Surface Modification Determines the Distribution and Toxicity of Quantum Dots during the Development of Early Staged Zebrafish.

Surface modifications are generally used to functionalize QDots to improve their properties for practical applications, but the relationship between QDot modification and biological activity is not well understood. Using an early staged zebrafish model, we investigated the biodistribution and toxicity of CdSe/ZnS QDots with four types of modifications, including anionic poly(ethylene glycol)-carboxyl ((PEG)n-COOH), anionic mercaptopropionic acid (MPA), zwitterionic glutathione (GSH), and cationic cysteamine (CA). None of the QDots showed obvious toxicity to zebrafish embryos prior to hatching because the zebrafish chorion is an effective barrier that protects against QDot exposure. The QDots were mainly absorbed on the epidermis of the target organs after hatching and were primarily deposited in the mouth and gastrointestinal tract when the zebrafish started feeding. CA-QDots possessed the highest adsorption capacity; however, (PEG)n-COOH-QDots showed the most severe toxicity to zebrafish, as determined by mortality, hatching rate, heartbeat, and malformation assessments. It shows that the toxicity of the QDots is mainly attributed to ROS generation rather than Cd2+ release. This study provides a comprehensive understanding of the environmental and ecological risks of nanoparticles in relation to their surface modification.

Tetrabromobisphenol A Perturbs Erythropoiesis and Impairs Blood Circulation in Zebrafish Embryos.

Tetrabromobisphenol A (TBBPA), a ubiquitous environmental pollutant, has been implicated in developmental toxicity of aquatic animals. However, the impact of TBBPA on development and the related mechanism have not been fully elucidated. In this study, using a live imaging technique and transgenic labeling of zebrafish embryos, we described the toxic effects of TBBPA on hematopoietic development in zebrafish. We demonstrated that TBBPA induced erythroid precursor expansion in the intermediate cell mass (ICM), which perturbed the onset of blood circulation at 24-26 hours postfertilization (hpf). Consequently, excessive blood cells accumulated in the posterior blood island (PBI) and vascular cells formed defective caudal veins (CVs), preventing blood cell flow to the heart at 32-34 hpf. We found that the one-cell to 50% epiboly stage was the most sensitive period to TBBPA exposure during hematopoietic development. Furthermore, our results demonstrated that PBI malformation induced by TBBPA resulted from effects on erythroid precursor cells, which might involve THR signaling in complex ways. These findings will improve the understanding of TBBPA-induced developmental toxicity in teleost.

Chronic exposure to polystyrene microplastics increased the chemosensitivity of normal human liver cells via ABC transporter inhibition.

Microplastics (MPs) are ubiquitous in environmental compartments and consumer products. Although liver is frequently reported to be a target organ of MP accumulation in mammals, few studies have focused on MP hepatoxicity in humans. In this study, we used normal human liver cells, THLE-2, to assess the acute and chronic toxicity of polystyrene (PS) MPs with sizes of 0.1 and 1 µm. The results showed that after 48 h of exposure, both kinds of PS MPs could enter THLE-2 cells and cause no obviously acute cytotoxicity at <20 µg/mL. In contrast, metabolomic analysis revealed that 90 days of PS MPs exposure at environmentally relevant dose (0.2 µg/mL) could significantly alter the metabolic profiles of the cells, especially the nanosized MPs. KEGG pathway analysis showed that the ATP-binding cassette (ABC) transporter pathway was the most significantly changed pathway. Cell functional tests confirmed that chronic PS MP treatment could inhibit the activity of the ABC efflux transporter and further increase the cytotoxicity of arsenic, indicating that the PS MPs had a chemosensitizing effect. These findings underline the chronic risk of MPs to human liver.

Highly sensitive response to the toxicity of environmental chemicals in transparent casper zebrafish.

