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Derivation and characterization of a zebrafish liver cell line
Abstract
ZF-L cells were derived from normal adult zebrafish liver, and have been growing in culture for more than 100 generations. The cells were derived in basal nutrient medium supplemented with fetal bovine serum (FBS), trout serum, trout embryo extract, bovine insulin and mouse epidermal growth factor. After 50 generations in culture, optimal growth of the cells was achieved in medium supplemented with FBS (5%) and trout serum (0.5%). ZF-L cells were hypodiploid (modal chromosome number = 46) and exhibited an epithelial morphology. ZF-L cell homogenates exhibited alanine and aspartate aminotransferase, glucose-6-phosphatase and alkaline phosphatase enzyme activities. The cells synthesized and released several proteins into the culture medium, including a 70 kDa protein recognized by anti-bovine serum albumin IgG.
... 32 Very few gene expression studies have been performed on RPE in zebrafish, [33][34][35] which is partly due to the difficulty in obtaining intact RPE tissue. Several workers have developed continuous stable cell cultures from zebrafish, embryo, and adult tissues, 36 ZF4 cell line derived from 1 day post fertilization (dpf) embryos, 37 ZFL cell line derived from normal adult livers, 38,39 zebrafish fibroblast-like cell lines ZF13 and ZF29 generated from 20 h embryos, 40 zebrafish spleen cell line, ZSSJ, 41 and PAC2 cell line from 24 h zebrafish embryos. 42 Laale 43 reported in vitro RPE migration and monolayer formation from blastoderm explants of zebrafish for the first time. ...
... Taju et al. 14 with 10% FBS. Ghosh et al. 39 reported that ZF-L cell line derived from adult zebrafish liver cultured in LDF (50% Leibovitz L-15/35% Dulbecco's modified Eagle's/15% Ham's F12 media) basal nutrient medium supplemented with insulin, epidermal growth factor, trout embryo extract, trout serum, and FBS had a doubling time of *72 h. ...
... 72 ( -1.17),39.95 ( -1.90), and 59.18 ( -1.21)% with no significant differences between replicates for 100, 500, and 1000 cells, respectively. ...
Abstract Danio rerio retinal pigmented epithelial (DrRPE) cell line, derived from the RPE tissue, was established and characterized. The cells were able to grow at a wide range of temperatures from 25°C to 32°C in Leibovitz's L-15 medium. The DrRPE cell line consists of epithelial cells with a diameter of 15-19 μm. The cell line was characterized by mitochondrial 12S rRNA gene, immunocytochemical analysis, and karyotyping. DrRPE cells treated with 10 μM of all-trans-retinol for 24 h readily formed lipid droplets. DrRPE cells were irradiated with narrowband ultraviolet-B (UV-B) radiation at different time periods of 0, 10, 20, and 40 min. The cells were subsequently examined for changes in morphology, cell viability, phagocytotic activity, mitochondrial distribution, nuclei morphology, generation of reactive oxygen species, and expression of apoptotic-related genes p53 and Cas3 by quantitative polymerase chain reaction. The results demonstrate that UV-B radiation can cause a considerable decrease in DrRPE cell viability as well as in phagocytotic activity. In addition, the results demonstrate that UV-B radiation can induce the degradation of mitochondria and DNA in cultured DrRPE cells.
... The first report of tissue-specific cell line (ZPF) and embryonic cell line (ZEM) featuring fibroblast morphology were derived from the fin tissue and mid-blastula, respectively [5]. Most in vitro research on zebrafish has been intensively carried out in the liver tissue-specific epithelial cell line ZFL [6]. Over the decades, very few tissue-specific cell lines, viz., ZSSJ (spleen), DrRPE (retinal pigment), DRM (muscle), DrG (Gills), DrF (caudal fin), and DRCF (caudal fin) [7][8][9][10][11][12] respectively have been reported from the genetically important model animal. ...
... The tissue-specific cell lines from zebrafish find their applications limited to wound healing, ultraviolet radiation biology, contaminants toxicity, and gene expression studies. Although zebrafish is a vital model organism, it lacks other in vitro experimental approaches due to the unavailability of suitable cell culture systems [6]. To exploit various in vitro research applications, establishing novel tissue-specific cell lines of higher genetic importance is foremost. ...
