Zebrafish Embryonic Cell Line Research Articles

Exploring Zebrafish Embryonic Cell Line PAC2 by Proteomics Profiling

Exploring Zebrafish Embryonic Cell Line PAC2 by Proteomics Profiling

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.

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).

Genomic and gene expression responses to genotoxic stress in PAC2 zebrafish embryonic cell line.

PAC2 cell line is, along most of the developed zebrafish cell lines, poorly characterized concerning its response to genotoxicants. To define the PAC2 cell line response to different forms of genotoxic stress, we exposed the cells to model genotoxic agents (benzo[a]pyrene, B[a]P, and ethyl methanesulfonate) and subsequently monitored DNA damage and alterations by using the battery of tests, including the Comet assay, quantitative random-amplified polymorphic DNA and amplified fragment length polymorphism. The expression of several DNA repair (xpc, xpd, hr23b, rad51, msh2) and oxidative stress response (sod (Cu/Zn)) genes was monitored as well. To obtain an indication of the PAC2 cell line metabolizing capacity, the expression of genes belonging to cyp1, cyp2 and cyp3 families was assessed upon exposure to B[a]P. Genotoxic responses were observed in all the used methods, and quantitative random-amplified polymorphic DNA and amplified fragment length polymorphism proved to be more sensitive by revealing DNA alterations even when the Comet assay indicated lack of significant damage. The PAC2 cell line demonstrated basal and B[a]P-induced expression of several cyp genes, suggesting its ability to metabolize indirect acting xenobiotics to a certain point. Based on these results, PAC2 cells seem to be sensitive zebrafish in vitro model in the genotoxicity assessment of the direct acting genotoxicant; however, they are less sensitive toward the indirect acting genotoxicant due to their limited metabolizing properties.

Cell Growth-Promoting Activity of Fluid from Eye Sacs of the Bubble-Eye Goldfish (Carassius auraitus)

The growth-promoting effects of fish body fluids, such as serum and embryonic extract, on fish cell cultures have been widely demonstrated. The bubble-eye variety of aquarium goldfish is characterized as having a large sac filled with fluid (sac fluid) under each eye. These sacs are believed to contain lymph, which is similar in composition to serum or blood plasma. In order to test whether the sac fluid can be used as an additive for fetal bovine serum (FBS) in growth factor supplements, we compared cell growth in media containing FBS together with different concentrations of sac fluid. A dose-dependent growth-promotion effect was observed in early passage caudal fin cells from both medaka and zebrafish. Cell-growth promotion was also confirmed in early passage medaka blastula cells and in a zebrafish embryonic cell line (ZF4). Replacement of the fluid in the eye sacs of bubble-eyes occurs within a couple of months after the sac fluid has been harvested, and the cell-growth promoting activity of the new fluid is similar to that of the fluid that was tapped initially. These findings suggest that sac fluid can be used as a growth-promoting supplement for fish cell culture. Importantly, the ability of the goldfish to replace the fluid combined with the fact that equipotent fluid can be repeatedly harvested from the eye sacs means that a sustainable source of the fluid can be obtained without needing to sacrifice the fish.

XBP-1, a key regulator of unfolded protein response, activates transcription of IGF1 and Akt phosphorylation in zebrafish embryonic cell line

The unfolded protein response (UPR) is a conserved and adaptive cellular response to increase cell survival during ER stress. XBP-1 spliced form (XBP-1S) generated by IRE1 endoribonuclease is a key transcriptional regulator in UPR to activate genes involved in protein folding and degradation to restore ER function. Although Akt activation was suggested to be a pro-survival pathway activated during ER stress, the signal to trigger Akt is still not clear. In this study, we report IGF1 transcription and Akt phosphorylation are enhanced in XBP-1S stably overexpressed clone of zebrafish embryonic cell line (ZF4). In addition, zebrafish IGF1 intron1 with predicted UPRE (XBP-1S binding sites) and ERSE (ATF6/XBP-1S binding site) linked with basal promoter could be activated by XBP-1S, not by XBP-1 unspliced form (XBP-1U). Furthermore, we demonstrate that expression of endogenous IGF1 is transiently induced as XBP-1 splicing during ER stress in parallel to ER chaperone GRP78/Hspa5 and ER resided E3 ubiquitin ligase Synoviolin in ZF4 cells by quantitative PCR. Our results suggest zebrafish XBP-1S not only activates genes responsible for protein folding, transporting, glycosylation and ER associated degradation but also activates anti-apoptosis signal via IGF1/Akt pathway in unfolded protein response to cope with ER stress.

Teleost multiple tissue ( tmt) opsin: a candidate photopigment regulating the peripheral clocks of zebrafish?

Isolated organs and cell lines from zebrafish exhibit circadian oscillations in clock gene expression that can be entrained to a 24-h light/dark cycle. The mechanism underlying this cellular photosensitivity is unknown. We report the identification of a novel opsin family, tmt-opsin, that has a genomic structure characteristic of vertebrate photopigments, an amino acid identity equivalent to the known photopigment opsins, and the essential residues required for photopigment function. Significantly, tmt-opsin is expressed in a wide variety of neural and non-neural tissues, including a zebrafish embryonic cell line that exhibits a light entrainable clock. Collectively the data suggest that tmt-opsin is a strong candidate for the photic regulation of zebrafish peripheral clocks.