Zebrafish Hepatocytes Research Articles

Computational Analysis of Calcium Flux Data Using R.

Calcium imaging has emerged as a powerful tool for studying cellular dynamics, with applications spanning neuroscience, cell biology, and beyond. In this chapter, we present a comprehensive guide to the computational analysis of calcium flux data using the R programming language. Using an example of in vivo live imaging of GCaMP signal in zebrafish hepatocytes, we demonstrate techniques for segmentation, normalization, and quantification of calcium transients. We provide a step-by-step code example showcasing extraction of meaningful information from calcium imaging datasets. The code allows insights into the number of oscillating cells, number of oscillations per cell within a time frame, and generation of publication-ready plots for showcasing calcium dynamics. This chapter serves as a valuable resource for researchers seeking to leverage freely available computational tools for analyzing calcium flux data at cellular resolution and uncovering novel insights into cellular physiology.

A ‘molecular chameleon’ mimic ZrIV-consolidated Fluoro-specific coordination complex and arseno-selective supramolecular metallogelator as a phase discriminatory sensing probe: Substantiation of in-vitro and in-silico approaches with the sensing response

ZrIV consolidated solvatochromic and halochromic coordination complex [ZrIV-ABH (C)] has been synthesized which exhibited promising selectivity and sensitivity towards aqueous phase F-. Upon addition of a trace amount of F-, ZrIV-ABH (C) showcased concentration-reliant chromogenic alteration and fluorogenic spectral shift. Sensing phenomena were corroborated through, UV–Vis spectroscopy, photoluminescence and cyclic voltammetry along with an in-silico approach. The equivalence-dependent chromo-fluorogenic alteration upon the addition of F- on ZrIV-ABH (C), further has been validated theoretically by means of diminution of energy of fluoro-adducts. The bio-compatibility of the probe has been validated through in-vitro detection of F- from hepatocytes of zebrafish and HepG2 cell line. The anti-interfering sensing response of F- among its geogenic lethal congeners has been further utilized to formulate a six-input-two-output logic gate circuitry. Furthermore, ZrIV epicentric coordination complex-based metallogelator probe namely, ZrIV-ABH (G) has been synthesized which can detect AsO2- selectively from the aqueous phase. ZrIV-ABH (G) has been utilized for the real-field validation of aquifer-genic AsO2-. The solvent as well as the temperature-specific formation of ZrIV-ABH (G) metallogelator has been substantiated by rheological behavior. The thermosensitive activity of ZrIV-ABH (G) has been professed by means of chromogenic alteration and fluorogenic turn-on response at a particular elevated temperature. Therefore, this phase-selective switching of sensing response from F- to AsO2- validated its endurance as a value-added contender in its category.

Pyridaben impaired cell cycle progression through perturbation of calcium homeostasis and PI3K/Akt pathway in zebrafish hepatocytes

Environmental pollution caused by pesticides is a growing concern. Pyridaben, a widely used organochlorine insecticide, is a representative water pollutant. Owing to its extensive usage, it has been detected in various aquatic ecosystems, including rivers and oceans. Pyridaben is highly toxic to aquatic organisms; however, the mechanism of its toxicity in the liver, which is important in toxicant metabolism, has not been studied. Therefore, we employed zebrafish and its well-characterized liver cell line, ZFL to assess pyridaben hepatotoxicity and explore its potential mechanisms of action. Pyridaben led to reduction of the liver size and fluorescence intensity of dsRed-labeled Tg (fabp10a:dsRed) zebrafish. It reduced the viability and proliferation of ZFL cells in vitro by inducing apoptosis and cell cycle arrest. These changes might be primarily linked to uncontrolled intracellular calcium flow in ZFL cells exposed to pyridaben. Additionally, it also downregulates the PI3K/Akt signaling cascade, leading to the inactivation of Gsk3β and nuclear translocation of β-catenin. Taken together, our findings suggest that pyridaben could have hepatotoxic effects on aquatic organisms. This study is the first to provide insight into the hepatotoxic mechanism of pyridaben using both in vivo and in vitro models.