Background
The available fully sequenced genome and genetic similarities compared to humans make zebrafish a prominent in vitro vertebrate model for drug discovery & screening, toxicology, and radiation biology. Zebrafish also possess well developed immune systems which is ideal for studying infectious diseases. Fish skin confers immunity by serving as a physical barrier against the invading pathogens in the aquatic habitat. Therefore in vitro models from the skin tissue of zebrafish help to study the physiology, functional genes in vitro, wound healing, and pathogenicity of microbes. Hence the study aimed to develop and characterize a skin cell line from the wild-type zebrafish Danio rerio.Methods and resultsA novel cell line designated as DRS (D. rerio skin) was established and characterized from the skin tissue of wild-type zebrafish, D. rerio, by the explant technique. The cells thrived well in the Leibovitz’s -15 medium supplemented with 15% FBS and routinely passaged at regular intervals. The DRS cells mainly feature fibroblast-like morphology. The culture conditions of the cells were determined by incubating the cells at varying concentrations of FBS and temperature; the optimum was 15% FBS and 28 °C, respectively. Cells were cryopreserved and revived with 70–75% viability at different passage levels. Two extracellular products from bacterial species Aeromonas hydrophila and Edwardsiella tarda were tested and found toxic to the DRS cells. Mitochondrial genes, namely COI and 16S rRNA PCR amplification and partial sequencing authenticated the species of origin of cells. The modal diploid (2n) chromosome number of the cells was 50. The cell line DRS was found to be free from mycoplasma. The cells were transfected with pMaxGFP plasmid and tested positive for green fluorescence at 24–48 h post-transfection.Conclusion
The findings from this study thus confirm the usefulness of the developed cell line in bacterial susceptibility and transgene expression studies.
... Esse complexo é codificado por um grupo de genes de composição e organização variadas, denominados de genes mcy (CARMICHAEL, 1992;CHRISTIANSEN et al., 2003;KURMAYER;CHRISTIANSEN, 2009;PEARSON et al., 2010 (ERIKSSON et al., 1990;HOOSER et al., 1991;TOIVOLA;ERIKSSON, 1999 ZHAO, 1993;GHOSH;COLLODI, 1994;HE, 2010). A linhagem celular ZFL é derivada da junção de aproximadamente 10 fígados de peixes adultos normais (Danio rerio), sendo esta a única linhagem que apresenta a morfologia epitelial típica deste órgão. ...
... Esse complexo é codificado por um grupo de genes de composição e organização variadas, denominados de genes mcy (CARMICHAEL, 1992;CHRISTIANSEN et al., 2003;KURMAYER;CHRISTIANSEN, 2009;PEARSON et al., 2010 (ERIKSSON et al., 1990;HOOSER et al., 1991;TOIVOLA;ERIKSSON, 1999 ZHAO, 1993;GHOSH;COLLODI, 1994;HE, 2010). A linhagem celular ZFL é derivada da junção de aproximadamente 10 fígados de peixes adultos normais (Danio rerio), sendo esta a única linhagem que apresenta a morfologia epitelial típica deste órgão. ...
... The ZFL cell line, derived from normal adult zebrafish [20], was obtained from American Type Culture Collection (N • CRL-2634). The cells were cultured under a humidified air atmosphere at 28 • C in a medium composed of 50% Leibovitz L-15 medium, 35% Dulbecco's modified Eagle's medium, and 15% Ham's F-12 medium, supplemented with HEPES, 15 mM; NaHCO 3 , 0.15 g/L; insulin, 0.01 mg/mL; epidermal growth factor, 50 ng/mL; penicillin/streptomycin, 100 U/mL; and 5% heat-inactivated FBS. ...
... After the incubation, the cells were treated with IM (0, 0.001, 0.01, 0.1 or 1 g/mL), for 4 and 24 h, and in the case of the ZFL cells, also for 72 h. The longer incubation time was chosen due to the longer doubling time of the ZFL cells (approximately 72 h, according to the American Type Culture Collection) compared to the HepG2 cells (24-28 h) [20] and because we observed that, in ZFL cells, DNA damage induced by B[a]P was detected only after 72 h exposure (unpublished data). After treatment, ZFL and HepG2 cells were harvested and the formation of DNA strand breaks was determined with the alkaline comet assay as described by Singh et al. [23]. ...
The selective tyrosine kinase inhibitor imatinib mesylate (IM) is a widely used anticancer drug. Recent studies showing that IM can induce DNA and chromosomal damage in crustaceans and higher plants prompted us to re-examine its potential genotoxicity. IM was not mutagenic in the Ames assay (Salmonella typhimurium). Cytotoxicity and genotoxicity were evaluated in vitro in zebrafish (Danio rerio) liver (ZFL), human hepatoma (HepG2), and human peripheral blood lymphocyte (HPBL) cells. Genotoxicity was determined with the comet assay and with the cytokinesis-block micronucleus cytome assay. ZFL and HPBL cells showed comparable sensitivity to IM cytotoxicity, while HepG2 cells were less sensitive. At non-cytotoxic concentrations, IM induced DNA strand breaks in ZFL and HepG2 cells. An increase in the number of micronuclei was observed in ZFL and HPBL cells. In HPBLs, IM also induced an increase in the number of nucleoplasmic bridges and nuclear buds. Based on the data of the consumption of IM in European countries the predicted environmental concentrations (PEC) were calculated to be in the range between 3.3 and 5.0 ng/L, which are several orders of magnitude lower from those that caused adverse effects in fish and human derived cells. However, based on the in vitro studies it is not possible to quantitatively predict the hazard for wildlife and humans, therefore further studies are warranted to explore the underlying molecular mechanisms of induced IM genotoxic effects as well as the studies of the occurrence of IM in the aquatic and occupational environment to establish the relevance of these observations for aquatic organisms and occupationally exposed personnel.
