Zebrafish Embryonic Development Research Articles

Zrsr2 Is Essential for the Embryonic Development and Splicing of Minor Introns in RNA and Protein Processing Genes in Zebrafish.

ZRSR2 (zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2) is an essential splicing factor involved in 3′ splice-site recognition as a component of both the major and minor spliceosomes that mediate the splicing of U2-type (major) and U12-type (minor) introns, respectively. Studies of ZRSR2-depleted cell lines and ZRSR2-mutated patient samples revealed its essential role in the U12-dependent minor spliceosome. However, the role of ZRSR2 during embryonic development is not clear, as its function is compensated for by Zrsr1 in mice. Here, we utilized the zebrafish model to investigate the role of zrsr2 during embryonic development. Using CRISPR/Cas9 technology, we generated a zrsr2-knockout zebrafish line, termed zrsr2hg129/hg129 (p.Trp167Argfs*9) and examined embryo development in the homozygous mutant embryos. zrsr2hg129/hg129 embryos displayed multiple developmental defects starting at 4 days post fertilization (dpf) and died after 8 dpf, suggesting that proper Zrsr2 function is required during embryonic development. The global transcriptome analysis of 3 dpf zrsr2hg129/hg129 embryos revealed that the loss of Zrsr2 results in the downregulation of essential metabolic pathways and the aberrant retention of minor introns in about one-third of all minor intron-containing genes in zebrafish. Overall, our study has demonstrated that the role of Zrsr2 as a component of the minor spliceosome is conserved and critical for proper embryonic development in zebrafish.

Ethalfluralin induces developmental toxicity in zebrafish via oxidative stress and inflammation

Ethalfluralin, of dinitroaniline herbicide family, is an effective weed controller. Following residue detection in herbicide-treated fields, ethalfluralin was reported to interfere with early stages of implantation in some vertebrate species. However, the role of ethalfluralin in the development of zebrafish embryos has not been elucidated yet. Therefore, in the present study, we investigated the morphological and physiological changes that occur in the embryonic development of zebrafish due to ethalfluralin exposure. Results indicated that ethalfluralin decreased survival rate along with reduction in the hatching ratio and heartbeat. It was observed to cause edema in the heart and yolk sac, and apoptosis in the anterior region of the developing zebrafish larvae; as visualized through acridine orange and TUNEL staining. In addition, ethalfluralin increased the expression of the apoptosis-associated genes including tp53, cyc1, casp8, casp9, and casp3. The Seahorse Mito Stress analysis revealed that ethalfluralin slightly reduced mitochondrial respiration in live zebrafish embryos. Reactive oxygen species (ROS) production was also observed to be elevated in zebrafish larvae in response to ethalfluralin. Treatment with ethalfluralin decreased blood vessel formation in brain and intestine in flk1 transgenic zebrafish embryos. The decrease in angiogenesis related gene expression was specifically observed in vegfc, flt1, and kdrl, and in the intestinal vasculature related genes apoa4a, aqp3, fabp2, and vil1. Moreover, an increase in inflammatory genes such as cox2a, cox2b, cxcl-c1c, il8, mcl1a, mcl1b, and nf-κb was observed using real-time PCR analysis. Collectively, these results indicate that oxidative stress generated by exposure to ethalfluralin induced ROS generation, apoptosis, inflammation and anti-angiogenic effects, and therefore, ethalfluralin may be toxic to the development of zebrafish embryos.

Patterns and Crucial Regulation of Alternative Splicing During Early Development in Zebrafish