Ronidazole Is a Superior Prodrug to Metronidazole for Nitroreductase-Mediated Hepatocytes Ablation in Zebrafish Larvae.

The liver plays a very important role in physiological processes of the human body. Liver regeneration has developed into an important area of study in liver disease. The Mtz (metronidazole)/NTR (nitroreductase)-mediated cell ablation system has been widely used to study the processes and mechanisms of liver injury and regeneration. However, high concentrations and toxic side effects of Mtz severely limit the application of the Mtz/NTR system. Therefore, screening new analogs to replace Mtz has become an important means to optimize the NTR ablation system. In this study, we screened five Mtz analogs including furazolidone, ronidazole, ornidazole, nitromide, and tinidazole. We compared their toxicity on the transgenic fish line Tg(fabp10a: mCherry-NTR) and their specific ablation ability on liver cells. The results showed that Ronidazole at a lower concentration (2 mM) had the same ability to ablate liver cells comparable with that of Mtz (10 mM), almost without toxic side effects on juvenile fish. Further study found that zebrafish hepatocyte injury caused by the Ronidazole/NTR system achieved the same liver regenerative effect as the Mtz/NTR system. The above results show that Ronidazole can replace Mtz with NTR to achieve superior damage and ablation effects in zebrafish liver.

Ecotoxicological assessment of guanitoxin-producing cyanobacteria in Danio rerio and Daphnia similis

Anthropogenic activity has dramatically deteriorated aquatic ecosystems in recent years. Such environmental alterations could change the primary producers’ composition, exacerbating the proliferation of harmful microorganisms such as cyanobacteria. Cyanobacteria can produce several secondary metabolites, including guanitoxin, a potent neurotoxin and the only naturally occurring anticholinesterase organophosphate ever reported in the literature. Therefore, this study investigated the acute toxicity of guanitoxin-producing cyanobacteria Sphaerospermopsis torques-reginae (ITEP-024 strain) aqueous and 50% methanolic extracts in zebrafish (Danio rerio) hepatocytes (ZF-L cell line), zebrafish embryos (fish embryo toxicity - FET) and specimens of the microcrustacean Daphnia similis. For this, hepatocytes were exposed to 1–500 mg/L of the ITEP-024 extracts for 24 h, the embryos to 31.25–500 mg/L for 96 h, and D. similis to 10–3000 mg/L for 48 h. Non-target metabolomics was also performed to analyze secondary metabolites produced by the ITEP-024 using LC-MS/MS. Metabolomics indicated the guanitoxin presence just in the aqueous extract of the ITEP-024 and the presence of the cyanopeptides namalides, spumigins, and anabaenopeptins in the methanolic extract. The aqueous extract decreased the viability of zebrafish hepatocytes (EC(I)50(24h) = 366.46 mg/L), and the methanolic extract was not toxic. FET showed that the aqueous extract (LC50(96) = 353.55 mg/L) was more toxic than the methanolic extract (LC50(96) = 617.91 mg/L). However, the methanolic extract had more sublethal effects, such as abdominal and cardiac (cardiotoxicity) edema and deformation (spinal curvature of the larvae). Both extracts immobilized daphnids at the highest concentration analyzed. However, the aqueous extract was nine times more lethal (EC(I)50(48h) = 108.2 mg/L) than the methanolic extract (EC(I)50(48h) = 980.65 mg/L). Our results showed an imminent biological risk for aquatic fauna living in an ecosystem surrounded by ITEP-024 metabolites. Our findings thus highlight the urgency of understanding the effects of guanitoxin and cyanopeptides in aquatic animals.

Atm Mutation and Oxidative Stress Enhance the Pre-Cancerous Effects of UHRF1 Overexpression in Zebrafish Livers.