The response sensitivity to toxic substances is the most concerned performance of animal model in chemical risk assessment. Casper (mitfaw2/w2;mpv17a9/a9), a transparent zebrafish mutant, is a useful in vivo model for toxicological assessment. However, the ability of casper to respond to the toxicity of exogenous chemicals is unknown. In this study, zebrafish embryos were exposed to five environmental chemicals, chlorpyrifos, lindane, α-endosulfan, bisphenol A, tetrabromobisphenol A (TBBPA), and an antiepileptic drug valproic acid. The half-lethal concentration (LC50) values of these chemicals in casper embryos were 62-87 % of that in the wild-type. After TBBPA exposure, the occurrence of developmental defects in the posterior blood island of casper embryos was increased by 67-77 % in relative to the wild-type, and the half-maximal effective concentration (EC50) in casper was 73 % of that in the wild-type. Moreover, the casper genetic background significantly increased the hyperlocomotion caused by chlorpyrifos and lindane exposure compared with the wild-type. These results demonstrated that casper had greater susceptibility to toxicity than wild-type zebrafish in acute toxicity, developmental toxicity and neurobehavioral toxicity assessments. Our data will inform future toxicological studies in casper and accelerate the development of efficient approaches and strategies for toxicity assessment via the use of casper.

Special Distribution of Nanoplastics in the Central Nervous System of Zebrafish during Early Development.

There is growing concern about the distribution of nanoplastics (NPs) in the central nervous system (CNS), whereas intrusion is poorly understood. In this study, fluorescent-labeled polystyrene NPs (PS-NPs) were microinjected into different areas of zebrafish embryo to mimic different routes of exposure. PS-NPs were observed in the brain, eyes, and spinal cord through gametal exposure. It indicated that maternally derived PS-NPs were specially distributed in the CNS of zebrafish during early development. Importantly, these NPs were stranded in the CNS but not transferred to other organs during development. Furthermore, using neuron GFP-labeled transgenic zebrafish, colocalization between NPs and the neuron cells revealed that NPs were mostly enriched in the CNS surrounded but not the neurons. Even so, the intrusion of NPs into the CNS induced the significant upregulation of some neurotransmitter receptors, leading to an inhibited effect on the movement of zebrafish larvae. This work provides insights into understanding the intrusion and distribution of NPs in the CNS and the subsequent potential adverse effects.

The germ cell nuclear proteins hnRNP G-T and RBMY activate a testis-specific exon.

The human testis has almost as high a frequency of alternative splicing events as brain. While not as extensively studied as brain, a few candidate testis-specific splicing regulator proteins have been identified, including the nuclear RNA binding proteins RBMY and hnRNP G-T, which are germ cell-specific versions of the somatically expressed hnRNP G protein and are highly conserved in mammals. The splicing activator protein Tra2beta is also highly expressed in the testis and physically interacts with these hnRNP G family proteins. In this study, we identified a novel testis-specific cassette exon TLE4-T within intron 6 of the human transducing-like enhancer of split 4 (TLE4) gene which makes a more transcriptionally repressive TLE4 protein isoform. TLE4-T splicing is normally repressed in somatic cells because of a weak 5' splice site and surrounding splicing-repressive intronic regions. TLE4-T RNA pulls down Tra2beta and hnRNP G proteins which activate its inclusion. The germ cell-specific RBMY and hnRNP G-T proteins were more efficient in stimulating TLE4-T incorporation than somatically expressed hnRNP G protein. Tra2b bound moderately to TLE4-T RNA, but more strongly to upstream sites to potently activate an alternative 3' splice site normally weakly selected in the testis. Co-expression of Tra2beta with either hnRNP G-T or RBMY re-established the normal testis physiological splicing pattern of this exon. Although they can directly bind pre-mRNA sequences around the TLE4-T exon, RBMY and hnRNP G-T function as efficient germ cell-specific splicing co-activators of TLE4-T. Our study indicates a delicate balance between the activity of positive and negative splicing regulators combinatorially controls physiological splicing inclusion of exon TLE4-T and leads to modulation of signalling pathways in the testis. In addition, we identified a high-affinity binding site for hnRNP G-T protein, showing it is also a sequence-specific RNA binding protein.

