Zebrafish Embryonic Cells Research Articles

Assessment of plant- and microbial-derived protein hydrolysates as sustainable for fetal bovine serum in seafood cell culture media

This study seeks to explore alternatives by substituting or reducing the conventional 10% serum concentration in zebrafish embryonic stem cell (ESC) growth media with protein hydrolysates sourced from peas, mushrooms, yeast, and algae. Notably, algae exhibited the highest protein content, optimal amino acid balance, and favorable functional properties. When applied at concentrations ranging from 1 to 10 mg/mL, all protein hydrolysates demonstrated pro-apoptotic effects and inhibited cell growth, particularly when used in conjunction with 10% serum. However, concentrations ranging from 0.001 to 0.1 mg/mL displayed anti-apoptotic properties and promoted cell proliferation. The study found that media containing 1% or 2.5% serum, along with 0.01 mg/mL of protein hydrolysates, supported cell growth effectively. Lactate Dehydrogenase (LDH) Activity served as an indicator of cell health and integrity under the specified conditions of protein hydrolysate supplementation. Cells cultured in serum-free media exhibited significantly decreased cell membrane integrity (P < 0.05) compared to those in regular media or media containing low serum (1% and 2.5%) along with low concentrations (0.01 mg/mL) of protein hydrolysates. Furthermore, analysis of Greenhouse Gas emissions (GHG) suggested that media formulations containing 1% serum combined with low concentrations of protein hydrolysates present a sustainable approach for cell-based seafood production.

Exploring Zebrafish Embryonic Cell Line PAC2 by Proteomics Profiling

Exploring Zebrafish Embryonic Cell Line PAC2 by Proteomics Profiling

Establishment and transcriptome analysis of single blastomere-derived cell lines from zebrafish

Maintaining chromosome euploidy in zebrafish embryonic cells is challenging because of the degradation of genomic integrity during cell passaging. In this study, we report the derivation of zebrafish cell lines from single blastomeres. These cell lines have a stable chromosome status attributed to BMP4 and exhibit continuous proliferation in vitro. Twenty zebrafish cell lines are successfully established from single blastomeres. Single-cell transcriptome sequencing analysis confirms the fidelity of gene expression profiles throughout long-term culturing of at least 45 passages. The long-term cultured cells are specialized into epithelial cells, exhibiting similar expression patterns validated by integrative transcriptomic analysis. Overall, this work provides a protocol for establishing zebrafish cell lines from single blastomeres, which can serve as valuable tools for in vitro investigations of epithelial cell dynamics in terms of life–death balance and cell fate determination during normal homeostasis.

Magnetic Soft Microrobot Design for Cell Grasping and Transportation

Manipulating cells at a small scale is widely acknowledged as a complex and challenging task, especially when it comes to cell grasping and transportation. Various precise methods have been developed to remotely control the movement of microrobots. However, the manipulation of micro-objects necessitates the use of end-effectors. This paper presents a study on the control of movement and grasping operations of a magnetic microrobot, utilizing only 3 pairs of electromagnetic coils. A specially designed microgripper is employed on the microrobot for efficient cell grasping and transportation. To ensure precise grasping, a bending deformation model of the microgripper is formulated and subsequently validated. To achieve precise and reliable transportation of cells to specific positions, an approach that combines an extended Kalman filter with a model predictive control method is adopted to accomplish the trajectory tracking task. Through experiments, we observe that by applying the proposed control strategy, the mean absolute error of path tracking is found to be less than 0.155 mm. Remarkably, this value accounts for only 1.55% of the microrobot’s size, demonstrating the efficacy and accuracy of our control strategy. Furthermore, an experiment involving the grasping and transportation of a zebrafish embryonic cell (diameter: 800 μm) is successfully conducted. The results of this experiment not only validate the precision and effectiveness of the proposed microrobot and its associated models but also highlight its tremendous potential for cell manipulation in vitro and in vivo.