The ataxia-telangiectasia mutated (atm) gene is activated in response to genotoxic stress and leads to activation of the tp53 tumor suppressor gene which induces either senescence or apoptosis as tumor suppressive mechanisms. Atm also serves non-canonical functions in the response to oxidative stress and chromatin reorganization. We previously reported that overexpression of the epigenetic regulator and oncogene Ubiquitin Like with PHD and Ring Finger Domains 1 (UHRF1) in zebrafish hepatocytes resulted in tp53-dependent hepatocyte senescence, a small liver and larval lethality. We investigated the role of atm on UHRF1-mediated phenotypes by generating zebrafish atm mutants. atm-/- adults were viable but had reduction in fertility. Embryos developed normally but were protected from lethality caused by etoposide or H2O2 exposure and failed to fully upregulate Tp53 targets or oxidative stress response genes in response to these treatments. In contrast to the finding that Tp53 prevents the small liver phenotype caused by UHRF1 overexpression, atm mutation and exposure to H2O2 further reduced the liver size in UHRF1 overexpressing larvae whereas treatment with the antioxidant N-acetyl cysteine suppressed this phenotype. We conclude that UHRF1 overexpression in hepatocytes causes oxidative stress, and that loss of atm further enhances this, triggering elimination of these precancerous cells, leading to a small liver.

In vivo imaging of calcium dynamics in zebrafish hepatocytes.

Hepatocytes were the first cell type for which oscillations of cytoplasmic calcium levels in response to hormones were described. Since then, investigation of calcium dynamics in liver explants and culture has greatly increased our understanding of calcium signaling. A bottleneck, however, exists in observing calcium dynamics in a noninvasive manner because of the optical inaccessibility of the mammalian liver. Here, we aimed to take advantage of the transparency of the zebrafish larvae to image hepatocyte calcium dynamics in vivo at cellular resolution. We developed a transgenic model expressing a calcium sensor, GCaMP6s, specifically in zebrafish hepatocytes. Using this, we provide a quantitative assessment of intracellular calcium dynamics during multiple contexts, including growth, feeding, ethanol-induced stress, and cell ablation. Specifically, we show that synchronized calcium oscillations are present in vivo , which are lost upon starvation. Starvation induces lipid accumulation in the liver. Feeding recommences calcium waves in the liver, but in a spatially restricted manner, as well as resolves starvation-induced hepatic steatosis. By using a genetically encoded scavenger for calcium, we show that dampening of calcium signaling accelerates the accumulation of starvation-related lipid droplets in the liver. Furthermore, ethanol treatment, as well as cell ablation, induces calcium flux, but with different dynamics. The former causes asynchronous calcium oscillations, whereas the latter leads to a single calcium spike. We demonstrate the presence of oscillations, waves, and spikes in vivo . Calcium waves are present in response to nutrition and negatively regulate starvation-induced accumulation of lipid droplets.

Esculetin Alleviates Nonalcoholic Fatty Liver Disease on High-Cholesterol-Diet-Induced Larval Zebrafish and FFA-Induced BRL-3A Hepatocyte.

Non-alcoholic fatty liver disease (NAFLD), defined in recent years as metabolic-associated fatty liver disease (MAFLD), is one of the most common liver diseases in the world, with no drugs on market. Esculetin (ESC) is an active compound discovered in a variety of natural products that modulates a wide range of metabolic diseases and is a potential drug for the treatment of NAFLD. In this study, we used an HCD-induced NAFLD larval zebrafish model in vivo and an FFA-induced BRL-3A hepatocyte model in vitro to evaluate the anti-NAFLD effect of ESC. Lipid lowering, anti-oxidation and anti-inflammation effects were revealed on ESC and related gene changes were observed. This study provides a reference for further study and development of ESC as a potential anti-NAFLD/MAFLD drug.