Assessing the toxicity of bisphenol A and its six alternatives on zebrafish embryo/larvae.

Bisphenol A (BPA) analogues are gradually replacing BPA in the plastics industry. Whether these alternatives are indeed safer than BPA itself, however, remains unclear. Here, we studied the toxicity of BPA and six of its alternatives-BPB, BPC, BPE, BPF, BPAF, and BPAP-using zebrafish embryos/larvae. According to their half lethal concentration (LC50) values, the acute toxicity of BPA and six alternative bisphenols to zebrafish embryos, from highest to lowest, was BPAP ≈ BPAF > BPC > BPB > BPA > BPE > BPF. Under nonlethal concentrations, the tested bisphenols had different toxic effects on development in terms of reducing the hatching rate, frequency of spontaneous movements, and heart rate in the embryo, as well as inducing yolk sac edema, pericardial edema, and spinal deformation in the larvae. The estrogenic activity of BPE, BPF, and BPAF was higher than that of BPA, as shown by vtg1 expression assays. Moreover, BPA and its alternatives increased SOD activity and cell apoptosis in embryos/larvae under nonlethal concentrations. Our findings indicate that BPA alternatives may not be safer than BPA in zebrafish, and that these BPA alternatives should be applied with caution.

Tetrabromobisphenol A perturbs cell fate decisions via BMP signaling in the early embryonic development of zebrafish.

Tetrabromobisphenol A (TBBPA) readily accumulates in the egg yolk of aquatic oviparous animals and is transferred to their embryos. Early embryogenesis is vital for organ formation and subsequent development. The developmental toxicity of TBBPA in aquatic animals has been extensively reported. However, few studies have assessed the toxic effects of TBBPA in the early embryonic development. In this work, we found that TBBPA perturbed cell fate decisions along the dorsal-ventral (DV) axis during gastrulation, further disrupting early organogenesis in the entire embryo. TBBPA exposure increased the number of embryonic cells that acquired a ventral cell fate, which formed epidermis, blood and heart tissues. In return, the number of embryonic cells that acquired a dorsal cell fate was greatly decreased, causing the TBBPA-exposed embryos to develop a small brain and small eyes. We revealed that TBBPA elevated the activity gradient of bone morphogenetic protein (BMP) signaling which is responsible for cell fate specification along the DV axis, with up-regulation of BMP ligands (bmp4, bmp7a) and target genes (szl) and promotion signal transduction through phosphorylation of Smad1/5. As the function of BMP signaling in embryogenesis is highly conserved among many vertebrates, these findings highlight the ecological and health risks of TBBPA.

Activation of GH signaling and GH-independent stimulation of growth in zebrafish by introduction of a constitutively activated GHR construct.

Growth hormone (GH) gene transfer can markedly increase growth in transgenic fish. In the present study we have developed a transcriptional assay to evaluate GH-signal activation (GHSA) in zebrafish embryos. By analyzing the transcription of c-fos and igf1, and the promoter activity of spi2.1, in zebrafish embryos injected with different constructs, we found that overexpression of either GH or growth hormone receptor (GHR) resulted in GHSA, while a synergetic overexpression of GH and GHR gave greater activation. Conversely, overexpression of a C-terminal truncated dominant-negative GHR (ΔC-GHR) efficiently blocked GHSA epistatic to GH overexpression, demonstrating the requirement for a full GHR homodimer in signaling. In view of the importance of signal-competent GHR dimerization by extracellular GH, we introduced into zebrafish embryos a constitutively activated GHR (CA-GHR) construct, which protein products constitutively dimerize the GHR productively by Jun-zippers to activate downstream signaling in vitro. Importantly, overexpression of CA-GHR led to markedly higher level of GHSA than the synergetic overexpression of GH and GHR. CA-GHR transgenic zebrafish were then studied in a growth trial. The transgenic zebrafish showed higher growth rate than the control fish, which was not achievable by GH transgenesis in these zebrafish. Our study demonstrates GH-independent growth by CA-GHR in vivo which bypasses normal IGF-1 feedback control of GH secretion. This provides a novel means of producing growth enhanced transgenic animals based on molecular protein design.