A Comprehensive Review on Zebrafish and Zebrafish Embryonic Stem Cells (Zescs) as the Versatile Biotechnological Green Tool for Detecting Environmental Pollutants

This article discusses the embryonic development of zebrafish, which are essential aquatic models for investigating neurotoxicity caused by environmental toxins. Zebrafish are one of the few fish species that can survive in highly toxic environments, making them an interesting model for assessing pollutants' performance and determining their toxicity. Zebrafish's limited life expectancy, ease of maintenance and growth, transparent embryos, and homology of genetic and biological features make them an interesting tool for studying the effects of common substances like insecticides, nanoparticles, and food supplements on human health. The study focuses on the regulation of morphology and behavior, stress caused by oxidative damage, transcription, neurogenesis, and neuron progression. Further research is needed at cellular and signaling levels to understand the detrimental processes of pollutants in relation to epigenetic toxicity, negative interference of the BB barrier, and manipulation of the brain-gut-microbiota axis. Environmental pollution is a significant issue that poses a severe threat to public health. Biotechnological principles are now being widely used to monitor environmental pollutants due to their versatile applications. In this regard, zebrafish has established as a prominent vertebrate prototype organism, which offers a unique platform for toxicity screening and efficacy testing of various chemicals. Studies have shown that exposure to toxicants during embryonic development can cause developmental, cardiovascular, and neurodevelopmental toxicity, as well as hepatic disorders in zebrafish. Therefore, zebrafish has been extensively used to study the neurotoxicity of environmental pollutants, including pesticides, nanoparticles, food additives, and other pollutants. Additionally, the review discusses the use of zebrafish-derived embryonic stem cells (ZESCs) for environmental pollutants monitoring. Stem cells are highly sensitive to the toxicity of environmental chemicals during embryogenesis, which can lead to adverse effects on embryonic and fetal development. Thus, ZESCs cultivation and utilization in toxicological assays have become a valuable tool for evaluating the toxicity of potential environmental toxicants. The application of green science principles to evaluate the toxicity of environmental pollutants using zebrafish and ZESCs has enormous potential. This approach offers a sustainable and environmentally friendly way to monitor pollutants, and it can contribute to the development of effective mitigation strategies. Overall, the use of zebrafish and ZESCs in biotechnological pollutants monitoring could have far-reaching effects for public health and environmental sustainability.

Copper overload impairs hematopoietic stem and progenitor cell proliferation via prompting HSF1/SP1 aggregation and the subsequently downregulating FOXM1-Cytoskeleton axis

Unbalanced Cu homeostasis has been suggested to be associated with hematopoietic disease, but the roles of Cu overload in the hematopoietic system and the potential mechanisms are obscure. Here, we report a novel association and the novel potential pathways for Cu overload to induce proliferation defects in zebrafish embryonic hematopoietic stem and progenitor cells (HSPCs) via down-regulating expression of foxm1-cytoskeleton axis, which is conserved from fish to mammals. Mechanistically, we show the direct binding of Cu to transcriptional factors HSF1 and SP1 and that Cu overload induces the cytoplasmic aggregation of proteins HSF1 and SP1. These result in the reduced transcriptional activities of HSF1 and SP1 on their downstream FOXM1 as well as the FOXM1 transcriptional activities on cytoskeletons in HSPCs, which leads to ultimately cell proliferation impairment. These findings unveil the novel linkage of Cu overload with specific signaling transduction as well as the subsequent HSPC proliferation defects.

Evaluating the Potential of Marine Invertebrate and Insect Protein Hydrolysates to Reduce Fetal Bovine Serum in Cell Culture Media for Cultivated Fish Production.