Ultrastructural evidence of four types of lipofuscins in the melanomacrophagic centers in hepatocytes of zebrafish (Denio rerio)

Melanomacrophagic centers (MMCs) were studied in the hepatocytes of zebrafish using transmission electron microscope (TEM). The MMCs with irregular or amoeboid nucleus were located in the hepatocytes adjacent to the bile canaliculi. Several engulfed structures were present in the cytoplasm of MMCs. The most frequent observation was the presence of mitochondria, ranging in size from small to giant, with distorted shape and inconspicuous cristae. Occasionally the fragments of erythrocytes were found. The rough endoplasmic reticulum (rER) showed whirling around the mitochondria and lipid droplets, forming membrane-like structures. The damaged mitochondria were invaded by the lysosomes, and this was covered by a membrane led to the formation of lipofuscin. Four different types of lipofuscins were observed; namely, (1) granular with/without vacuoles of high electron-density, (2) homogenous surrounded by indistinct limiting membrane, (3) lamellated structures similar to inner matrix and cristae of mitochondria, and, (4) compound structure made by the combinations of first 3 types, (granular and homogenous, granular and lamellated, homogenous and lamellated). The present evidence suggests that MMCs in the hepatocytes of zebrafish perform continuous functions of removal of the damaged cellular organelles. The lipofuscin formation work in coordination with the cellular players of immune system and remove pathogens and maintain the internal homeostasis of cells.

Berberine Regulation of Cellular Oxidative Stress, Apoptosis and Autophagy by Modulation of m6A mRNA Methylation through Targeting the Camk1db/ERK Pathway in Zebrafish-Hepatocytes.

Berberine (BBR) ameliorates cellular oxidative stress, apoptosis and autophagy induced by lipid metabolism disorder, however, the molecular mechanism associated with it is not well known. To study the mechanism, we started with m6A methylation modification to investigate its role in lipid deposition zebrafish hepatocytes (ZFL). The results showed that BBR could change the cellular m6A RNA methylation level, increase m6A levels of Camk1db gene transcript and alter Camk1db gene mRNA expression. Via knockdown of the Camk1db gene, Camk1db could promote cellular ERK phosphorylation levels. Berberine regulated the expression level of Camk1db mRNA by altering the M6A RNA methylation of the Camk1db gene, which further affected the synthesis of calmodulin-dependent protein kinase and activated ERK signaling pathway resulting in changes in downstream physiological indicators including ROS production, cell proliferation, apoptosis and autophagy. In conclusion, berberine could regulate cellular oxidative stress, apoptosis and autophagy by mediating Camk1db m6A methylation through the targeting of the Camk1db/ERK pathway in zebrafish-hepatocyte.

Environmentally relevant fluoride alters nuclear integrity in erythrocytes and induces DNA damage in hepatocytes of zebrafish

Environmentally relevant fluoride alters nuclear integrity in erythrocytes and induces DNA damage in hepatocytes of zebrafish

Concentration- and time-dependence toxicity of graphene oxide (GO) and reduced graphene oxide (rGO) nanosheets upon zebrafish liver cell line.

Graphene oxide (GO) and reduced graphene oxide (rGO) are carbon-based nanomaterials that have a wide range of applicability. Therefore, it is expected that their residual traces reach the aquatic environment, accumulate, and interact with its different compartments and the biota living in them. The concentration- and time-dependency response to GO and rGO in aquatic organisms are still poorly known. In the present study, the effects of GO and rGO on zebrafish hepatocytes were investigated using in vitro assays performed with established liver cell lines from zebrafish (ZFL). GO and rGO nanosheets were applied on ZFL cells at a concentration range of 1-100 µg mL-1 for 24 and 72 h. The internalization of GO and rGO nanosheets, reactive oxygen species (ROS) production, cell viability, and cell death were evaluated. The internalization of GO increased as the concentrations of GO increased. The rGO nanosheets were smaller than GO nanosheets, and their hydrophobic characteristic favors their interaction with the cell membrane. However, the rGO nanosheets were not observed in the uptake assay. Exposure for 72 h was found to cause harmful effects in ZFL cells, causing higher ROS production in cells exposed to rGO and stopping cell replication. Nevertheless, GO did not stop cell replication, but exposed cells had higher levels of apoptosis and necrosis. After 72 h, both GO and rGO were toxic, but with different mechanisms of toxicity.

Prostaglandin 2α Promotes Autophagy and Mitochondrial Energy Production in Fish Hepatocytes.