Many vertebrate genes generate multiple transcript variants that may encode functionally distinct protein isoforms, but the transcriptomes of various developmental stages are poorly defined. Identifying the transcriptome and its regulation during the normal developmental process is the key to deciphering the developmental stage-specific functions of genes. Here we presented a systematic assessment of the temporal alternative splicing (AS) events during the critical development stages to capture the dynamic gene expression changes and AS in zebrafish. An unexpected transcriptome complexity generated by AS was observed during zebrafish development. The patterns of AS events varied substantially among developmental stages despite the similarities in the total proportion of AS genes. We further found that AS afforded substantial functional diversification of genes through the generation of stage-specific AS events from broadly protein-coding genes as an essential developmental regulatory mechanism. Skipped exon (SE) showed the strongest signals among developmental AS (devAS), suggesting that devAS events generated by SE may be necessary for the normal development of zebrafish. Most developmental genes regulated by AS mechanisms were not modulated in terms of their overall expression levels, indicating that AS shaped the transcriptome independently from transcriptional regulation during development. 128-cell stage was a critical stage for gene transcription during embryonic development. Splicing factors as an essential developmental regulator underwent AS in the potential autoregulatory feedback loop and expressed multiple isoforms. Thus, zebrafish development was shaped by an interplay of programs controlling gene expression levels and AS. Overall, we provided a global view of developmental patterns of AS during zebrafish development and revealed that AS transitions were the crucial regulatory component of zebrafish embryonic development.

Mixture toxicity of cadmium and acetamiprid to the early life stages of zebrafish (Danio rerio)

Aquatic organisms are often exposed to contaminants that occur in the natural environment. Nevertheless, the toxic effects of chemical combinations on aquatic animals and their underlying toxic mechanisms for dealing with such exposures are still not fully understood. In this study, we investigated the combined effects of cadmium (Cd) and acetamiprid (ACE) on zebrafish (Danio rerio) using various endpoints. Cd exhibited a 96-h LC50 value of 4.77 mg a.i. L−1 against zebrafish embryos, which was lower than that of ACE (152.6 mg a.i. L−1). In contrast, the 96-h LC50 value of the mixture of Cd and ACE was 157.4 mg a.i. L−1. The mixture of Cd and ACE had a synergetic effect on the organisms. The activities of T-SOD, POD, and CarE were significantly changed in most exposures compared with the control group. In addition, five genes (TRα, crh, Tnf, IL, and P53) involved in oxidative stress, cellular apoptosis, the immune system, and the endocrine system exhibited more remarkable changes when exposed to chemical mixtures relative to their individual counterparts, demonstrating variations in the cellular and mRNA expression levels induced by the mixture exposure of ACE and Cd during the embryonic development of zebrafish. Therefore, these results indicated that the combined pollution of ACE and Cd could be a potentially hazardous factor, and further investigation is necessary for the safety evaluation and application of ACE. Moreover, further investigation on the combined toxicities of various chemicals must be performed to determine the chemical mixtures with synergistic responses.

Perfluorononanoate and Perfluorobutane Sulfonate Induce Cardiotoxic Effects in Zebrafish.

Globally, per- and polyfluoroalkyl substances are common artificial ingredients in industrial and consumer products. Recently, they have been shown to be an emerging human health risk. Perfluorononanoic acid (PFNA)/perfluorononanoate and perfluorobutane sulfonic acid (PFBS)/perfluorobutane sulfonate cause reproductive toxicity and hepatotoxicity, disrupt thyroid functions, and damage embryonic development in zebrafish. However, the cardiotoxic effects of PFNA and PFBS have not been fully established. We found that PFNA and PFBS exposures repress hatchability while increasing malformation and mortality in zebrafish embryos. Hematoxylin and eosin staining as well as assessment of the transgenic zebrafish line Tg(myl7:nDsRed) revealed that exposure of embryos to PFNA increases the occurrence of severe cardiac malformations relative to exposure to PFBS. Moreover, we evaluated the differential expressions of cardiac development-associated genes in response to PFNA and PFBS, which validated the potential cardiotoxic effects, consistent with cardiac dysfunctions. Overall, our findings reveal novel cardiotoxic effects of PFNA and PFBS in zebrafish, implying that they may exert some cardiotoxic effect in humans. To the best of our knowledge, ours is the first study to show that PFNA exerts more severe cardiotoxic effects in zebrafish when compared with PFBS. Based on these findings, studies should evaluate the mechanisms of their cardiotoxic effects. Environ Toxicol Chem 2022;41:2527-2536. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Bioreactor-grown exo- and endo-β-glucan from Malaysian Ganoderma lucidum: An in vitro and in vivo study for potential antidiabetic treatment.