The use of fetal bovine serum (FBS) and the price of cell culture media are the key constraints for developing serum-free cost-effective media. This study aims to replace or reduce the typical 10% serum application in fish cell culture media by applying protein hydrolysates from insects and marine invertebrate species for the growth of Zebrafish embryonic stem cells (ESC) as the model organism. Protein hydrolysates were produced from black soldier flies (BSF), crickets, oysters, mussels, and lugworms with a high protein content, suitable functional properties, and adequate amino-acid composition, with the degree of hydrolysis from 18.24 to 33.52%. Protein hydrolysates at low concentrations from 0.001 to 0.1 mg/mL in combination with 1 and 2.5% serums significantly increased cell growth compared to the control groups (5 and 10% serums) (p < 0.05). All protein hydrolysates with concentrations of 1 and 10 mg/mL were found to be toxic to cells and significantly reduced cell growth and performance (p < 0.05). However, except for crickets, all the hydrolysates were able to restore or significantly increase cell growth and viability with 50% less serum at concentrations of 0.001, 0.01, and 0.1 mg/mL. Although cell growth was enhanced at lower concentrations of protein hydrolysates, the cell morphology was altered due to the lack of serum. The lactate dehydrogenase (LDH) activity results indicated that BSF and lugworm hydrolysates did not alter the cell membrane. In addition, light and fluorescence imaging revealed that the cell morphological features were comparable to those of the 10% serum control group. Overall, lugworm and BSF hydrolysates reduced the serum by up to 90% while preserving excellent cell health.

Iron supplementation inhibits hypoxia-induced mitochondrial damage and protects zebrafish liver cells from death.

Acute hypoxia in water has always been a thorny problem in aquaculture. Oxygen and iron play important roles and are interdependent in fish. Iron is essential for oxygen transport and its concentration tightly controlled to maintain the cellular redox homeostasis. However, it is still unclear the role and mechanism of iron in hypoxic stress of fish. In this study, we investigated the role of iron in hypoxic responses of two zebrafish-derived cell lines. We found hypoxia exposed zebrafish liver cells (ZFL) demonstrated reduced expression of Ferritin and the gene fth31 for mitochondrial iron storage, corresponding to reduction of both intracellular and mitochondrial free iron and significant decrease of ROS levels in multiple cellular components, including mitochondrial ROS and lipid peroxidation level. In parallel, the mitochondrial integrity was severely damaged. Addition of exogenous iron restored the iron and ROS levels in cellular and mitochondria, reduced mitochondrial damage through enhancing mitophagy leading to higher cell viability, while treated the cells with iron chelator (DFO) or ferroptosis inhibitor (Fer-1) showed no improvements of the cellular conditions. In contrast, in hypoxia insensitive zebrafish embryonic fibroblasts cells (ZF4), the expression of genes related to iron metabolism showed opposite trends of change and higher mitochondrial ROS level compared with the ZFL cells. These results suggest that iron homeostasis is important for zebrafish cells to maintain mitochondrial integrity in hypoxic stress, which is cell type dependent. Our study enriched the hypoxia regulation mechanism of fish, which helped to reduce the hypoxia loss in fish farming.

Wnt Signaling in Heart Development and Regeneration.

Cardiovascular diseases are the leading cause of death worldwide, largely due to the limited regenerative capacity of the adult human heart. In contrast, teleost zebrafish hearts possess natural regeneration capacity by proliferation of pre-existing cardiomyocytes after injury. Hearts of mice can regenerate if injured in a few days after birth, which coincides with the transient capacity for cardiomyocyte proliferation. This review tends to elaborate the roles and mechanisms of Wnt/β-catenin signaling in heart development and regeneration in mammals and non-mammalian vertebrates. Studies in zebrafish, mice, and human embryonic stem cells demonstrate the binary effect for Wnt/β-catenin signaling during heart development. Both Wnts and Wnt antagonists are induced in multiple cell types during cardiac development and injury repair. In this review, we summarize composites of the Wnt signaling pathway and their different action routes, followed by the discussion of their involvements in cardiac specification, proliferation, and patterning. We provide overviews about canonical and non-canonical Wnt activity during heart homeostasis, remodeling, and regeneration. Wnt/β-catenin signaling exhibits biphasic and antagonistic effects on cardiac specification and differentiation depending on the stage of embryogenesis. Inhibition of Wnt signaling is beneficial for cardiac wound healing and functional recovery after injury. Understanding of the roles and mechanisms of Wnt signaling pathway in injured animal hearts will contribute to the development of potential therapeutics for human diseased hearts.

Mitochondrial changes in fish cells in vitro in response to serum deprivation.