... EPC cells were maintained at 28°C, 4% CO2 in Minimum Essential Medium (MEM) (GIBCO-Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO-Invitrogen, Carlsbad, CA) and antibiotics. ZFL (zebrafish liver) cells were derived from normal adult zebrafish liver (Ghosh et al., 1994). ZFL cells display an epithelial morphology and it has been demonstrated that the cells exhibit properties in culture that are normally associated with liver cell function in vivo (Ghosh et al., 1994). ...
... ZFL (zebrafish liver) cells were derived from normal adult zebrafish liver (Ghosh et al., 1994). ZFL cells display an epithelial morphology and it has been demonstrated that the cells exhibit properties in culture that are normally associated with liver cell function in vivo (Ghosh et al., 1994). ZFL cells were maintained at 28°C, 0% CO2 in LDF culture medium (50% Leibovitz's L-15 Medium, 35% Dulbecco's modified Eagle's Medium, and 15% F-12 Medium) supplemented with heat inactivated fetal bovine serum. ...
... Cell culture. The ZFL cell line was purchased from American Type Culture Collection and cultured according to established protocols (27). All media were obtained from Corning, Inc. ...
Background: Palmitic acid (PA) is the main saturated fatty acid naturally occurring in animal fats and vegetable oils. In
... Cultura de células de fígado (hepatócitos) é um valioso modelo em estudos de toxicologia in vitro. Estas células podem ser utilizadas para estudar fatores que influenciam a expressão e regulação do sistema de oxidação do citocromo P450, bem como verificar a produção de metabólitos bioativos por compostos xenobióticos(COLLODI et al., 1992) e hepatócitos de zebrafish apresentam propriedades em cultura que são associadas com diferentes células de fígado, in vivo(GOSH et al., 1994).Os resultados deste trabalho são semelhantes aos estudos de Loh e colaboradores (2010), que investigaram a citotoxicidade de nanopartículas de quitosana e tripolifosfato de sódio (porém, sem lecitina de soja em sua formulação) em cultivo de hepatócitos humanos (linhagem BHAL). Em uma concentração superior, de 5 mg/mL, em relação a utilizada neste trabalho (na ordem de microgramas), hepatócitos humanos toleraram estas nanopartículas por um período de 4 horas, utilizando-se do teste colorimétrico MTT. ...
... Current Protocols discussed above (accessibility, variability, and derivation difficulties). Fortunately, quite a few hepatocyte cell lines from different species have been derived and established as immortalized cell lines, such as the human hepatoma cell line HepG2, the human hepatocyte line cBAL111 (Deurholt et al., 2009), and the zebrafish liver cell line ZF-L (Ghosh et al., 1994) as well as a rainbow trout liver cell line (Lee et al., 1993). These established cell lines have been used for research in metabolism, virus biology, toxicity, and hepatic cell biology. ...
The tetra fish species Astyanax mexicanus comprises two morphotypes: cavefish that live in caves and surface fish that inhabit rivers and lakes. Because cavefish have adapted to the nutrient‐poor conditions in their habitat whereas the surface fish populations can be used as a proxy for the ancestral condition, this species has become a powerful model system for understanding genetic variation underlying metabolic adaptation. The liver plays a critical role in glucose and fat metabolism in the body and hence is an important tissue for studying altered metabolism in health and disease. Cavefish morphs of A. mexicanus have been shown to develop fatty livers and exhibit massive differences in gene expression and chromatin architecture. Primary cell lines from various tissues have become invaluable tools for biochemical, toxicology, and cell biology experiments, as well as genetic and genomic analyses. To enhance the utility of the model system by enabling an expanded set of biochemical and in vitro experiments, we developed protocols for the isolation and maintenance of primary liver cells from A. mexicanus surface fish and cavefish. We also describe methods that can be used for primary cell characterization, including cloning, characterization of cell growth pattern, and lentivirus transduction. © 2023 Wiley Periodicals LLC.