Fatty liver, characterized by excessive lipid droplet (LD) accumulation in hepatocytes, is a common physiological condition in humans and aquaculture species. Lipid mobilization is an important strategy for modulating the number and size of cellular LDs. Cyclooxygenase (COX)-mediated arachidonic acid derivatives are known to improve lipid catabolism in fish; however, the specific derivatives remain unknown. In the present study, we showed that serum starvation induced LD degradation via autophagy, lipolysis, and mitochondrial energy production in zebrafish hepatocytes, accompanied by activation of the COX pathway. The cellular concentration of PGF2α, but not other prostaglandins, was significantly increased. Administration of a COX inhibitor or interference with PGF2α synthase abolished serum deprivation-induced LD suppression, LD–lysosome colocalization, and expression of autophagic genes. Additionally, exogenous PGF2α suppressed the accumulation of LDs, promoted the accumulation of lysosomes with LD and the autophagy marker protein LC3A/B, and augmented the expression of autophagic genes. Moreover, PGF2α enhanced mitochondrial accumulation and ATP production, and increased the transcript levels of β-oxidation- and mitochondrial respiratory chain-related genes. Collectively, these findings demonstrate that the COX pathway is implicated in lipid degradation induced by energy deprivation, and that PGF2α is a key molecule triggering autophagy, lipolysis, and mitochondrial development in zebrafish hepatocytes.

Development and In Vitro Cytotoxicity of Citrus sinensis Oil-Loaded Chitosan Electrostatic Complexes

Electrostatic complexes based on chitosan, lecithin, and sodium tripolyphosphate were produced and evaluated with respect to their encapsulation capacity and cytotoxicity. Physical chemical properties were determined by zeta potential values and size distributions. For encapsulation assays, the emulsification method was followed, and Citrus senensis peel oil was utilized as volatile compound model. Morphology of complexes with oil incorporated was observed by scanning electron microscopy. The cytotoxicity of complexes was related to cell viability of zebrafish hepatocytes. The complexes produced presented positive Zeta potential values and size distributions dependent on the mass ratio between compounds. Higher concentrations of sodium tripolyphosphate promote significant changes (p < 0.05) in zeta values, which did not occur at smaller concentrations of the crosslinking agent. These complexes were able to encapsulate Citrus sinensis peel oil, with encapsulation efficiency higher than 50%. Cytotoxicity profiles showed that in a range of concentrations (0.1–100 μg/mL) studied, they did not promote cellular damage in zebrafish liver cells, being potential materials for food and pharmaceutical applications.

Proliferative response avoids mutagenic effects of titanium dioxide (TiO2) nanoparticles in a zebrafish hepatocyte cell line

Titanium dioxide nanoparticles (TiO2NP) have been widely applied in numerous industrial products, and their discharge into wastewater continues to increase, representing a risk hazard for aquatic biota. In the aquatic environment, NP enter fish cells, accumulate in vesicles, and interact with organelles and DNA. This study evaluated the acute (24 h) toxic potential of TiO2NP in the emergent Neotropical model zebrafish liver (ZFL) cell lines at concentrations of 0, 250, 500, 750, and 1000 ng mL−1. The main aim was to determine if TiO2NP causes ROS production and affects cell function due to membrane damage, mitochondrial activity impairment, lysosome malfunction, and DNA damage, which may lead to cell death. High concentrations (750 and 1000 ng mL−1) of TiO2NP increased ROS production and caused cell membrane damage, cell proliferation, lysosome proliferation or swelling, DNA strand breaks, and a low frequency of micronucleus formation. Cytotoxicity and genotoxicity were likely related to ROS production and NP uptake, which impair cell function and integrity and activate necrosis and apoptosis. This study contributes to the understanding of TiO2NP cytotoxicity in fish cells and provides new insights into proliferative hepatocyte responses that allow ZFL cells to lower TiO2NP toxicity and avoid its mutagenic potential.

Development, scrutiny, and modulation of transient reporter gene assays of the xenobiotic metabolism pathway in zebrafish hepatocytes.