Mitochondria are critical to cellular activity that implicated in expansive networks to maintain organismal homeostasis under external stimuli of nutrient variability, a common and severe stress to fish performance during the intensive culture conditions. In the present study, zebrafish embryonic fibroblast cells were used to investigate the fish mitochondrial changes upon serum deprivation. Results showed that mitochondrial content and membrane potential were significantly reduced with increased intracellular ROS level in the serum deprivation treated fish cells. And the impaired mitochondria were characterized by rough and fracted outer membrane, and more fused mitochondria were frequently observed with the upregulated mRNA expressions of mitochondrial fusion genes (mfn1b, mfn2, and opa1). Besides, the mitochondrial DNA (mtDNA) copy numbers of mtatp6, mtcox1, mtcytb, mtnd4, and mtnd6 were overall showing the highly significant reduction, together with the mRNA expressions of these genes significantly increased, exhibiting the compensatory effects in mitochondria. Furthermore, the methyl-cytosine of whole mtDNA was compared and the methyl-reads numbers were distinctly increased in the treatment group, reflecting the instability of fish mtDNA with mitochondrial dysfunction under nutrient fluctuations. Collectively, current findings could facilitate the integrated research between fish mitochondrial response and external variables that indicates the potentially profound and durative deficits in fish health during the aquaculture processes.

New Findings on LMO7 Transcripts, Proteins and Regulatory Regions in Human and Vertebrate Model Organisms and the Intracellular Distribution in Skeletal Muscle Cells.

LMO7 is a multifunctional PDZ–LIM protein that can interact with different molecular partners and is found in several intracellular locations. The aim of this work was to shed light on LMO7 evolution, alternative transcripts, protein structure and gene regulation through multiple in silico analyses. We also explored the intracellular distribution of the LMO7 protein in chicken and zebrafish embryonic skeletal muscle cells by means of confocal fluorescence microscopy. Our results revealed a single LMO7 gene in mammals, sauropsids, Xenopus and in the holostean fish spotted gar while two lmo7 genes (lmo7a and lmo7b) were identified in teleost fishes. In addition, several different transcripts were predicted for LMO7 in human and in major vertebrate model organisms (mouse, chicken, Xenopus and zebrafish). Bioinformatics tools revealed several structural features of the LMO7 protein including intrinsically disordered regions. We found the LMO7 protein in multiple intracellular compartments in chicken and zebrafish skeletal muscle cells, such as membrane adhesion sites and the perinuclear region. Curiously, the LMO7 protein was detected within the nuclei of muscle cells in chicken but not in zebrafish. Our data showed that a conserved regulatory element may be related to muscle-specific LMO7 expression. Our findings uncover new and important information about LMO7 and open new challenges to understanding how the diverse regulation, structure and distribution of this protein are integrated into highly complex vertebrate cellular milieux, such as skeletal muscle cells.

Cu-based nanoparticle toxicity to zebrafish cells regulated by cellular discharges

Cellular transport of metal nanoparticles (NPs) is critical in determining their potential toxicity, but the transformation of metal ions released from the internalized NPs is largely unknown. Cu-based NPs are the only metallic-based NPs that are reported to induce higher toxicity compared with their corresponding ions, likely due to their unique cellular turnover. In the present study, we developed a novel gold core to differentiate the particulate and ionic Cu in the Cu2O microparticles (MPs), and the kinetics of bioaccumulation, exocytosis, and cytotoxicity of Au@Cu2O MPs to zebrafish embryonic cells were subsequently studied. We demonstrated that the internalized MPs were rapidly dissolved to Cu ions, which then undergo lysosome-mediated exocytosis. The uptake rate of smaller MPs (130 nm) was lower than that of larger ones (200 nm), but smaller MPs were dissolved much quickly in cells and therefore activated the exocytosis more quickly. The rapid release of Cu ions resulted in an immediate toxic action of Cu2O MPs, while the cell deaths mainly occurred by necrosis. During this process, the buffering ability of glutathione greatly alleviated the Cu toxicity. Therefore, although the turnover of intracellular Cu at a sublethal exposure level was hundred times faster than the basal values, labile Cu(I) concentration increased by only 2 times at most. Overall, this work provided new insights into the toxicity of copper NPs, suggesting that tolerance to Cu-based NPs depended on their ability to discharge the released Cu ions.