Basic Protocol 1 : Primary culture of liver cells
Support Protocol 1 : Maintenance of A. mexicanus primary liver cells
Support Protocol 2 : Banking of A. mexicanus primary liver cells
Support Protocol 3 : Recovery of A. mexicanus primary liver cells
Support Protocol 4 : Primary liver cell cloning
Support Protocol 5 : Characterization of A. mexicanus primary liver cell growth pattern
Basic Protocol 2 : Lentiviral transduction of A. mexicanus primary liver cells
... Among alternative approaches, in vitro cell-based systems represent a promising technology for the predictive assessment of in vivo toxicity, allowing for more controlled manipulation of cellular functions and biological processes. Both freshly isolated primary hepatocytes and the zebrafish liver (ZFL) cell line are commonly cultured on two-dimensional (2D) substrates to study liver response dynamics to EDCs at the molecular and cellular level (Ghosh et al., 1994;Chen and Chan, 2011;Park et al., 2020). In particular, the ZFL cell line offers continued cell proliferation and adheres to the 3Rs (EU, 2010). ...
... Water quality parameters are maintained within safe limits (upper limit of total ammonia nitrogen range 1 mg/L; upper limit of nitrite range 0.5 mg/L; upper limit of nitrate range 60 mg/L; temperature set-point of 22 Media composition. The complete media (ZFL-c) composition was adopted from the media used for a zebrafish liver cell line with minor changes 17 , consisting of 45% Leibovitz's L-15, 30% Dulbecco's modified Eagle's and 15% Ham's F12, 15 mM Hepes-NaOH, 0.01 mg/ml bovine insulin, 50 ng/ml mouse epidermal growth factor, 5% heat-inactivated fetal bovine serum, 0.5% Trout serum and 1% embryo extract. The embryo extract was used for the initial ~ 10-15 passages until the cell lines stabilized. ...
Cell lines have become an integral resource and tool for conducting biological experiments ever since the Hela cell line was first developed (Scherer et al. in J Exp Med 97:695–710, 1953). They not only allow detailed investigation of molecular pathways but are faster and more cost-effective than most in vivo approaches. The last decade saw many emerging model systems strengthening basic science research. However, lack of genetic and molecular tools in these newer systems pose many obstacles. Astyanax mexicanus is proving to be an interesting new model system for understanding metabolic adaptation. To further enhance the utility of this system, we developed liver-derived cell lines from both surface-dwelling and cave-dwelling morphotypes. In this study, we provide detailed methodology of the derivation process along with comprehensive biochemical and molecular characterization of the cell lines, which reflect key metabolic traits of cavefish adaptation. We anticipate these cell lines to become a useful resource for the Astyanax community as well as researchers investigating fish biology, comparative physiology, and metabolism.
... The zebrafish is an excellent model, both for in vivo (Magyary, 2018;Trigueiro et al., 2020;Verma et al., 2021) and in vitro assessment (Lungu-Mitea, 2020;Morozesk et al., 2020Morozesk et al., , 2018, and the established zebrafish liver cell line (ZFL) has a great potential for studies on cell biology. ZFL cells show general hepatocyte morphology and have been previously used in toxicological studies (Bonomo et al., 2021(Bonomo et al., , 2020Ghosh et al., 1994;He, 2010). This study was performed using the ZFL cells to evaluate the toxicity of GO and rGO nanosheets addressing four main questions: 1) Can GO and rGO be internalized by the cells? ...
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.
... The ZFL cell line was purchased from American Type Culture Collection (Manassas, VA, USA), and cultured according to established protocols (24,36). All media were obtained from Corning, Inc. Penicillin-Streptomycin solution and bovine insulin were purchased from Sigma (St. Louis, MO, USA). ...
Being highly unsaturated, n-3 long-chain polyunsaturated fatty acids (LC-PUFAs) are prone to lipid peroxidation. In this study, zebrafish were fed with low-fat diet (LFD), high-fat diet (HFD), or 2% DHA-supplemented HFD (HFDHA2.0). To study the possible negative effects of the high level of dietary DHA, growth rates, blood chemistry, liver histology, hepatic oxidative stress, apoptosis, and inflammatory processes were assessed. The cell studies were used to quantify the effects of DHA and antioxidant on cellular lipid peroxidation and viability. The possible interaction between gut microbiota and zebrafish host was evaluated in vitro. HFDHA2.0 had no effect on hepatic lipid level but induced liver injury, oxidative stress, and hepatocellular apoptosis, including intrinsic and death receptor-induced apoptosis. Besides, the inclusion of 2% DHA in HFD increased the abundance of Proteobacteria in gut microbiota and serum endotoxin level. In the zebrafish liver cell model, DHA activated intrinsic apoptosis while the antioxidant 4-hydroxy-Tempo (tempo) inhibited the pro-apoptotic negative effects of DHA. The apoptosis induced by lipopolysaccharide (LPS) was unaffected by the addition of tempo. In conclusion, the excess DHA supplementation generates hepatocellular apoptosis-related injury to the liver. The processes might propagate along at least two routes, involving lipid peroxidation and gut microbiota-generated LPS.
