Zebrafish Liver Cell Line Cells Research Articles

Hif1α/Dhrs3a Pathway Participates in Lipid Droplet Accumulation via Retinol and Ppar-γ in Fish Hepatocytes.

Excessive hepatic lipid accumulation is a common phenomenon in cultured fish; however, its underlying mechanisms are poorly understood. Lipid droplet (LD)-related proteins play vital roles in LD accumulation. Herein, using a zebrafish liver cell line (ZFL), we show that LD accumulation is accompanied by differential expression of seven LD-annotated genes, among which the expression of dehydrogenase/reductase (SDR family) member 3 a/b (dhrs3a/b) increased synchronously. RNAi-mediated knockdown of dhrs3a delayed LD accumulation and downregulated the mRNA expression of peroxisome proliferator-activated receptor gamma (pparg) in cells incubated with fatty acids. Notably, Dhrs3 catalyzed retinene to retinol, the content of which increased in LD-enriched cells. The addition of exogenous retinyl acetate maintained LD accumulation only in cells incubated in a lipid-rich medium. Correspondingly, exogenous retinyl acetate significantly increased pparg mRNA expression levels and altered the lipidome of the cells by increasing the phosphatidylcholine and triacylglycerol contents and decreasing the cardiolipin, phosphatidylinositol, and phosphatidylserine contents. Administration of LW6, an hypoxia-inducible factor 1α (HIF1α) inhibitor, reduced the size and number of LDs in ZFL cells and attenuated hif1αa, hif1αb, dhrs3a, and pparg mRNA expression levels. We propose that the Hif-1α/Dhrs3a pathway participates in LD accumulation in hepatocytes, which induces retinol formation and the Ppar-γ pathway.

Untargeted lipidomics reveals the toxicity of bisphenol A bis(3-chloro-2- hydroxypropyl) ether and bisphenols A and F in zebrafish liver cells

Given the opposing responses reported for bisphenol A (BPA) in terms of induction of obesogenic effects and impaired lipid metabolism, the increasing use of bisphenol F (BPF), and the relatively low information available regarding the effects of bisphenol A bis(3-chloro-2- hydroxypropyl) ether (BADGE·2HCl) in aquatic organisms, this work aims to use the zebrafish liver cell line (ZFL) as an alternative model to characterize the toxicity and the lipid metabolism disruptive potential of the selected compounds in fish. All three bisphenols increased intracellular levels of dihydroceramides and ether-triacylglycerides (ether-TGs), suggestive of inhibited cell growth. However, while BPA and BADGE·2HCl caused an increase of saturated and lower unsaturated TGs, BPF caused oxidative stress and the decrease of TGs containing polyunsaturated fatty acids (PUFAs). Analysis by qPCR highlighted the up-regulation of the lipogenic genes scd and elovl6 by BPA and BPF in line with an increase of lipids containing saturated and monounsaturated FA and a decrease of lipids containing PUFAs. This study shows that BPA, BPF and BADGE·2HCl target lipid homeostasis in ZFL cells through different mechanisms, and highlights the higher lipotoxicity of BADGE·2HCl compared to BPA and BPF.

Toxic effects of octocrylene on zebrafish larvae and liver cell line (ZFL).

Octocrylene (OC) is a broad-spectrum ultraviolet-absorbing chemical used in sunscreen and other personal care products. Its health effects are a concern because it has been detected in water, fish, humans, and food chains. In vivo and in vitro investigations were performed in zebrafish (Danio rerio) larvae and a zebrafish liver cell line (ZFL), respectively, to understand the potential risks and molecular mechanisms of OC toxicity. The 96-h median lethal concentration (LC50) of OC was determined to be 251.8μM in larvae and 5.5μM in ZFL cells. Quantitative real-time PCR (qRT-PCR) showed that OC induced the expression of genes for CYPs (CYP1A, CYP3A65), estrogen receptors (ERα, ERβ1, GPER), vitellogenin (VTG1), and sex determination (BRCA2, CYP19A, DMRT1, SOX9A), both in vitro and in vivo. A whole-transcriptome sequencing method was used to evaluate the gene expression profile of larvae exposed to OC. OC was found to mediate the biosynthesis of estrogens (such as estriol) and affect the antioxidant pathway (glutathione transferases and peroxisome). These findings clarify the toxic effects and molecular mechanisms of OC and support banning its use in cosmetics.

