatment led to a significant reduction in both larval survival percentage and heart rate. Furthermore, HFPO-TA exposure caused severe pericardial edema and elongation of the sinus venous to bulbus arteriosus distance (SV-BA) in Tg (myl7: GFP) transgenic larvae, disrupting the expression of genes involved in heart development and thus causing abnormal heart looping. Obvious sprouting angiogenesis was observed in the 120 and 240 mg/L exposed Tg (fli: GFP) transgenic larvae. HFPO-TA treatment also impacted the mRNA levels of genes involved in the vascular endothelial growth factor (VEGF) pathway and embryonic vascular development. HFPO-TA exposure significantly decreased erythrocyte number in Tg (gata1: DsRed) transgenic embryos and influenced gene expression associated with the heme metabolism pathway. HFPO-TA also induced oxidative stress and altered the transcriptional levels of genes related to cell cycle and apoptosis, inhibiting cell proliferation while promoting apoptosis. Therefore, HFPO-TA exposure may induce abnormal development of the cardiovascular and hematopoietic systems in zebrafish embryos, suggesting it may not be a suitable or safe alternative for PFOA.

Sanguinarine exposure induces immunotoxicity and abnormal locomotor behavior in zebrafish.

Sanguinarine (C20H14NO4+), a plant alkaloid and pesticide, works well a fungicidal and insecticidal applications. The prospect that sanguinarine may have potentially toxic effects on aquatic organisms has been brought to light by its use in agriculture. The first evaluation of the immunotoxic and behavioral effects of sanguinarine exposure on larval zebrafish was done in this work. Firstly, zebrafish embryos exposed to sanguinarine had shorter body length, larger yolk sacs, and slower heart rates. Secondly, the number of innate immune cells was significantly reduced. Thirdly, alterations in locomotor behavior were observed as exposure concentrations increased. Total distance travelled, travel time, and mean speed were all reduced. We also found significant changes in oxidative stress-related indicators and a significant increase in apoptosis in the embryos. Further studies revealed aberrant expression of some key genes in the TLR immune signaling pathway including CXCL-c1c, IL8, MYD88, and TLR4. At the same time, the expression of the pro-inflammatory cytokine IFN-γ was upregulated. To sum up, our results suggest that sanguinarine exposure may cause immunotoxicity and aberrant behavior in larval zebrafish.

Pexidartinib hydrochloride exposure induces developmental toxicity and immunotoxicity in zebrafish embryos via activation of Wnt signaling.

Pexidartinib, a macrophage colony-stimulating factor receptor (CSF-1R) inhibitor, is indicated for the treatment of tendon sheath giant cell tumor (TGCT). However, few studies on the toxicity mechanisms of pexidartinib for embryonic development. In this study, the effects of pexidartinib on embryonic development and immunotoxicity in zebrafish were investigated. Zebrafish embryos at 6 h post fertilization (6 hpf) were exposed to 0, 0.5, 1.0, and 1.5 µM concentrations of pexidartinib, respectively. The results showed that different concentrations of pexidartinib induced the shorter body, decreased heart rate, reduced number of immune cells and increase of apoptotic cells. In addition, we also detected the expression of Wnt signaling pathway and inflammation-related genes, and found that these genes expression were significantly upregulated after pexidartinib treatment. To test the effects of embryonic development and immunotoxicity due to hyperactivation of Wnt signaling after pexidartinib treatment, we used IWR-1, Wnt inhibitor, for rescue. Results show that IWR-1 could not only rescue developmental defects and immune cell number, but also downregulate the high expression of Wnt signaling pathway and inflammation-related caused by pexidartinib. Collectively, our results suggest that pexidartinib induces the developmental toxicity and immunotoxicity in zebrafish embryos through hyperactivation of Wnt signaling, providing a certain reference for the new mechanisms of pexidartinib function.

Hexafluoropropylene oxide trimer acid (HFPO-TA) disturbs embryonic liver and biliary system development in zebrafish.