This study aims to identify the roles of exo-β-glucan (EPS-BG) and endo-β-glucan (ENS-BG) extracted from Ganoderma lucidum (GL) in inhibiting the alpha-glucosidase enzyme, a target mechanism for postprandial hyperglycaemia regulation. Upscale production of GL was carried out using a 10 L bioreactor. The zebrafish embryo toxicity test (ZFET) was carried out based on OECD guidelines. The hatching rate, survival rate, heart rate, morphological malformation, and teratogenic defects were observed and determined every 24 h from 0–120 h of post-exposure (hpe). For diabetes induction, adult zebrafish (3–4 months of age) were overfed and induced with three doses of 350 mg/kg streptozotocin (STZ) by intraperitoneal injection (IP) on three different days (days 1, 3, and 5). The oral sucrose tolerance test (OSTT) and anti-diabetic activity of EPS-BG and ENS-BG were evaluated (day 7) using the developed model (n = 15). This study showed that EPS is the most potent compound with the highest inhibitory effect toward the alpha-glucosidase enzyme with an IC50 value of 0.1575 mg/ml compared to ENS extracts (IC50 = 0.3479 mg/ml). Both EPS-BG and ENS-BG demonstrated a strong inhibition of alpha-glucosidase activity similar to the clinically approved alpha-glucosidase inhibitor, acarbose (IC50 = 0.8107 mg/ml). ENS-BG is non-toxic toward zebrafish embryos with LC50 of 0.92 mg/ml and showed no significant changes in ZE hatching and normal heart rate as compared to untreated embryos (161 beats/min). Teratogenic effects of ENS-BG (<1.0 mg/ml) on zebrafish embryonic development were not observed. The DM model of zebrafish was acquired after the third dose of STZ with a fasting BGL of 8.98 ± 0.28 mmol/L compared to the normal healthy group (4.23 ± 0.62 mmol/L). The BGL of DM zebrafish after 30 min treated with EPS-BG and ENS-BG showed a significant reduction (p < 0.0001). Both EPS-BG and ENS-BG significantly reduced DM zebrafish’s peak blood glucose and the area under the curve (AUC) in OSTT. Hence, EPS-BG and ENS-BG extracted from GL showed promising inhibition of the alpha-glucosidase enzyme and are considered non-toxic in ZE. Moreover, EPS-BG and ENS-BG reduced blood glucose levels and inhibited hyperglycemia in DM zebrafish.

Lead Exposure Causes Spinal Curvature during Embryonic Development in Zebrafish.

Lead (Pb) is an important raw material for modern industrial production, they enter the aquatic environment in several ways and cause serious harm to aquatic ecosystems. Lead ions (Pb2+) are highly toxic and can accumulate continuously in organisms. In addition to causing biological deaths, it can also cause neurological damage in vertebrates. Our experiment found that Pb2+ caused decreased survival, delayed hatching, decreased frequency of voluntary movements at 24 hpf, increased heart rate at 48 hpf and increased malformation rate in zebrafish embryos. Among them, the morphology of spinal malformations varied, with 0.4 mg/L Pb2+ causing a dorsal bending of the spine of 72 hpf zebrafish and a ventral bending in 120 hpf zebrafish. It was detected that spinal malformations were mainly caused by Pb2+-induced endoplasmic reticulum stress and apoptosis. The genetic changes in somatic segment development which disrupted developmental polarity as well as osteogenesis, resulting in uneven myotomal development. In contrast, calcium ions can rescue the series of responses induced by lead exposure and reduce the occurrence of spinal curvature. This article proposes new findings of lead pollution toxicity in zebrafish.

Lamprey immune protein triggers the ferroptosis pathway during zebrafish embryonic development

BackgroundPreviously, a novel lamprey immune protein (LIP) was identified, which plays an important role in immunity and the regulation of growth and development in lampreys. However, the mechanism of how LIP regulates growth and development remains unclear.MethodsIn this study, a zebrafish model of LIP overexpression was established by delivering a transgenic plasmid to the fertilized egg. The biological function of LIP was explored in vivo through phenotypic characterization, comparative transcriptome sequencing, and physiological and biochemical analyses.ResultsLIP caused developmental toxicity in zebrafish, increased embryo mortality and exhibited strong teratogenic, lethal, and developmental inhibitory effects. Comparative transcriptome analysis showed that LIP-induced large-scale cell death by triggering ferroptosis. Furthermore, LIP-induced lipid peroxidation and caused pericardial edema. Direct inhibition of acsl4a and tfr1a, or silencing of acsl4a and tfr1a with specific siRNA suppressed ferroptosis and pericardial edema.ConclusionsTaken together, we confirmed that LIP can participate in growth and development via the regulation of lipid peroxidation and ferroptosis. This lays the foundation for future studies on the function of LIP in lampreys.FULq97gHbZiSwv22VJ3jSJVideo