Toxicity assessment of synthesized titanium dioxide nanoparticles in fresh water algae Chlorella pyrenoidosa and a zebrafish liver cell line

This study aims to assess the toxicity of the commonly-spread titanium dioxide nanoparticles (TiO2 NPs) by evaluating the exposure impact of the particles on both freshwater algae Chlorella pyrenoidosa and zebrafish liver cell line (ZFL), the two common in vitro models in toxicological studies. To compare the toxic effects of TiO2 NPs with different physiochemical properties, three types of manufactured TiO2 were used: bulk TiO2, Degussa P25 TiO2, and ultrafine TiO2 NPs. Both short and long-term biological responses of green algae, such as the effect on the cell growth rate, pigment autofluorescence, and esterase activity were investigated. The dosage, physical property of TiO2 particles, and their interactions with algal cells affect cellular growth, especially after short-term exposure. The hydrodynamic size plays a critical role in determining the acute toxicity to C. pyrenoidosa in terms of autofluorescence and esterase activity, while all types of TiO2 NPs show toxic effects after exposure for 14 days. However, this observation is not seen when studying the effect of introduced particles in ZFL, for the precipitated Degussa P25 TiO2 showed the highest cellular inhibition. Interestingly, despite the obvious overall toxicity toward C. pyrenoidosa, the photocatalytical properties of TiO2 NPs may contribute to the enhanced photosynthesis in the low concentration range (<40 µg mL−1). Overall, we found that the physical interactions between TiO2 particles and the cells, particles’ size and dispersibility play critical role in the cytotoxic effect for both algal and ZFL cells, while the photocatalytical properties of TiO2 particles may produce mixed effects on the cytotoxicity of green algae.

Effects of multiwalled carbon nanotubes co-exposure with cadmium on zebrafish cell line: Metal uptake and accumulation, oxidative stress, genotoxicity and cell cycle

Carbon nanotubes presence in the environment increases every year because of exponential industrial production around the world. In aquatic environments, carbon nanotubes can interact with other pollutants based on their adsorbent surface chemistry properties. Heavy metal ions represent one of the biggest concerns in water resources nowadays due to anthropogenic activities, in which cadmium (Cd) is one of the most harmful metal for aquatic organisms. This study investigated the influence of two co-exposure protocols differing by the order of interaction of oxidized multiwalled carbon nanotubes (ox-MWCNT) with Cd in zebrafish liver cell line (ZFL). The ox-MWCNT was characterized, Cd content in culture medium and uptake by cells were quantified using ICP-MS and, the reactive oxygen species (ROS), the biotransformation enzymes activity of phase I and II as well as the antioxidants defenses and oxidative damage were analyzed. The effects on the cell cycle were investigated by flow cytometry and DNA damage by comet assay. The exposure to ox-MWCNT alone decreased the activity of catalase, glutathione peroxidase, and glutathione S-transferase and altered the cell cycle with a reduction of cells in the G2/M phase. Cd exposure alone decreased the activity of catalase and glutathione S-transferase, increased ROS, metallothionein, and lipid peroxidation content and causes genotoxicity in the cells. Despite different incubation protocol, the co-exposure ox-MWCNT-Cd increased the Cd content in ZFL cells after 24 h exposure, increased ROS production and DNA damage without differences between them. Our results showed the modulation of ox-MWCNT on Cd effects and contributed to future co-exposure toxicity investigations and nanosafety regulations involving carbon nanomaterials and aquatic pollutants.

Interference of goethite in the effects of glyphosate and Roundup® on ZFL cell line