The effect of Activin pathway modulation on the expression of both pluripotency and differentiation markers during early zebrafish development compared with other vertebrates.

Activin-like factors control many developmental processes, including pluripotency maintenance and differentiation. Although Activin-like factors' action in mesendoderm induction has been demonstrated in zebrafish, their involvement in preserving the stemness remains unknown. To investigate the role of maternal Activin-like factors, their effects were promoted or blocked using synthetic human Activin A or SB-431542 treatments respectively until the maternal to zygotic transition. To study the role of zygotic Activin-like factors, SB-431542 treatment was also applied after the maternal to zygotic transition. The effect of the pharmacological modulations of the Activin/Smad pathway was then studied on the mRNA expressions of the ndr1, ndr2, tbxta (no tail/ntl) as the differentiation index, mych, nanog, and oct4 (pou5f3) as the pluripotency markers of the zebrafish embryonic cells as well as sox17 as a definitive endoderm marker. Expression of the target genes was measured at the 16-cell, 256-cell, 1K-cell, oblong, dome, and shield stages using the real-time quantitative polymerase chain reaction (RT-qPCR). Activation of the maternal Activin signaling pathway led to an increase in zygotic expression of the tbxta, particularly marked at the oblong stage. In other words, promotion of the maternal Activin/Smad pathway induced differentiation by advancing the major peaks of ndr1 and nanog, thereby eliciting tbxta expression. Whereas suppression of the maternal or zygotic Activin/Smad pathway sustained the pluripotency by preventing the major peaks of ndr1 and nanog as well as tbxta encoding.

Detection of N6-Methyladenine in Eukaryotes.

DNA N6-methyladenine (6mA) is a chemical modification at the N6-positon of adenine. In the last decades, 6mA had been found in genome from numerous prokaryotic species, but only existed in a few lower eukaryotes. In prokaryotes, 6mA plays an important role in restriction-modification, DNA replication, and DNA mismatch repair. Because of the too low abundance of 6mA, it was long-stalled whether 6mA existed in multicellular eukaryotes and playing any functions, particularly in mammals. In recent years, partially benefitting from the advances in analytical methods, 6mA was found in the genomes from Drosophila melanogaster, Chlamydomonas algae, Caenorhabditis elegans, zebrafish, Xenopus laevis and mouse embryonic stem cells and even in the human genome. The 6mA was dynamic changed in early embryonic development of fly and zebrafish and much more enriched in gene body of transposons in fly, repetitive regions in zebrafish, around the transcription start sites in Chlamydomonas, and widespread distribution in C. elegans, indicating 6mA probably playing different functions in different species. Meanwhile, 6mA methylases and demethylases were found in fly, worm, and Chlamydomonas. In this chapter, we will briefly review the distribution, regulation, and function of 6mA in eukaryotes and focus on the advances of 6mA analysis methods, especially LC-MS/MS, immunoprecipitation, next-generation sequencing, and single-molecule real-time sequencing technology.

Comparison of zebrafish in vitro and in vivo developmental toxicity assessments of perfluoroalkyl acids (PFAAs)