... However, there are few studies of their action on liver cell lines. Cultures of hepatocytes, such as zebrafish liver (ZFL cell line), are interesting because they reproduce in vitro the action of xenobiotics that are metabolized by this organ [16]. ...
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.
... All in vitro experiments were performed using the ZFL cell line. ZFL cells derived from normal liver of adult zebrafish were obtained from American Type Culture Collection (ATCC number: CRL-2634) through the Banco de Células do Rio de Janeiro, Brazil (BCRJ-0256) (Ghosh et al., 1994). Cells were tested for mycoplasma detection to exclude contamination and authenticated by the supplier. ...
Anticancer agents pose a great environmental risk due to their high toxicity. The aim of the current study is to assess the toxicity of trabectedin, a cytotoxic but atypical DNA binder, to liver cell line (ZFL) and embryo-larvae of the zebrafish Danio rerio employing an innovative approach. In ZFL cells, trabectedin cytotoxicity was measured using MTT and Trypan blue exclusion assay, and cell morphology was evaluated by fluorescence-activated cell sorting (FACS) and by immunofluorescence analysis. Trabectedin was 60-fold more toxic to ZFL cells than to zebrafish embryo-larvae in terms of mortality/cell viability, with mortality being observed in 42.7 µg.L⁻¹ for embryo-larvae and non-viability in 0.04 µg.L⁻¹ for cultured cells. Immunofluorescence staining showed morphology alterations of ZFL-cells exposed to trabectedin in a dose-dependent manner, from 0.04 to 0.15 µg.L⁻¹. Furthermore, trabectedin induced morphological abnormalities to zebrafish embryo-larvae, such as tail malformations, pericardial edema and lack of equilibrium at concentrations lower than 50.3 µg.L⁻¹. Regarding larvae behavior analysis, trabectedin increased velocity and total distance covered by zebrafish exposed to 42.7 µg.L⁻¹ under dark conditions. These results reveal trabectedin to be toxic in both in vitro and in vivo zebrafish models, and thus the occurrence and persistence of this anticancer agent in the environment may represent a potential risk factor to the biota.
... Among alternative approaches, in vitro cell-based systems represent a promising technology for the predictive assessment of in vivo toxicity, allowing for more controlled manipulation of cellular functions and biological processes. Both freshly isolated primary hepatocytes and the zebrafish liver (ZFL) cell line are commonly cultured on two-dimensional (2D) substrates to study liver response dynamics to EDCs at the molecular and cellular level (Ghosh et al., 1994;Chen and Chan, 2011;Park et al., 2020). In particular, the ZFL cell line offers continued cell proliferation and adheres to the 3Rs (EU, 2010). ...
In recent decades, extensive efforts have focused on developing in vitro platforms mimicking fish livers to better understand the acute or chronic effects of toxicants on lower aquatic vertebrates. Fish liver cell lines have emerged as a promising culture system for these in vitro platforms because they complement the currently limited in vitro tools that mostly consist of mammalian cell lines and adhere to the 3Rs: replacement, reduction, and refinement of living animal tests. However, monolayer cell lines have lower transcriptional and physiological responses upon exposure to toxic chemicals than freshly isolated primary cells. To overcome this challenge, we utilized a three-dimensional (3D) spheroid-based in vitro platform, in which hepatocyte cells had self-organized into spheroid forms via E-cadherin bonds. This platform exhibited augmented transcriptomic and phenotypic regulation of liver cells in comparison to monolayer cells. We examined the organoid platform using the zebrafish liver (ZFL) cell line as a model system. ZFL cells spontaneously clustered into 3D spheroids with long-term viability by optimizing cell seeding density on a non-adherent substrate. Interestingly, 3D ZFL spheroids treated with estrogenic chemicals were activated to synthesize a higher level of vitellogenin (Vtg) than monolayer cells. Whole-transcriptome sequencing analysis confirmed that 3D ZFL spheroids had greater transcriptional regulation of genes related to reproductive toxicological response and liver functions, such as the urea cycle, estrogen receptors, and vitellogenin, compared to monolayer cells. These results may contribute to the engineering of novel 3D in vitro platforms for screening harmful chemicals and improving understanding of the underlying liver toxicity mechanisms at the molecular and cellular levels.
... The ZFL cell line was purchased from American Type Culture Collection (Manassas, VA, USA), and cultured according to established protocols (37). For cell cycle and cell apoptosis analysis, ZFL cells were seeded at a density of 1 × 10 5 cells/well on a six-well plate. ...