Hexafluoropropylene oxide trimer acid (HFPO-TA), a novel alternative to perfluorooctanoic acid (PFOA), has emerged as a potential environmental pollutant. Here, to investigate the toxic effects of HFPO-TA on liver and biliary system development, zebrafish embryos were exposed to 0, 50, 100, or 200 mg/L HFPO-TA from 6 to 120 h post-fertilization (hpf). Results showed that the 50 % lethal concentration (LC50) of HFPO-TA was 231 mg/L at 120 hpf, lower than that of PFOA. HFPO-TA exposure decreased embryonic hatching, survival, and body length. Furthermore, HFPO-TA exerted higher toxicity at the specification stage than during the differentiation and maturation stages, leading to small-sized livers in Tg(fabp10a: DsRed) transgenic larvae and histopathological changes. Significant decreases in the mRNA expression of genes related to liver formation were observed. Alanine transaminase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and direct bilirubin (DBIL) levels were significantly increased. HFPO-TA decreased total cholesterol (TCHO) and triglyceride (TG) activities, disturbed lipid metabolism through the peroxisome proliferator-activated receptor (PPAR) pathway, and induced an inflammatory response. Furthermore, HFPO-TA inhibited intrahepatic biliary development in Tg(Tp1:eGFP) transgenic larvae and interfered with transcription of genes associated with biliary duct development. HFPO-TA reduced bile acid synthesis but increased bile acid transport, resulting in disruption of bile acid metabolism. Therefore, HFPO-TA influenced embryonic liver and biliary system morphogenesis, caused liver injury, and may be an unsafe alternative for PFOA.

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.

Cysteamine affects skeletal development and impairs motor behavior in zebrafish.

Cysteamine is a kind of feed additive commonly used in agricultural production. It is also the only targeted agent for the treatment of cystinosis, and there are some side effects in clinical applications. However, the potential skeletal toxicity remains to be further elucidated. In this study, a zebrafish model was for the first time utilized to synthetically appraise the skeletal developmental defects induced by cysteamine. The embryos were treated with 0.35, 0.70, and 1.05 mM cysteamine from 6 h post fertilization (hpf) to 72 hpf. Substantial skeletal alterations were manifested as shortened body length, chondropenia, and abnormal somite development. The results of spontaneous tail coiling at 24 hpf and locomotion at 120 hpf revealed that cysteamine decreased behavioral abilities. Moreover, the level of oxidative stress in the skeleton ascended after cysteamine exposure. Transcriptional examination showed that cysteamine upregulated the expression of osteoclast-related genes but did not affect osteoblast-related genes expression. Additionally, cysteamine exposure caused the downregulation of the Notch signaling and activating of Notch signaling partially attenuated skeletal defects. Collectively, our study suggests that cysteamine leads to skeletal developmental defects and reduces locomotion activity. This hazard may be associated with cysteamine-mediated inhibition of the Notch signaling and disorganization of notochordal cells due to oxidative stress and apoptosis.

Transcriptome analysis of 3D primary mouse liver spheroids shows that long-term exposure to hexafluoropropylene oxide trimer acid disrupts hepatic bile acid metabolism.

Hexafluoropropylene oxide trimer acid (HFPO-TA), an alternative to perfluorooctanoic acid (PFOA), has been detected in various environmental and human matrices. However, information regarding its toxicity remains limited. Here, we established a three-dimensional (3D) primary mouse liver spheroid model to compare the hepatotoxicity of HFPO-TA and PFOA. The 3D spheroids were repeatedly exposed to 25-, 50-, or 100-µM HFPO-TA and PFOA for 28 d. Compared with the PFOA groups, the HFPO-TA groups showed higher bioaccumulation potential, higher lactate dehydrogenase (LDH) leakage, and lower adenosine triphosphate (ATP), albumin, and urea secretion. Transcriptome analysis identified 1603 and 772 differentially expressed genes in the 100-µM HFPO-TA- and PFOA-treated groups, respectively. Bioinformatics analysis indicated that cholesterol metabolism, bile acid metabolism, and inflammatory response were significantly altered. Exposure to 100-µM HFPO-TA increased triglyceride content but decreased total cholesterol content, while no changes were observed in the 100-µM PFOA-treated group. Total bile acids in the re-polarized 3D spheroids increased significantly after 100-µM HFPO-TA and PFOA treatment, which did not affect bile acid synthesis but inhibited the expression levels of Bsep and Mrp2 related to bile acid transport. Thus, HFPO-TA exhibited more serious hepatotoxicity than PFOA in 3D primary liver spheroids and may not be a safe alternative.