Goethite (α-FeOOH) brings important perspectives in environmental remediation, as, due to its physicochemical properties, this iron oxide can adsorb a wide variety of compounds, including glyphosate. This study aimed to evaluate the effects of goethite nanoparticles (NPs), glyphosate (Gly), Roundup® (Rd), and co-exposures (Gly + NPs and Rd + NPs) on zebrafish liver cell line (ZFL). ZFL cells were exposed to NPs (1, 10, and 100 mg L−1), Gly (3.6 mg L−1), Rd (10 mg L−1), and co-exposures (Gly + NPs and Rd + NPs), or only to saline for 1, 6, and 12 h. Cell viability was assessed by Trypan blue, MTT, and neutral red assays. The generation of reactive oxygen species and total antioxidant capacity were also determined, while genotoxicity was quantified by the comet assay. Both NPs and Rd in isolation produced cytotoxic effects at 6 h and genotoxic effects at 1 and 6 h. Rd + NPs resulted in synergistic effects, intensifying the toxicity. Cells exposed to Gly did not present toxic effects and Gly + NPs resulted in the suppression of toxic effects observed for NPs. The presence of other components in Roundup® seems to favor its toxicity compared to the active ingredient. In conclusion, according to the in vitro model, the concentrations used were not safe for the ZFL lineage.

Inhibited Lipophagy Suppresses Lipid Metabolism in Zebrafish Liver Cells.

Lipophagy degrades lipid droplets (LDs) through the lysosomal degradative pathway, thus plays important roles in regulating lipid metabolism in mammals. However, information on the existence and functions of lipophagy in fish lipid metabolism is still limited. In the present study, we confirmed the existence of lipophagy by observing the structures of LDs sequestered in autophagic vacuoles in the zebrafish liver cell line (ZFL) via electronic microscopy. Moreover, starved cells increased the mRNA expression of the microtubule-associated protein 1A/1B light chain 3 beta (LC3), which is a marker protein for autophagy and protein conversion from LC3-I to LC3-II. Inhibiting autophagy with chloroquine increased significantly the LDs content and decreased fatty acid β-oxidation and esterification activities in the ZFL cells cultured in the fed state. Furthermore, inhibiting autophagy function downregulated the mRNA expression of the genes and their proteins related to lipid metabolism. Altogether, the present study verified the existence of lipophagy and its essential regulatory roles in lipid metabolism in fish cells.

Functional characterization of copper transporters zCtr1, zAtox1, zAtp7a and zAtp7b in zebrafish liver cell line ZFL.

Copper (Cu) is an essential element for all organisms, serving as an enzyme cofactor to maintain cellular activity and vitality. However, Cu homeostasis must be maintained at the physiological and cellular levels as Cu ions can be highly toxic. In mammals, ATP7A is expressed in most tissues, but relatively lower expression is found in the liver, and is responsible for the intestinal uptake of Cu, while ATP7B is highly expressed in the liver, kidneys and placenta, and is responsible for removal of Cu in the liver. CTR1 and ATOX1 are responsible for cellular Cu uptake and intracellular Cu transport, respectively. Here, using a zebrafish liver cell line (ZFL), we studied the cellular functions of four zebrafish Cu transporters. In zebrafish, zAtp7a is expressed mainly in the liver and zAtp7b is expressed mainly in the intestines, different from that of humans which have a high ATP7b level in the liver and high ATP7a level in the intestines. We here found that zctr1 or zatox1 overexpression increased Cu accumulation in ZFL cells. Moreover, zctr1 overexpression made ZFL cells more sensitive to Cu and Zn exposure, and overexpression of zatox1 or zatp7b increased Cu uptake and Cu tolerance in ZFL cells. Overexpression of zatp7a made ZFL cells more sensitive to Zn. Taken together, our findings suggest that zatp7b is responsible for Cu export despite its expression level being much lower than zatp7a in ZFL cells.

Microcystin-LR-regulated transcriptome dynamics in ZFL cells

Microcystin-LR (MC-LR) is a highly toxic hepatotoxin that poses great hazards to aquatic organisms and even human health. The zebrafish liver cell line (ZFL) is a valuable model for investigating toxicity and metabolism of toxicants. However, the toxicity of MC-LR and its effects on gene transcription of ZFL cells remains to be characterized. In this study, we determined the toxicity of MC-LR for ZFL cells and investigated the effects of MC-LR on cellular transcriptome dynamics. The EC50 of MC-LR for ZFL cells was 80.123 μg/mL. The ZFL cells were exposed to 10 μg/mL MC-LR for 0, 1, 3, 6, 12 or 24 h, and RNA-sequencing was performed to analyze gene transcription. A total of 10,209 genes were found to be regulated by MC-LR. The numbers of up- and down-regulated genes at different time points ranged from 2179 to 3202 and from 1501 to 2597, respectively. Furthermore, 1543 genes underwent differential splicing (AS) upon MC-LR exposure, of which 620 were not identified as differentially expressed gene (DEG). The effects of MC-LR on cellular functions were highly time-dependent. MAPK (mitogen-activated protein kinase) and FoxO (forkhead box O) signaling pathways were the most prominent pathways activated by MC-LR. Steroid biosynthesis and terpenoid backbone biosynthesis were the most enriched for the down-regulated genes. A gene regulatory network was constructed from the expression profile datasets and the candidate master transcription factors were identified. Our results shed light on the molecular mechanisms of MC-LR cellular toxicity and the transcriptome landscapes of ZFL cells upon MC-LR toxicity.