With the widespread use of high-fat diets (HFDs) in aquaculture, fatty livers are frequently observed in many fish species. The aim of this study was to investigate if docosahexaenoic acid (DHA) could be used to reduce the fatty liver in zebrafish generated by a 16% soybean oil-HFD over 2 weeks of feeding. The DHA was added to iso-lipidic HFD at 0.5, 1.0, and 2.0% of diet. Supplementation of DHA reduced growth and feed efficiency in a dose dependent manner being lowest in the HFDHA2.0 group. Hepatic triglyceride (TG) in zebrafish fed 0.5% DHA-supplemented HFD (HFDHA0.5) was significantly lower than in the HFD control. Transcriptional analyses of hepatic genes showed that lipid synthesis was reduced, while fatty acid β-oxidation was increased in the HFDHA0.5 group. Furthermore, the expression of Cyclin D1 in liver of zebrafish fed HFDHA0.5 was significantly reduced compared to that in fish fed HFD. In zebrafish liver cells, Cyclin D1 knockdown and blocking of Cyclin D1-CDK4 signal led to inhibited lipid biosynthesis and elevated lipid β-oxidation. Besides, DHA-supplemented diet resulted in a rich of Proteobacteria and Actinobacteriota in gut microbiota, which promoted lipid β-oxidation but did not alter the expression of Cyclin D1 in germ-free zebrafish model. In conclusion, DHA not only inhibits hepatic lipid synthesis and promotes lipid β-oxidation via Cyclin D1 inhibition, but also facilitates lipid β-oxidation via gut microbiota. This study reveals the lipid-lowering effects of DHA and highlights the importance of fatty acid composition when formulating fish HFD.
... In brief, 30,000 cells were seeded in each well of a 96-well plate a day prior to the day of experiment. Each cell line had [8][9][10][11][12][13][14][15][16] replicates on the same plate. On the day of experiment, the plate was placed in the BioTek Cytation system for brightfield imaging to count cells for normalization of the results. ...
Cell lines have become an integral resource and tool for conducting biological experiments ever since the Hela cell line was first developed (1). They not only allow detailed investigation of molecular pathways but are faster and more cost-effective than most in vivo approaches. The last decade saw many emerging model systems strengthening basic science research. However, lack of genetic and molecular tools in these newer systems pose many obstacles. Astyanax mexicanus is proving to be an interesting new model system for understanding metabolic adaptation. To further enhance the utility of this system, we developed liver-derived cell lines from both surface-dwelling and cave-dwelling morphotypes. In this study, we provide detailed methodology of the derivation process along with a comprehensive biochemical and molecular characterization of the cell lines, which reflects key metabolic traits of cavefish adaptation. We anticipate these cell lines to become a useful resource for the Astyanax community as well as researchers investigating fish biology, comparative physiology, and metabolism.
... The zebrafish liver cell line (ZFL) (obtained from the China Zebrafish Resource Center, Wuhan, China) were maintained in modified limit dilution factor (LDF) medium (50% Leibowitz-15, 35% Dulbecco's modified Essential medium, 15% HAM's F12, 15 mM HEPES, 0.15 g/L NaHCO 3 , and 10 µg/mL bovine insulin) supplemented with 5% (v/v) fetal bovine serum (FBS, Gibco) and 2% antibiotic (100 U/mL penicillin, 100 µg/mL streptomycin), and kept at 28 • C in a 0.5% CO 2 incubator [49]. ...
Elongation of very long-chain fatty acid (Elovl) proteins are key enzymes that catalyze the rate-limiting step in the fatty acid elongation pathway. The most recently discovered member of the Elovl family, Elovl8, has been proposed to be a fish-specific elongase with two gene paralogs described in teleosts. However, the biological functions of Elovl8 are still to be elucidated. In this study, we showed that in contrast to previous findings, elovl8 is not unique to teleosts, but displays a rather unique and ample phylogenetic distribution. For functional determination, we generated elovl8a (elovl8a−/−) and elovl8b (elovl8b−/−) zebrafish using CRISPR/Cas9 technology. Fatty acid composition in vivo and zebrafish liver cell experiments suggest that the substrate preference of Elovl8 overlapped with other existing Elovl enzymes. Zebrafish Elovl8a could elongate the polyunsaturated fatty acids (PUFAs) C18:2n-6 and C18:3n-3 to C20:2n-6 and C20:3n-3, respectively. Along with PUFA, zebrafish Elovl8b also showed the capacity to elongate C18:0 and C20:1. Gene expression quantification suggests that Elovl8a and Elovl8b may play a potentially important role in fatty acid biosynthesis. Overall, our results provide novel insights into the function of Elovl8a and Elovl8b, representing additional fatty acid elongases not previously described in chordates.