ABSTRACT Perfluoroalkyl acids (PFAAs) are persistent environmental contaminants that are associated with various adverse health outcomes. Perfluorooctanoic acid (PFOA) is one of the most prominently detected PFAAs in the environment, which is now replaced with shorter chain carbon compounds including perfluorohexanoic acid (PFHxA) and perfluorobutyric acid (PFBA). The aim of this study was to compare the toxicity of four PFAAs as a function of chain length and head group (carboxylate versus sulfonate) with in vitro and in vivo zebrafish assessments, which were subsequently compared to other cell and aquatic models. Mortality rate increased with chain length (PFOA > PFHxA ≫ PFBA) in both whole embryo/larvae and embryonic cell models. The sulfonate group enhanced toxicity with perfluorobutane sulfonate (PFBS) showing higher toxicity than PFBA and PFHxA in both larvae and cells. Toxicity trends were similar among different aquatic models, but sensitivities varied. Discrepancies with other zebrafish studies were confirmed to be associated with a lack of neutralization of acidic pH of dosing solutions in these other investigations, demonstrating the need for rigor in reporting pH of exposure solutions in all experiments. The zebrafish embryonic cell line was also found to be similar to most other cell lines regardless of exposure length. Overall, results agree with findings in other cell lines and organisms where longer chain length and sulfonate group increase toxicity, except in investigations not neutralizing the exposure solutions for these acidic compounds.

Characterization of the Mercapturic Acid Pathway, an Important Phase II Biotransformation Route, in a Zebrafish Embryo Cell Line.

In view of the steadily increasing number of chemical compounds used in various products and applications, high-throughput toxicity screening techniques can help meeting the needs of 21st century risk assessment. Zebrafish (Danio rerio), especially its early life stages, are increasingly used in such screening efforts. In contrast, cell lines derived from this model organism have received less attention so far. A conceivable reason is the limited knowledge about their overall capacity to biotransform chemicals and the spectrum of expressed biotransformation pathways. One important biotransformation route is the mercapturic acid pathway, which protects organisms from harmful electrophilic compounds. The fully functional pathway involves a succession of several enzymatic reactions. To investigate the mercapturic acid pathway performance in the zebrafish embryonic cell line, PAC2, we analyzed the biotransformation products of the reactions comprising this pathway in the cells exposed to a nontoxic concentration of the reference substrate, 1-chloro-2,4-dinitrobenzene (CDNB). Additionally, we used targeted proteomics to measure the expression of cytosolic glutathione S-transferases (GSTs), the enzyme family catalyzing the first reaction in this pathway. Our results reveal that the PAC2 cell line expresses a fully functional mercapturic acid pathway. All but one of the intermediate CDNB biotransformation products were identified. The presence of the active mercapturic acid pathway in this cell line was further supported by the expression of a large palette of GST enzyme classes. Although the enzymes of the class alpha, one of the dominant GST classes in the zebrafish embryo, were not detected, this did not seem to affect the capacity of the PAC2 cells to biotransform CDNB. Our data provide an important contribution toward using zebrafish cell lines, specifically PAC2, for animal-free high- throughput screening in toxicology and chemical hazard assessment.

Genomic responses of DNA methylation and transcript profiles in zebrafish cells upon nutrient deprivation stress

Environmental stress such as nutrient deprivation across multiple fields in nature causes physiological and biochemical changes in organism. Understanding the potential epigenetic modulations to phenotypic variation upon nutrient deprivation stress is crucial for environmental assessments. Here, the methyl-cytosine at single-base resolution was mapped across the whole genome and the methylation patterns and methylation levels coordinated with transcript analysis were systemically elaborated in zebrafish embryonic fibroblast cells under serum starvation stress. The down-regulated genes mainly annotated to the pathways of DNA replication and cell cycle that were consistent with cell physiological changes. Vast differentially methylated regions were identified in genomic chromosome and showed enrichment in the intron and intergenic regions. In an integrated transcriptome and DNA methylation analyses, 135 negatively correlated genes were determined, wherein the hub genes of gins2, cdca5, fbxo5, slc29a2, suv39h1b, and zgc:174160 were predominant responsive to the nutrient condition changes. Besides, nutrient recovery and DNA methyltransferases inhibitor supplements partly rescued cell proliferation with decrease of DNA methylation and reactivation of several depressed genes, implying the possible intrinsic relationships among cell physiological state, mRNA expression, and DNA methylation. Collectively, current study proved the broad role of DNA methylation in governing cellular responses to nutrient deprivation and revealed the epigenetic risk of starvation stress in zebrafish.