Comparing different surface modifications of zinc oxide nanoparticles in the developmental toxicity of zebrafish embryos and larvae

Nanotechnology allows for a greater quality of life, but may also cause environmental and organismic harm. Zinc oxide nanoparticles (ZnONPs) are one of the most commonly used metal oxide nanoparticles for commercial and industrial products. Due to its extensive use in various fields, there has already been much concern raised about the environmental health risks of ZnONPs. Many studies have investigated the toxicological profile of ZnONPs in zebrafish embryonic development; however, the specific characteristics of ZnONPs in zebrafish embryonic/larval developmental damage and their molecular toxic mechanisms of liver development are yet to be fully elucidated. This study aimed to reveal the hazard ranking of different surface modifications of ZnONPs on developing zebrafish and the toxicological mechanisms of these modified ZnONPs in liver tissue. The ~30 nm ZnONPs with amino- (NH2- ZnONPs) or carboxyl- (COOH-ZnONPs) modification were incorporated during the embryonic/larval stage of zebrafish. Severe toxicity was observed in both ZnONP groups, especially NH2-ZnONPs, which presented a higher toxicity in the low concentration groups. After prolonging the exposure time, the long-term toxicity assay showed a greater retardation in body length of zebrafish in the NH2-ZnONP group. Response data from multiple toxicity studies was integrated for the calculation of the EC50 values of bulk ZnO and ZnONPs, and the hazard levels were found to be decreasing in the order of NH2-, COOH-ZnONPs and bulk ZnO. Notably, NH2-ZnONPs induced ROS burden in the developing liver tissue, which activated autophagy-related gene and protein expression and finally induced liver cell apoptosis to reduce liver size. In conclusion, our findings are conducive to understanding the hazard risks of different surface modifications of ZnONPs in aquatic environments and will also be helpful for choosing the type of ZnONPs in future industrial applications.

Biocompatibility Study of Curcumin-Loaded Pluronic F127 Nanoformulation (NanoCUR) against the Embryonic Development of Zebrafish (Danio rerio).

Curcumin (CUR) has been studied for its biomedical applications due to its active biological properties. However, CUR has limitations such as poor solubility, low bioavailability, and rapid degradation. Thus, CUR was nanoformulated with the application of polymeric micelle. Previous studies of CUR-loaded Pluronic F127 nanoformulation (NanoCUR) were generally prioritized toward cancer cells and its therapeutic values. There are reports that emphasize the toxicity of CUR, but reports on the toxicity of NanoCUR on embryonic developmental stages is still scarce. The present study aims to investigate the toxicity effects of NanoCUR on the embryonic development of zebrafish (Danio rerio). NanoCUR was synthesized via thin film hydration method and then characterized using DLS, UV-Vis, FTIR, FESEM, and XRD. The toxicity assessment of NanoCUR was conducted using zebrafish embryos, in comparison to native CUR, as well as Pluronic F127 (PF) as the controls, and ROS assay was further carried out. It was revealed that NanoCUR showed an improved toxicity profile compared to native CUR. NanoCUR displayed a delayed toxicity response and showed a concentration- and time-dependent toxicity response. NanoCUR was also observed to generate a significantly low reactive oxygen species (ROS) compared to native CUR in ROS assay. Overall, the results obtained highlight the potential of NanoCUR to be developed in clinical settings due to its improved toxicity profile compared to CUR.

Cytotoxicity of lycopene-mediated silver nanoparticles in the embryonic development of zebrafish-An animal study.