Hepatotoxicity of perfluorooctanoic acid and two emerging alternatives based on a 3D spheroid model.

Perfluorooctanoic acid (PFOA) toxicity is of considerable concern due to its wide application, environmental persistence, and bioaccumulation. In the current study, we used a scaffold-free three-dimensional (3D) spheroid model of mouse liver cells (AML12) to explore the toxicity of PFOA and emerging alternatives (HFPO-DA and PFO4DA). Comparing the short-term (24 and 72 h treatment) toxicity of PFOA between conventional 2D monolayer cells and 3D spheroids, we found that spheroids had higher EC50 values and lower ROS levels after treatment, indicating their greater resistance to PFOA. Cell viability (i.e., adenosine triphosphate (ATP) content and lactate dehydrogenase (LDH) leakage) and liver-specific function (i.e., albumin secretion) were stable in spheroids through 28 day of culture. However, under 100 and 200 µM-PFOA treatment for 28 day, ROS levels, LDH leakage, and caspase3/7 activity all increased significantly. As a sensitive parameter, ROS showed a significant increase at 21 day, even in the 50 µM-PFOA group. Consistent with the elevation of ROS and caspase3/7, the expressions of oxidative stress- and apoptosis-related genes, including Gsta2, Nqo1, Ho-1, caspase3, p53, and p21, were induced in dose- and time-dependent manners after PFOA exposure. The peroxisome proliferator-activated receptor alpha (PPARα) pathway was also activated after treatment, with significant induction of its target genes, Fabp4 and Scd1. Similar to PFOA, both HFPO-DA and PFO4DA activated the PPARα pathway, induced ROS levels, and initiated cell damage, though at a relatively lower extent than that of PFOA. Our results imply that the 3D spheroid model is a valuable tool in chronic toxicological studies.

Comparative Hepatotoxicity of Novel PFOA Alternatives (Perfluoropolyether Carboxylic Acids) on Male Mice.

As novel alternatives to perfluorooctanoic acid (PFOA), perfluoropolyether carboxylic acids (multiether PFECAs, CF3(OCF2) nCOO-, n = 2-4) have been detected in various environmental matrices; however, public information regarding their toxicities remains unavailable. To compare the hepatotoxicity of multiether PFECAs (e.g., PFO2HxA, PFO3OA, and PFO4DA) with PFOA, male mice were exposed to 0.4, 2, or 10 mg/kg/d of each chemical for 28 d, respectively. Results demonstrated that PFO2HxA and PFO3OA exposure did not induce marked increases in relative liver weight; whereas 2 and 10 mg/kg/d of PFO4DA significantly increased relative liver weight. Furthermore, PFO2HxA and PFO3OA demonstrated almost no accumulation in the liver or serum; whereas PFO4DA was accumulated but with weaker potential than PFOA. Exposure to 10 mg/kg/d of PFO4DA led to 198 differentially expressed liver genes (56 down-regulated, 142 up-regulated), with bioinformatics analysis highlighting the urea cycle disorder. Like PFOA, 10 mg/kg/d of PFO4DA decreased the urea cycle-related enzyme protein levels (e.g., carbamoyl phosphate synthetase 1) and serum ammonia content in a dose-dependent manner. Both PFOA and PFO4DA treatment (highest concentration) caused a decrease in glutamate content and increase in both glutamine synthetase activity and aquaporin protein levels in the brain. Thus, we concluded that PFO4DA caused hepatotoxicity, as indicated by hepatomegaly and karyolysis, though to a lesser degree than PFOA, and induced urea cycle disorder, which may contribute to the observed toxic effects.