... The permanent ZF-L cell line [44] was cultured in L15 medium (Leibovitz, with L-glutamine, Sigma Aldrich, L4386), supplemented with 10% fetal calf serum (FCS, Biowest, Cholet, France) in 75-cm 2 flasks at 28 • C. Cells were passaged regularly when reaching 90% of confluence. ...
Genotoxicity assessment is of high relevance for crude and refined petroleum products, since oil compounds are known to cause DNA damage with severe consequences for aquatic biota as demonstrated in long-term monitoring studies. This study aimed at the optimization and evaluation of small-scale higher-throughput assays (Ames fluctuation, micronucleus, Nrf2-CALUX®) covering different mechanistic endpoints as first screening tools for genotoxicity assessment of oils. Cells were exposed to native and chemically dispersed water-accommodated fractions (WAFs) of three oil types varying in their processing degree. Independent of an exogenous metabolic activation system, WAF compounds induced neither base exchange nor frame shift mutations in bacterial strains. However, significantly increased chromosomal aberrations in zebrafish liver (ZF-L) cells were observed. Oxidative stress was indicated for some treatments and was not correlated with observed DNA damage. Application of a chemical dispersant increased the genotoxic potential rather by the increased bioavailability of dissolved and particulate oil compounds. Nonetheless, the dispersant induced a clear oxidative stress response, indicating a relevance for general toxic stress. Results showed that the combination of different in vitro assays is important for a reliable genotoxicity assessment. Especially, the ZF-L capable of active metabolism and DNA repair seems to be a promising model for WAF testing.
... Zebrafish liver cell line ZFL (Ghosh et al. 1994;Eide et al. 2014) (CVCL_3276) was cultured in a medium consisting of 50% (v/v) Leibovitz's L-15 (Sigma-Aldrich, Steinheim, Germany), 35% (v/v) Dulbecco's modified Eagle's medium (Gibco, Paisley, UK), 15% (v/v) Ham's Nutrient Mixture F-12 (Gibco, Paisley, UK), and phenol red. Additionally, 150 mg/L sodium bicarbonate (Gibco, Paisley, UK), 15 mM HEPES (Gibco, Paisley, UK), 10 µg/mL bovine insulin (Sigma-Aldrich, Steinheim, Germany), 50 ng/mL mouse EGF (Sigma-Aldrich, Steinheim, Germany), and 5% (v/v) fetal bovine serum (Gibco, Paisley, UK) were supplemented. ...
The water framework directive re-evaluation proposes the integration of effect-based tools, increasing the need for alternative methods. Especially within aquatic toxicology, coverage of specific toxicity pathways is scarce, and most applications are based on mammalian or bacterial models, not reflecting realistic exposure scenarios. The use of transient reporter gene assays in cells from organisms of interest could be a quick and inexpensive solution. However, interference with cellular homeostasis may impact the system beyond the function of the manipulated gene and thus lead to non-specific results. We describe how varying vector geometry and different regulatory gene elements on plasmids used for transfection in zebrafish hepatocytes and embryonic fibroblasts may lead up to a tenfold difference in potency. Cells were transiently co-transfected
with an Nrf2-responsive Firefly luciferase reporter plasmid and eight different Renilla luciferase normalization plasmids. Transfected cells were exposed to two different regimes (0.1–100 μM and 7.8–250 μM) of the oxidative stress-inducing
compounds, sulforaphane, tertbutylhydroquinone, and metazachlor. Nrf2 activity was measured in dual-luciferase assays. In parallel, cytotoxicity was assessed for different endpoints (energy metabolism, protein amount, membrane stability, and cell proliferation) in non-transfected cells and cells co-transfected with constructs of increasing size, to be used for normalization. Transfected cells were more susceptible to cytotoxicity in a vector size-dependent manner. Conclusively, we report that vector geometries (size, backbones, gene-regulatory units), cell line (tissue origin), applied transfection methods, and
signal normalization may alter the sensitivity of reporter bioassays in a synergistic manner. Further, we propose that thorough bioassay design is needed to ensure reliability and regulatory acceptance.
... 4 The zebrafish liver (ZFL) cell line is an adherent hepatocyte line isolated from zebrafish (Brachydanio rerio). 5 Because heavy metal detoxification, including Cu, Zn and Cd, occurs in the liver, we have used ZFL cells to study the expression of metallothionein (MT) and metal regulatory element (MRE)binding transcription factor 1 (MTF-1) messenger RNA in response to various metal ions. 6,7 Cd ion cytotoxicity has also been studied in ZFL cells, using a proteomic approach with 77 proteins identified to express differentially compared with the controls. ...