Copper induce zebrafish retinal developmental defects via triggering stresses and apoptosis

BackgroundThe disorder of copper homeostasis is linked with disease and developmental defects, and excess copper_nanoparticles (CuNPs) and ion (Cu2+) will induce developmental malformation and disease in organisms. However, little knowledge is available regarding its potential regulation mechanisms, and little study links excess copper with retinal developmental malformation and disease.MethodsEmbryos were stressed with copper (CuNPs and Cu2+), and cell proliferation and apoptosis assays, reactive oxygen species (ROS) and endoplasmic reticulum (ER) signaling detections, and genetic mutants cox17−/− and atp7a−/− application, were used to evaluate copper induced retinal developmental malformation and the underlying genetic and biological regulating mechanisms.ResultsCopper reduced retinal cells and down-regulated expression of retinal genes, damaged the structures of ER and mitochondria in retinal cells, up-regulated unfold protein responses (UPR) and ROS, and increased apoptosis in copper-stressed retinal cells. The copper induced retinal defects could be significantly neutralized by ROS scavengers reduced Glutathione (GSH) & N-acetylcysteine (NAC) and ER stress inhibitor 4- phenylbutyric acid (PBA). Blocking the transportation of copper to mitochondria, or to trans-Golgi network and to be exported into plasma, by deleting gene cox17 or atp7a, could alleviate retinal developmental defects in embryos under copper stresses.ConclusionsThis is probably the first report to reveal that copper nanoparticles and ions induce retinal developmental defects via upregulating UPR and ROS, leading to apoptosis in zebrafish embryonic retinal cells. Integrated function of copper transporter (Cox17 and Atp7a) is necessary for copper induced retinal defects.Graphical abstract

LC-APCI(-)-MS Determination of 1-Chloro-2,4-dinitrobenzene, a Model Substrate for Glutathione S-Transferases.

1-Chloro-2,4-dinitrobenzene (CDNB) is widely used as a model substrate for measuring the enzyme activity of glutathione S-transferases in toxicity studies and in studies focusing on the metabolic capacity of different test systems. To allow the quantification of CDNB at low, nontoxic concentrations, we developed a sensitive liquid chromatography-mass spectrometry (LC-MS) technique, which is based on electron capture ionization using atmospheric pressure chemical ionization (APCI) in negativeion mode. Gas-phase reactions occurring under atmospheric pressure produce specific ions that allow direct CDNB quantification down to 17 ng/mL in water. Using the new technique, we were able to verify CDNB exposure concentrations applied in two typical toxicity studies with early life stages of the common model organisms, zebrafish (Danio rerio) and a zebrafish embryonic cell line (PAC2).

Identification and characterization of miRNAs involved in cold acclimation of zebrafish ZF4 cells.

MicroRNAs (miRNAs) play vital roles in various biological processes under multiple stress conditions by leading to mRNA cleavage or translational repression. However, the detailed roles of miRNAs in cold acclimation in fish are still unclear. In the present study, high-throughput sequencing was performed to identify miRNAs from 6 small RNA libraries from the zebrafish embryonic fibroblast ZF4 cells under control (28°C, 30 days) and cold-acclimation (18°C, 30 days) conditions. A total of 414 miRNAs, 349 known and 65 novel, were identified. Among those miRNAs, 24 (19 known and 5 novel) were up-regulated, and 23 (9 known and 14 novel) were down-regulated in cold acclimated cells. The Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses indicated that the target genes of known differentially expressed miRNAs (DE-miRNA) are involved in cold acclimation by regulation of phosphorylation, cell junction, intracellular signal transduction, ECM-receptor interaction and so on. Moreover, both miR-100-3p inhibitor and miR-16b mimics could protect ZF4 cells under cold stress, indicating the involvement of miRNA in cold acclimation. Further study showed that miR-100-3p and miR-16b could regulate inversely the expression of their target gene (atad5a, cyp2ae1, lamp1, rilp, atxn7, tnika, btbd9), and that overexpression of miR-100-3p disturbed the early embryonic development of zebrafish. In summary, the present data show that miRNAs are closely involved in cold acclimation in zebrafish ZF4 cells and provide information for further understanding of the roles of miRNAs in cold acclimation in fish.