Interactions of oxidized multiwalled carbon nanotube with cadmium on zebrafish cell line: The influence of two co-exposure protocols on in vitro toxicity tests

The widespread production and application of carbon nanotubes (CNT) have raising concerns about their release into the environment and, the joint toxicity of CNT with pre-existing contaminants needs to be assessed. This is the first study that investigated the co-exposure of oxidized multiwalled carbon nanotubes (ox-MWCNT) and cadmium (Cd) using a zebrafish liver cell line (ZFL). Two in vitro co-exposure protocols differing by the order of ox-MWCNT interaction with Cd and fetal bovine serum (FBS) proteins were evaluated. Ox-MWCNT was physical and chemical characterized and its adsorption capacity and colloidal stability in cell culture medium was determined in both protocols. Cytotoxicity was investigated by MTT, neutral red, trypan blue, lactate dehydrogenase assays and the necrosis and apoptosis events were determined using flow cytometer. The Cd presence in medium did not interfere in the protein corona composition of MWCNT but the order of interaction of FBS and Cd interfered in its colloidal stability and metal adsorption rate. The ox-MWCNT increased Cd toxicity at low concentration probably by a “Trojan horse” and/or synergistic effect, and induced apoptosis and necrosis in ZFL cells. Although it was not observed differences of toxicity between protocols, the interaction of ox-MWCNT first with Cd led to its precipitation in cell culture medium and, as a consequence, to a possible false viability result by neutral red assay. Taken together, it was evident that the order of compounds interactions disturbs the colloidal stability and affects the in vitro toxicological assays. Considering that Protocol A showed more ox-MWCNT stability after interaction with Cd, this protocol is recommended to be adopted in future studies.

Transcriptional inhibition of TCDD-mediated induction of cytochrome P450 1A1 and alteration of protein expression in a zebrafish hepatic cell line following the administration of TCDD and Cd2+

We studied the effects of Cd2+ on TCDD-mediated induction of the cytochrome P450 1A1 (cyp1a1) gene using a zebrafish liver cell line (ZFL). Our results showed that Cd2+ inhibited the TCDD-mediated induction of the cyp1a1 protein, enzyme activity, and mRNA expression level. Cd2+ also down-regulated levels of the aryl hydrocarbon receptor (ahr2) and the aryl hydrocarbon receptor nuclear translocator 2b (arnt2b) mRNAs. Compared with TCDD (3nM) treatment alone, co-treatment with Cd2+ (0–30μM) and TCDD (3nM) significantly inhibited the activity of the luciferase reporter gene constructs harboring the distal promoter region (P-2626/-2009) of CYP1A1 and the synthetic 3XRE gene promoter. This indicates that Cd2+ decreased the level of TCDD-induced cyp1a1 through transcriptional inhibition. Proteomic analysis was also used to evaluate the effect of Cd2+ on TCDD-altered protein expression in ZFL cells. The identified proteins are mainly enzymes of the glycolysis pathway and proteasomes, and have anti-oxidative and anti-stress effects.