Toxicity of silver nanoparticles on wound healing: A case study of zebrafish fin regeneration model.

Dressings coated with silver nanoparticle (AgNP) are widely used in the management of acute and chronic wounds. However, whether AgNP exerts toxicity on wound healing remains ambiguous. To demonstrate the effects of AgNP on wound healing, we precisely quantified the recovery speed of wound by taking advantage of the fin regeneration of zebrafish. This method also enabled assessment of the adverse effect of AgNP on various steps of wound healing in vivo. We revealed that AgNP treatment at the concentration of 2 µg/ml impaired fin regeneration when exposure was performed at the phases of epithelialization and the beginning of blastema formation. Cell proliferation of regenerative blastema was significantly decreased after AgNP exposure. But the canonical signals including Wingless/Integrated (Wnt), Notch and Fibroblast growth factor (Fgf) which play important roles in cell proliferation during fin regeneration were not modulated at 36 hours post amputation (hpa). Further study showed that AgNP impaired fin regeneration through declining amputation-induced ROS as early as epithelialized phase at 18 hpa, rather than inducing ROS generation. AgNP exposure also promoted recruitment of neutrophils in the early phase of wound healing, which suggests that this event dampened amputation-induced ROS. Overall, this study suggested that application of AgNP-coated dressings should be carefully considered at the beginning stage of wound healing.

A Novel Dietary Source of EPA and DHA: Metabolic Engineering of an Important Freshwater Species-Common Carp by fat1-Transgenesis.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), are essential for neural development and human health. The n-3 PUFAs are mainly obtained from marine fish by dietary intake. Freshwater fish species usually contain low level of n-3 PUFAs due to the lack of n-3 PUFAs in their food chain. In this study, we report on the substantial production of EPA and DHA in a globally important freshwater fish species, common carp (Cyprinus carpio). This was achieved by introducing an "all-fish" transgene CA:fat1 containing the fish codon-optimized omega-3 desaturase gene (fat1) driven by the common carp ß-actin promoter (CA). Through a sperm sample screening method, we successfully generated fat1-positive F1 transgenic population with high efficiency. In F1 population, the muscle contents of ALA (18:3n-3), EPA and DHA were significantly increased when compared with non-transgenic siblings. Thereafter, four independent F2 heterozygous lines were obtained from 4 F1 transgenic males and a detailed comparison of fatty acids profile and growth performance was carried out for these 4 lines. All fat1-transgenic common carps from 4 lines showed an evident decrease in n-6 PUFA contents and a substantial increase in n-3 PUFA contents, among which line 4 stands out, showing a statistically significant increase in all 4 types of n-3 PUFAs including ALA (4.4-fold increase, p < 0.001), EPA (4.8-fold increase, p < 0.01), C22:5n-3 (DPA, 2.4-fold increase, p < 0.05), and DHA (1.9-fold increase, p < 0.05). Therefore, the line 4 was selected as the optimized breeding stock for further study, and the proximate nutrition composition and PUFA synthesis pathway were analyzed. Our study demonstrates that in the transgenic group, the muscular lipid content did not change, while fat accumulations in the internal organs and especially in the liver were significantly decreased as a result of hyperactivation of fatty acid oxidation process. Finally, we conclude that the "all-fish" CA:fat1-transgenic freshwater fish-common carp-can serve as a novel healthy dietary source of n3-PUFAs, especially EPA and DHA.

Evaluating estrogenic and anti-estrogenic effect of endocrine disrupting chemicals (EDCs) by zebrafish (Danio rerio) embryo-based vitellogenin 1 (vtg1) mRNA expression.