The "toxicology in the twenty-first century" paradigm shift demands the development of alternative in vitro test systems. Especially in the field of ecotoxicology, coverage of aquatic species-specific assays is relatively scarce. Transient reporter gene assays could be a quick, economical, and reliable bridging technology. However, the user should be aware of potential pitfalls that are influenced by reporter vector geometry. Here, we report the development of an AhR-responsive transient reporter-gene assay in the permanent zebrafish hepatocytes cell line (ZFL). Additionally, we disclose how viral, constitutive promoters within reporter-gene assay cassettes induce squelching of the primary signal. To counter this, we designed a novel normalization vector, bearing an endogenous zebrafish-derived genomic promoter (zfEF1aPro), which rescues the squelching-delimited system, thus, giving new insights into the modulation of transient reporter systems under xenobiotic stress. Finally, we uncovered how the ubiquitously used ligand BNF promiscuously activates multiple toxicity pathways of the xenobiotic metabolism and cellular stress response in an orchestral manner, presumably leading to a concentration-related inhibition of the AhR/ARNT/XRE-toxicity pathway and non-monotonous concentration-response curves. We named such a multi-level inhibitory mechanism that might mask effects as "maisonette squelching." A transient reporter gene assay in zebrafish cell lines utilizing endogenous regulatory gene elements shows increased in vitro toxicity testing performance. Synthetic and constitutive promotors interfere with signal transduction ("squelching") and might increase cellular stress (cytotoxicity). The squelching phenomenon might occur on multiple levels (toxicity pathway crosstalk and normalization vector), leading to a complete silencing of the reporter signal.

Identification of NQO2 As a Protein Target in Small Molecule Modulation of Hepatocellular Function.

The utility of in vitro human disease models is mainly dependent on the availability and functional maturity of tissue-specific cell types. We have previously screened for and identified small molecules that can enhance hepatocyte function in vitro. Here, we characterize the functional effects of one of the hits, FH1, on primary human hepatocytes in vitro, and also in vivo on primary hepatocytes in a zebrafish model. Furthermore, we conducted an analogue screen to establish the structure-activity relationship of FH1. We performed affinity-purification proteomics that identified NQO2 to be a potential binding target for this small molecule, revealing a possible link between inflammatory signaling and hepatocellular function in zebrafish and human hepatocyte model systems.

Farnesoid X Receptor Activation Impairs Liver Progenitor Cell-Mediated Liver Regeneration via the PTEN-PI3K-AKT-mTOR Axis in Zebrafish.

Following mild liver injury, pre-existing hepatocytes replicate. However, if hepatocyte proliferation is compromised, such as in chronic liver diseases, biliary epithelial cells (BECs) contribute to hepatocytes through liver progenitor cells (LPCs), thereby restoring hepatic mass and function. Recently, augmenting innate BEC-driven liver regeneration has garnered attention as an alternative to liver transplantation, the only reliable treatment for patients with end-stage liver diseases. Despite this attention, the molecular basis of BEC-driven liver regeneration remains poorly understood. By performing a chemical screen with the zebrafish hepatocyte ablation model, in which BECs robustly contribute to hepatocytes, we identified farnesoid X receptor (FXR) agonists as inhibitors of BEC-driven liver regeneration. Here we show that FXR activation blocks the process through the FXR-PTEN (phosphatase and tensin homolog)-PI3K (phosphoinositide 3-kinase)-AKT-mTOR (mammalian target of rapamycin) axis. We found that FXR activation blocked LPC-to-hepatocyte differentiation, but not BEC-to-LPC dedifferentiation. FXR activation also suppressed LPC proliferation and increased its death. These defects were rescued by suppressing PTEN activity with its chemical inhibitor and ptena/b mutants, indicating PTEN as a critical downstream mediator of FXR signaling in BEC-driven liver regeneration. Consistent with the role of PTEN in inhibiting the PI3K-AKT-mTOR pathway, FXR activation reduced the expression of pS6, a marker of mTORC1 activation, in LPCs of regenerating livers. Importantly, suppressing PI3K and mTORC1 activities with their chemical inhibitors blocked BEC-driven liver regeneration, as did FXR activation. FXR activation impairs BEC-driven liver regeneration by enhancing PTEN activity; the PI3K-AKT-mTOR pathway controls the regeneration process. Given the clinical trials and use of FXR agonists for multiple liver diseases due to their beneficial effects on steatosis and fibrosis, the detrimental effects of FXR activation on LPCs suggest a rather personalized use of the agonists in the clinic.