Toxic effects of triclosan on a zebrafish (Danio rerio) liver cell line, ZFL

Triclosan (TCS, 5-chloro-2-(2,4-dichlorophenoxy) phenol) is an antimicrobial agent widely used in personal care products. It has been detected in surface water, soil, aquatic species, and even humans. In this study, we used zebrafish (Danio rerio) as a model to test the hypothesis that TCS exhibits toxic effects by interacting with thyroid hormone receptor β (TRβ) and aryl hydrocarbon receptor (AhR) and by inducing the transcription of thyroid hormone (TH)-associated genes and affecting phase I and phase II enzymes. The median lethal concentrations (LC50) of TCS in zebrafish embryos/larvae and a zebrafish liver cell line (ZFL) were first determined. Hatched larvae were most sensitive to TCS exposure, with LC50 values ranging from 1.26 to 1.46μM for 96h after hatching exposure. The major effect of TCS was delayed hatching which occurred from 1.13μM. The constructed GFP–zfTRβ fusion protein revealed the subcellular location of zfTRβ as the nucleus in both T3-induced and uninduced states, adding to the difficulty of studying TCS action on thyroid hormone receptors in ZFL cells. TCS had neither agonistic nor antagonistic effects on zfTRβLBD or AhR from the reporter gene systems. Ethoxyresorufin-o-deethylase (EROD) assay suggested that TCS is a weak P4501a (Cyp1a) agonist at 5μM and that it inhibits cytochrome Cyp1a activity induced by benzo(a)pyrene (BaP). In time course-based mRNA profiling in ZFL cells, 4-h exposure to TCS caused a significant (up to 37.5-fold) inhibition of Cyp1a at 2.5μM. An overall inhibition of liver phase I and II gene transcription at 4h exposure indicates the possible quick catabolism of TCS. Our findings suggest that TCS is not a TH mimic that affects TH-related gene expression. The impairment of Cyp1a mRNA expression could be due to stimulation by other stressors such as oxidative stress, warranting further investigation into the underlying mechanism in zebrafish.

Taurine Biosynthesis in a Fish Liver Cell Line (ZFL) Adapted to a Serum-Free Medium.

Although taurine has been shown to play multiple important physiological roles in teleosts, little is known about the molecular mechanisms underlying dietary requirements. Cell lines can provide useful tools for deciphering biosynthetic pathways and their regulation. However, culture media and sera contain variable taurine levels. To provide a useful cell line for the investigation of taurine homeostasis, an adult zebrafish liver cell line (ZFL) has been adapted to a taurine-free medium by gradual accommodation to a commercially available synthetic medium, UltraMEM™-ITES. Here we show that ZFL cells are able to synthesize taurine and be maintained in medium without taurine. This has allowed for the investigation of the effects of taurine supplementation on cell growth, cellular amino acid pools, as well as the expression of the taurine biosynthetic pathway and taurine transporter genes in a defined fish cell type. After taurine supplementation, cellular taurine levels increase but hypotaurine levels stay constant, suggesting little suppression of taurine biosynthesis. Cellular methionine levels do not change after taurine addition, consistent with maintenance of taurine biosynthesis. The addition of taurine to cells grown in taurine-free medium has little effect on transcript levels of the biosynthetic pathway genes for cysteine dioxygenase (CDO), cysteine sulfinate decarboxylase (CSAD), or cysteamine dioxygenase (ADO). In contrast, supplementation with taurine causes a 30% reduction in transcript levels of the taurine transporter, TauT. This experimental approach can be tailored for the development of cell lines from aquaculture species for the elucidation of their taurine biosynthetic capacity.

Nanoparticles as a novel delivery system for vitamin C administration in aquaculture

Abstract The potential of chitosan-based nanoencapsulation as a tool for delivering ascorbic acid (AA) to marine and freshwater organisms was investigated. Polymeric non-loaded and loaded vitamin C nanoparticles (NPs) were made by ionic gelation and the particles were characterized. In vitro performance of nanoparticles was evaluated in a zebrafish liver cell-line (ZFL) and in vivo studies were carried out in fish (post-metamorphic larvae of Solea senegalensis) and rotifers (Brachionus plicatilis) to assess the potential use of these NPs to be used as a tool in nutritional aquaculture studies. The results showed that NPs are suitable to trap hydrosoluble compounds such as AA by forming positively charged complexes (30–35 mV), in a nanosize range ( 90% of loaded AA remained within nanoparticles after 2 h in seawater). The potential cytotoxicity of the NPs was evaluated in ZFL cells and no decrease in cell viability was noted up to 2.5 mg/ml of nanoparticle concentration. The NP uptake was analyzed in ZFL cells by FACS cytometry and confocal laser scanning microscopy (CLSM). Time course and dose–response experiments were performed using fluorescein isothiocyanate labeled NPs (FITC-NPs). The in vitro endocytosis assays with ZFL cells showed a maximum uptake after 6 h of incubation and a dose-dependent increase of fluorescence intensity directly proportional to the FITC-NP concentration. The antioxidant properties of vitamin C nanoparticles (AA-NPs) were also analyzed in ZFL cell extracts. Lipopolysaccharide (LPS) was added to ZFL cells to induce oxidative stress. The total antioxidant capacity of the AA-NP-treated cells showed a statistically significant increase with respect to the control with non-loaded nanoparticles (71.00 ± 9.6 and 25.36 ± 3.96 μM Trolox equivalent; p