By measuring the vitellogenin 1 (vtg1) expression through quantitative PCR and in situ hybridization, we used the zebrafish embryo as an in vivo model to access the estrogenic or anti-estrogenic effects of several endocrine disrupting chemicals (EDCs), such as natural estrogen 17ß-estradiol (E2), estriol (E3), synthetic hormones including diethylstilbestrol (DES), 4-octyl phenol (OP), bisphenol A (BPA), tamoxifen (TMX) and 3-(2,3-dibromopropyl) isocyanurate (TBC). According to our data, the estrogenic effect of the tested chemicals was ranked as: DES>E2>E3>OP>BPA, which is consistent with various in vivo and in vitro models. Therefore, the measurement of vtg1 gene expression in zebrafish embryos would be a valuable method for screening EDCs including both environmental estrogens and anti-estrogens.

Transcriptional Activity and DNA Methylation Dynamics of the Gal4/UAS System in Zebrafish.

The Gal4/upstream activating sequence (UAS) system is a powerful genetic tool for the temporal and spatial expression of target genes. In this study, the dynamic activity of the Gal4/UAS system was monitored in zebrafish throughout the entire lifespan and during germline transmission, using an optimized Gal4/UAS, KalTA4/4xUAS, which is driven by two muscle-specific regulatory sequences. We found that UAS-linked gene expression was transcriptionally amplified by Gal4/UAS during early developmental stages and that the amplification effects tended to weaken during later stages and even disappear in subsequent generations. In the F2 generation, the transcription of a UAS-linked enhanced green fluorescent protein (EGFP) reporter was transcriptionally silent from 16 days post-fertilization (dpf) into adulthood, yet offspring of this generation showed reactivation of the EGFP reporter in some strains. We further show that the transcriptional silencing and reactivation of UAS-driven EGFP correlated with the DNA methylation levels of the UAS regulatory sequences. Notably, asymmetric DNA methylation of the 4xUAS occurred in oocytes and sperm. Moreover, the paternal and maternal 4xUAS sequences underwent different DNA methylation dynamics after fertilization. Our study suggests that the Gal4/UAS system may represent a powerful tool for tracing the DNA methylation dynamics of paternal and maternal loci during zebrafish development and that UAS-specific DNA methylation should be seriously considered when the Gal4/UAS system is applied in zebrafish.

Double transgenesis of humanized fat1 and fat2 genes promotes omega-3 polyunsaturated fatty acids synthesis in a zebrafish model.

Omega-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are essential nutrients for human health. However, vertebrates, including humans, have lost the abilities to synthesize EPA and DHA de novo, majorly due to the genetic absence of delta-12 desaturase and omega-3 desaturase genes. Fishes, especially those naturally growing marine fish, are major dietary source of EPA and DHA. Because of the severe decline of marine fishery and the decrease in n-3 LC-PUFA content of farmed fishes, it is highly necessary to develop alternative sources of n-3 LC-PUFA. In the present study, we utilized transgenic technology to generate n-3 LC-PUFA-rich fish by using zebrafish as an animal model. Firstly, fat1 was proved to function efficiently in fish culture cells, which showed an effective conversion of n-6 PUFA to n-3 PUFA with the n-6/n-3 ratio that decreased from 7.7 to 1.1. Secondly, expression of fat1 in transgenic zebrafish increased the 20:5n-3 and 22:6n-3 contents to 1.8- and 2.4-fold, respectively. Third, co-expression of fat2, a fish codon-optimized delta-12 desaturase gene, and fat1 in fish culture cell significantly promoted n-3 PUFA synthesis with the decreased n-6/n-3 ratio from 7.7 to 0.7. Finally, co-expression of fat1 and fat2 in double transgenic zebrafish increased the 20:5n-3 and 22:6n-3 contents to 1.7- and 2.8-fold, respectively. Overall, we generated two types of transgenic zebrafish rich in endogenous n-3 LC-PUFA, fat1 transgenic zebrafish and fat1/fat2 double transgenic zebrafish. Our results demonstrate that application of transgenic technology of humanized fat1 and fat2 in farmed fishes can largely improve the n-3 LC-PUFA production.