Corticosteroid-binding globulin, induced in testicular Leydig cells by perfluorooctanoic acid, promotes steroid hormone synthesis.

Perfluorooctanoic acid (PFOA) is an abundant perfluoroalkyl substance widely applied in industrial and consumer products. It is a ubiquitous environmental pollutant and suspected endocrine disruptor. Corticosteroid-binding globulin (CBG) is a monomeric glycoprotein that can bind specifically to anti-inflammatory steroids, such as glucocorticoids and progesterone, in circulation. Our previous proteomic profile analysis revealed that CBG levels increased in testes after PFOA treatment. In the present study, we verified its increase in mouse testes following oral exposure to PFOA (0, 1.25 and 5 mg/kg/day for 28 days) by immunohistochemical analysis and Western blotting. In addition, RNA fluorescence in situ hybridization (FISH) confirmed that testicular CBG was specifically expressed in Leydig cells. Serum CBG levels in all three PFOA groups also increased, accompanied by increased corticosterone in the 5 and 20 mg/kg/day groups and decreased adrenocorticotropic hormone in the 20 mg/kg/day group. Thus, the influence of PFOA on blood CBG may change free steroid hormone concentrations, thereby serving as an endocrine disruptor. A stimulation effect of PFOA on CBG was also observed in vitro using the Leydig tumor mLTC-1 cell line. Overexpression of CBG in mLTC-1 cells increased progesterone release in culture media. In addition, CBG-induced proteins involved in steroidogenesis in mLTC-1 cells, including steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (CYP11A1), 17α-hydroxylase/17,20 lyase (CYP17A1), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD), which may be the mechanism behind increased progesterone. Furthermore, the production and release of CBG in mLTC-1 cells were also induced by luteinizing hormone, though this mechanism requires further exploration.

6:2 Chlorinated polyfluorinated ether sulfonate, a PFOS alternative, induces embryotoxicity and disrupts cardiac development in zebrafish embryos.

As an alternative to perfluorooctanesulfonate (PFOS), 6:2 chlorinated polyfluorinated ether sulfonate (commercial name: F-53B) has been used as a mist suppressant in Chinese electroplating industries for over 30 years. It has been found in the environment and fish, and one acute assay indicated F-53B was moderately toxic. However, the toxicological information on this compound was incomplete and insufficient for assessment of their environment impact. The object of this study was to examine the developmental toxicity of F-53B using zebrafish embryos. Zebrafish embryos were incubated in 6-well plates with various concentrations of F-53B (1.5, 3, 6, and 12mg/L) from 6 to 132h post fertilization (hpf). Results showed that F-53B exposure induced developmental toxicity, including delayed hatching, increased occurrence of malformations, and reduced survival. Malformations, including pericardial and yolk sac edemas, abnormal spines, bent tails, and uninflated swim bladders, appeared at 84 hpf, and increased with time course and dose. A decrease in survival percentages was noted in the 6 and 12mg/L F-53B-treated groups at 132 hpf. Continuous exposure to 3mg/L F-53B resulted in high accumulation levels in zebrafish embryos, suggesting an inability for embryos to eliminate this compound and a high cumulative risk to fish. We also examined the cardiac function of embryos at specific developmental stages following exposure to different concentrations, and found that F-53B induced cardiac toxicity and reduced heart rate. Even under low F-53B concentration, o-dianisidine staining results showed significant decrease of relative erythrocyte number at 72 hpf before the appearance of observed effects of F-53B on the heart. To elucidate the underlying molecular changes, genes involved in normal cardiac development were analyzed using real-time qPCR in the whole-body of zebrafish embryos. F-53B inhibited the mRNA expression of ß-catenin (ctnnb2) and wnt3a. The mRNA levels of ß-catenin targeted genes (nkx2.5 and sox9b), which play critical roles in cardiogenesis, were also reduced after exposure. Thus, exposure to F-53B impaired the development of zebrafish embryos and disrupted cardiac development, which might be mediated by effects on the Wnt signaling pathway and decrease of erythrocyte numbers.