Nano-silver (AgNP) has biological properties which are significant for consumer products, food technology, and medical applications (e.g., wound care products, implantable medical devices, in diagnosis, drug delivery, and imaging). Thus, the use of nano-silver is becoming more and more widespread in medicine. However, the effect of these AgNPs on zebrafish remains unclear. Whereas, lycopene, a fat-soluble carotenoid acts as a potent antioxidant has proved its major role in treating many oral health problems such as lichen planus, and periodontitis. Usage of these lycopene AgNPs would yield better results in wound healing. In this study, we investigated the cytotoxic effects of AgNPs on fibroblasts cells of humans.To investigate the toxic effects of the lycopene-mediated AgNPs on the zebrafish (Danio rerio) and to examine these effects on the embryonic development of the fetus of this species.In this study, zebrafish embryos were treated with AgNPs 0.5, 1, 2, 3, 4 or 5 L nanoparticles/ml for 24 to 96 h post fertilization.Our results showed that with the increasing concentration of nanoparticles, there was a very mild toxic effect. Less toxic effects were observed with 1μl.Our results show that exposure to silver nanoparticles is less toxic to embryonic zebrafish at lower concentrations. The results will contribute to the current understanding of the potential biotoxic effects of nanoparticles and will aid in the safety assessment and synthesis of silver oxide nanoparticles.

Temporal Dynamic Analysis of Alternative Splicing During Embryonic Development in Zebrafish.

Alternative splicing is pervasive in mammalian genomes and involved in embryo development, whereas research on crosstalk of alternative splicing and embryo development was largely restricted to mouse and human and the alternative splicing regulation during embryogenesis in zebrafish remained unclear. We constructed the alternative splicing atlas at 18 time-course stages covering maternal-to-zygotic transition, gastrulation, somitogenesis, pharyngula stages, and post-fertilization in zebrafish. The differential alternative splicing events between different developmental stages were detected. The results indicated that abundance alternative splicing and differential alternative splicing events are dynamically changed and remarkably abundant during the maternal-to-zygotic transition process. Based on gene expression profiles, we found splicing factors are expressed with specificity of developmental stage and largely expressed during the maternal-to-zygotic transition process. The better performance of cluster analysis was achieved based on the inclusion level of alternative splicing. The biological function analysis uncovered the important roles of alternative splicing during embryogenesis. The identification of isoform switches of alternative splicing provided a new insight into mining the regulated mechanism of transcript isoforms, which always is hidden by gene expression. In conclusion, we inferred that alternative splicing activation is synchronized with zygotic genome activation and discovered that alternative splicing is coupled with transcription during embryo development in zebrafish. We also unveiled that the temporal expression dynamics of splicing factors during embryo development, especially co-orthologous splicing factors. Furthermore, we proposed that the inclusion level of alternative splicing events can be employed for cluster analysis as a novel parameter. This work will provide a deeper insight into the regulation of alternative splicing during embryogenesis in zebrafish.

Aquaporin 8ab is required in zebrafish embryonic intestine development.

The aquaporin 8 (AQP8) is a small integral membrane protein that selectively transports water and other small uncharged solutes across cell plasma membranes. It has been demonstrated that AQP8 is ubiquitously present in various tissues and organs of mammals, and participates in many physiological and pathological processes. Recent studies showed that AQP8 is highly expressed in the columnar epithelial cells of mammalian colonic mucosa facing lumen, indicating that AQP8 plays potential roles in the physiology and pathophysiology of gastrointestinal tract. However, the role of AQP8 during gastrointestinal tract development is unclear. In the present study, RT-PCR results reveal that the zebrafish genome encodes three kinds of aqp8s ( aqp8aa, aqp8ab, and aqp8b). We use whole mount in situ hybridization to describe aqp8 genes spatiotemporal expression pattern, and the results show that aqp8ab mRNA is detectable mainly in the zebrafish embryonic intestine. To reveal the details of aqp8ab distribution, histological sections are employed. Transverse sections indicate that aqp8ab mRNA expression is more intense in the layer lining the intestinal cavity. Knockout of aqp8ab using the CRISPR/Cas9 system induces intestine development defects and abnormal formation of intestinal lumen. In addition, aqp8ab mRNA significantly rescues the intestine defects in the aqp8ab mutant. These results indicate that aqp8ab is required in the intestine development of zebrafish.