Distinct Roles of Two Zebrafish AHR Repressors (AHRRa and AHRRb) in Embryonic Development and Regulating the Response to 2,3,7,8-Tetrachlorodibenzo-p-dioxin

The aryl hydrocarbon receptor (AHR) repressor (AHRR), an AHR-related basic helix-loop-helix/Per-AHR nuclear translocator-Sim protein, is regulated by an AHR-dependent mechanism and acts as a transcriptional repressor of AHR function. Resulting from a teleost-specific genome duplication, zebrafish have two AHRR genes (AHRRa and AHRRb), but their functions in vivo are not well understood. We used antisense morpholino oligonucleotides (MOs) in zebrafish embryos and a zebrafish liver cell line (ZF-L) to characterize the interaction of AHRRs and AHRs in normal embryonic development, AHR signaling, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. Zebrafish embryos exposed to TCDD (2 and 8nM) during early development showed strong induction of CYP1A, AHRRa, and AHRRb at 48 and 72 hours post-fertilization (hpf). An MO targeting AHR2 inhibited TCDD-induced expression of CYP1A, AHRRa, and AHRRb by 84-95% in 48 hpf embryos, demonstrating a primary role for AHR2 in mediating AHRR induction. Dual MO knockdown of both AHRRs in ZF-L cells enhanced TCDD induction of CYP1A, but not other CYP1 genes. In embryos, dual knockdown of AHRRs, or knockdown of AHRRb alone, enhanced the induction of CYP1A, CYP1B1, and CYP1C1 by TCDD and decreased the constitutive expression of Sox9b. In contrast, knockdown of AHRRa did not affect Sox9b expression or CYP1 inducibility. Embryos microinjected with each of two different MOs targeting AHRRa and exposed to dimethyl sulfoxide (DMSO) displayed developmental phenotypes resembling those typical of TCDD-exposed embryos (pericardial edema and lower jaw malformations). In contrast, no developmental phenotypes were observed in DMSO-exposed AHRRb morphants. These data demonstrate distinct roles of AHRRa and AHRRb in regulating AHR signaling in vivo and suggest that they have undergone subfunction partitioning since the teleost-specific genome duplication.

Functional characterization of aryl hydrocarbon receptor (zfAHR2) localization and degradation in zebrafish ( Danio rerio)

The basic-helix-loop-helix/PAS (bHLH/PAS) family of proteins is a group of transcription factors that regulate key pathways during normal development and in the response to stress. The aryl hydrocarbon receptor (AHR) is a member of this family. Recently, Danio rerio (zebrafish) has become an important model system in the study of the signal transduction pathway and complements the results seen in mammalian models. However, studies of the AHR protein have been limited by the lack of antibody reagents and thus, little is known concerning the localization and degradation of the zebrafish AHR (zfAHR). In this report, we describe the production and characterization of specific polyclonal antibodies to the zfAHR2 protein and the analysis of AHR-mediated signal transduction in the zebrafish liver cell line (ZFL). The results show that the zfAHR2 is degraded via the 26S proteasome following exposure of cells to β-naphthoflavone (BNF). Interestingly, the time course is slower and the magnitude of zfAHR2 degradation is not as great as seen for the mammalian AHR. Studies also show that the zfAHR2 is rapidly degraded in a ligand-independent manner by exposure of cells to geldanamycin (GA) to levels consistent with mammalian AHR. Finally, immunohistochemical staining of the ZFL cells suggest that the unliganded AHR resides in both the cytoplasm and nucleus and undergoes active nucleocytoplasmic shuttling in the absence of ligand. These results suggest that there is conservation of function between fish and mammals with respect to ligand-dependent and -independent degradation of the AHR and that the zfAHR2 is degraded via the 26S proteasome.