Attenuating the Epidermal Growth Factor Receptor-Extracellular Signal-Regulated Kinase-Sex-Determining Region Y-Box 9 Axis Promotes Liver Progenitor Cell-Mediated Liver Regeneration in Zebrafish.

The liver is a highly regenerative organ, but its regenerative capacity is compromised in severe liver injury settings. In chronic liver diseases, the number of liver progenitor cells (LPCs) correlates proportionally to disease severity, implying that their inefficient differentiation into hepatocytes exacerbates the disease. Moreover, LPCs secrete proinflammatory cytokines; thus, their prolonged presence worsens inflammation and induces fibrosis. Promoting LPC-to-hepatocyte differentiation in patients with advanced liver disease, for whom liver transplantation is currently the only therapeutic option, may be a feasible clinical approach because such promotion generates more functional hepatocytes and concomitantly reduces inflammation and fibrosis. Here, using zebrafish models of LPC-mediated liver regeneration, we present a proof of principle of such therapeutics by demonstrating a role for the epidermal growth factor receptor (EGFR) signaling pathway in differentiation of LPCs into hepatocytes. We found that suppression of EGFR signaling promoted LPC-to-hepatocyte differentiation through the mitogen-activated ERK kinase (MEK)-extracellular signal-regulated kinase (ERK)-sex-determining region Y-box 9 (SOX9) cascade. Pharmacological inhibition of EGFR or MEK/ERK promoted LPC-to-hepatocyte differentiation as well as genetic suppression of the EGFR-ERK-SOX9 axis. Moreover, Sox9b overexpression in LPCs blocked their differentiation into hepatocytes. In the zebrafish liver injury model, both hepatocytes and biliary epithelial cells contributed to LPCs. EGFR inhibition promoted the differentiation of LPCs regardless of their origin. Notably, short-term treatment with EGFR inhibitors resulted in better liver recovery over the long term. The EGFR-ERK-SOX9 axis suppresses LPC-to-hepatocyte differentiation during LPC-mediated liver regeneration. We suggest EGFR inhibitors as a proregenerative therapeutic drug for patients with advanced liver disease.

ABC proteins activity and cytotoxicity in zebrafish hepatocytes exposed to triclosan.

Chemicals such as triclosan are a concern because of their presence on daily products (soap, deodorant, hand sanitizers …), consequently this compound has an ubiquitous presence in the environment. Little is known about the effect of this bactericide on aquatic life. The aim of this study is to analyze triclosan exposure (24h) to an invitro model, zebrafish hepatocytes cell line (ZF-L), if it can be cytotoxic (mitochondrial activity, membrane stability and apoptosis) and if can activate ATP-binding cassette (ABC) proteins (activity, expression and protein/compound affinity). Triclosan was cytotoxic to hepatocytes when exposed to concentrations (1-4mg/L). The results showed impaired mitochondria function, as well, plasma membrane rupture and an increase of apoptotic cells. We observed an ABC proteins activity inhibition in cells exposed to 0.5 and 1mg/L. When ABCBs and ABCC2 proteins expression were analyzed, there was an increase of protein expression in both ABC proteins families on cells exposed to 1mg/L of triclosan. On molecular docking results, triclosan and the fluorescent used as substrate (rhodamine) presented high affinity with all ABC proteins family tested, showing a greater affinity with ABCC2. In conclusion, this study showed that triclosan can be cytotoxic to ZF-L. Molecular docking indicated high affinity between triclosan and the tested pumps.