Expression analysis of nel during zebrafish embryonic development

Nel is a multimeric extracellular glycoprotein which predominantly expressed in the nervous system and play an important role in neural development and functions. There are three nel paralogues included nell2a, nell2b, and nell3 in zebrafish, while systematic expression analysis of the nel family is still lacking. In this study, we performed a phylogenetic analysis on 7 species, in different species the nell2a are highly conserved, as is nell2b. Then, the expression profiles of nell2a, nell2b and nell3 were detected by in situ hybridization in zebrafish embryo, and the result showed that nel genes highly enriched in the central nervous system, but distributed in different regions of the brain. In addition, nell2a is also expressed in the olfactory pit, spinal cord, otic vesicle and retina (ganglion cell layer), nell2b was detected to express in gill arches, olfactory epithelium, olfactory pit, spinal cord, photoreceptor and retina (ganglion cell layer), it should be noted that the expression of nell3 is special, was only detected at 96 hpf in the brain and spinal cord of zebrafish. Overall, our results indicate that nell2a and nell2b genes are expressed in the nervous system and eyes of zebrafish embryo, while nell3 is expressed in different regions in the nervous system. The phylogenetic analysis also shows that nell3 sequences are significantly different from nell2a and nell2b. This study provides new evidence to better understand the role of nel in zebrafish embryo development.

Degradation of the nonylphenol aqueous solution by strong ionization discharge: evaluation of degradation mechanism and the water toxicity of zebrafish.

Nonylphenol (NP) is a typical environmental endogenous disrupter with low concentration and high toxicity. This paper describes the mechanism of NP degradation in solution by strong ionization dielectric barrier discharge (SIDBD). Furthermore, the degradation performance of NP by SIDBD was tested by changing the equipment voltage, the initial concentration of NP in aqueous solution, pH, and inorganic ions. Degradation pathways of NP were detected using a high-performance liquid chromatography-mass spectrometer. The biological effects of NP degradation were assessed by detecting indicators of embryonic development in zebrafish (survival rate, fetal movement, heartbeat, the body length, behavior, deformity) and adult fish (sex differentiation, weight, ovarian testes pathological section analysis). The results showed when the input O2 was 5 L/min and the voltage was 3.2 kV, the degradation efficiency of NP can reach 99.0% after 60 min of experiment. Equipment voltage, initial concentration of NP in solution, pH, inorganic ions and other factors can influence the degradation efficiency of NP by DBD. At the higher concentration of NP, the greater influence on embryonic development in zebrafish was noticed. Although the effects of NP on zebrafish sex differentiation were not obvious, it showed significant male weight inhibition and decrease in sperm number.

Kdm4aa is required for reproduction and development of zebrafish

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

Study on the preparation and activity of intelligent response poly(lactic-co-glycolic acid)-ss-polyethylene glycol copolymer micelles.

Amphiphilic polymer micellar carriers are the most commonly used nanocarriers for oral delivery of hydrophobic drugs because their hydrophilic shell can avoid the recognition of the reticuloendothelial system (RES), has excellent drug-carrying capacity, and protect the drug from inactivation in the gastrointestinal fluid. The polymer micelle shell can enter cancer cells by endocytosis, and autophagy in cells, degradation by lysosomal pathway, so as to release drugs, prolong the circulation time of drugs in vivo, and then achieve the effect of drug sustained release. In this study, the glutathione-responsive PLGA-ss-PEG loaded paclitaxel (PTX) micelles (PLGA-ss-PEG-PTX) were developed for anticancer therapy. With its long-term circulation and EPR (enhanced permeability and retention) effect, and the micelle had disulfide bond, which could be used as the recognition group of tumor microenvironment, so that the PLGA-ss-PEG-PTX could specifically accumulate at the tumor site, so as to produce better anti-tumor effect. The PLGA-ss-PEG-PTX was formulated by the emulsification method in this study. The drug loading was about 21.54%, the entrapment efficiency was about 94.2%, and the particle size range was about 90nm with narrow particle size distribution. Cytotoxicity and embryonic toxicity experiments were carried out using mouse lung cancer cells (LLC) and zebrafish fertilized eggs. It was proved that the low concentration of blank micelles had little cytotoxicity, but high concentration of blank micelles had adverse effects on zebrafish embryonic development, resulting in embryonic malformation. The uptake of drugs by cancer cells was studied by a high connotation cell imaging analysis system. The experiments showed that the drug molecules encapsulated in micelles could achieve higher uptake by cells compared with free drug molecules. In addition, in the in vivo evaluation experiment of drugs, the PLGA-ss-PEG-PTX could significantly enhance the therapeutic effect of the PTX, improve its water solubility, and improve its oral bioavailability.

Quizalofop-P-ethyl induced developmental toxicity and cardiotoxicity in early life stage of zebrafish (Danio rerio)

Quizalofop-P-ethyl (QpE), a highly efficient selective herbicide, has good control effect on annual and perennial weeds. However, its excessive use will pose a threat to the ecological environment. QpE has been proven harmful to aquatic organisms, but there is little evidence on the adverse effects of QpE in the early life of aquatic organisms. In this work, zebrafish (Danio rerio) embryos were treated with 0.10, 0.20, 0.30, 0.40, and 0.50 mg/L of QpE for 120 h. The findings revealed that the LC50 value of QpE to zebrafish embryos was 0.23 mg/L at 96 hpf. QpE exposure significantly increased the mortality rate, decreased the hatching rate and caused morphological defects during zebrafish embryonic development, with a concentration dependent manner. QpE also caused severe morphological changes in the cardiovascular system, as well as resulted in a dysfunction in cardiovascular performance. Meanwhile, both histopathological examination and neutrophil observations showed inflammatory response occurred in the heart. Furthermore, several genes associated with heart development and inflammation were significantly altered following QpE exposure. A protein-protein interaction (PPI) network analysis proved that there was a connection between the changed heart development-relevant and inflammation-related genes. Taken together, our findings suggest that QpE causes cardiotoxicity in zebrafish embryos by altering the expression of genes in the regulatory network of cardiac development, which might be aggravated by inflammatory reactions, thereby affecting embryo development. These findings generated here are useful for in-depth assessment of the effects of QpE on early development of aquatic organisms and providing theoretical foundation for risk management measures.

Transcriptome analysis of developing zebrafish (Danio rerio) embryo following exposure to Gaudichaudione H reveals teratogenicity and cardiovascular defects caused by abnormal iron metabolism

Gaudichaudione H (GH), a caged polyprenylated xanthone from Garcinia plants, showed anti-cancer and anti-inflammatory effects in vitro. However, the in vivo toxicity of this compound has never been reported. The present study was aimed to address the toxic effects of Gaudichaudione H using zebrafish embryos and larvae as an in vivo test model. The zebrafish embryos were treated with GH having different concentrations (0, 0.28, 0.38 and 0.57 μg/mL). The results revealed that GH induces significant embryonic mortality, decreased heartbeat, cardiotoxicity, cardiovascular defects, increased apoptosis and decreased hemoglobinization in zebrafish embryos and larvae. According to transcriptome analysis, 1841 genes were significantly differentially expressed (1185 down-regulated and 656 up-regulated) after GH treatment. The main functions of these genes were related to iron metabolism pathways. The toxicity of GH on zebrafish embryonic development and cardiovascular may due to large amounts of downregulated genes involved in metabolic pathways and DEGs related to ‘Iron ion binding’ and ‘Heme binding’ functions.

Exposure to diclofenac alters thyroid hormone levels and transcription of genes involved in the hypothalamic–pituitary–thyroid axis in zebrafish embryos/larvae

Diclofenac (DCF), one of typical non-steroidal anti-inflammatory drugs (NSAIDs), has been frequently detected in various environmental media. Nevertheless，the potential endocrine disrupting effects of DCF on fish were poorly understood. In the present study, zebrafish embryos/larvae were used as a model to evaluate the adverse effects of DCF on development and thyroid system. The results demonstrated that DCF only significantly decreased the heart rate at 72 h post-fertilization (hpf), exhibiting limited influence on the embryonic development of zebrafish. Treatment with DCF significantly reduced whole-body thyroxine (T4) levels, and changed transcriptional levels of several genes related to the hypothalamic-pituitary-thyroid (HPT) axis. These findings provide important information regarding to the mechanisms of DCF-induced developmental toxicity and thyroid disruption in fish.