All cells require Cu as a cofactor, but Cu2+ induces toxicity and oxidative damage. A strict system is thus needed to maintain Cu homeostasis. Using the ZFL zebrafish liver cell line as a model, we studied the cellular responses after exposure to Cu2+, using whole-transcriptome shotgun sequencing (RNA-seq) to screen nearly all transcriptomes in cell samples and identify changes in gene expression. ZFL cells were treated with 100, 200, or 400-μM CuCl2 and harvested after 4 and 24 h. RNA was then extracted and subjected to RNA-Seq and qPCR validation. Exposure to 400-μM CuCl2 for 4 h and 24 h led to the regulation of 5993 and 4235 genes, respectively. In a gene ontology enrichment analysis, Cu2+ exposure enriched the nitrogen compound metabolic process and antioxidant activity but did not significantly affect cellular copper, zinc, iron and calcium ion homeostasis. In a KEGG pathway enrichment analysis, anti-oxidative stress induced the glutathione metabolism pathway. Furthermore, Cu2+ also induced genes related to apoptosis and arrested the cell cycle in the G2 phase. This study was based on full gene expression profile combine with pathways analysis details, provided a full cellular response picture on Cu.
... It exhibits two forms of cytochrome p450 functionally related to rainbow trout p4501A1 and has some properties characteristic of liver parenchymal cells. 19 The cells have been used for studying the induction of micronuclei by polycyclic aromatic hydrocarbons, [20][21][22] anticancer drugs 20-22 and complex mixtures. 21 Another zebrafish cell line, ZF4, which was established from 1 day old zebrafish embryos, has been used for the detection of micronuclei induced by gamma irradiation 23 and uranium compounds. ...
Concerns about the adverse health effects of chemicals and radiation present in the environment and at workplaces have created the need for better detection systems to assess their potential to cause DNA damage in humans and other organisms across ecosystems. The Micronucleus Assay in Toxicology is the first comprehensive volume concerning the use of micronucleus assays in genetic toxicology. It succinctly explains the mechanisms by which genotoxins cause micronucleus formation and its relation to diseases. Furthermore, it describes the methods which are currently used for the analyses of micronuclei in different types of cells in human in vivo biomonitoring studies, routine in vivo tests with rodents, in vitro studies with human and mammalian cells, environmental monitoring with invertebrates and vertebrates such as molluscs, fish and, also, in plant bioassays. Moreover, this book also focuses on the use of the micronucleus technique in other research areas, including the detection of DNA damage caused by important groups of genotoxic carcinogens (heavy metals, industrial chemicals, cytotoxic drugs, pesticides, ionising radiation, etc.) as well as study designs, statistical analyses, international regulatory guidelines, and the development of automated scoring devices for this assay. This book will serve as both, a reference and a guide to students, and investigators in biomedical, biochemical and pharmaceutical sciences interested in gaining a better understanding of the biology of micronuclei and their application in measuring DNA damage caused by natural or man-made genotoxins.
... The foremost advantage of this technique is that it mimics to some extent cell migration in vivo (Liang, Park, & Guan, 2007). Several researchers have developed permanent cell lines from zebrafish, embryo and adult tissues (Collodi, Kamei, Sharps, Ernst, & Barnes, 1992), ZF4 cell line generated from 1 day post-fertilization (dpf) embryos (Driever & Rangini, 1993), ZFL cell line derived from normal adult livers (Ghosh, Zhou, & Collodi, 1994;Miranda, Collodi, Zhao, Barnes, & Buhler, 1993), zebrafish fibroblast-like cell lines ZF13 and ZF29 derived from 20h embryos (Peppelenbosch, Tertoolen, Laat, & Zivkovic, 1995), zebrafish spleen cell line ZSSJ (Xing et al., 2009), PAC2 cell line from 24h zebrafish embryos (Senghaas & Koster, 2009), fibroblast cell lines AB.9 and SJD.1 derived from the amputated caudal fin of AB and SJD strains, respectively (Paw & Zon, 1999) and wild-type zebrafish cell lines DrRPE and DrG derived from the retinal pigmented epithelium and gill tissue, respectively (Nathiga Nambi et al., 2017, 2015. ...
The goal of this study was to develop and characterize a cell line from the caudal fin tissue of zebrafish and also its application as an in vitro model to study the effect of H2O2 in wound healing. Fibroblastic cell line was developed using explant culture method from caudal fin tissue of zebrafish and characterized. This cell line was named as DrF cell line. The DrF cells treated with 0–10 µM/ml H2O2 were tested for viability, proliferation and motility by MTT assay, trypan blue assay and chemotaxis assay, respectively. Among the different concentrations of H2O2, 4 µM was found to be nontoxic to study cell migration in in vitro scratch wound assay. Furthermore, the expression of proliferating cell nuclear antigen (PCNA) and chemokine receptor (CXCR4) genes was carried by qPCR. The cell survival, proliferation and migration were extremely enriched at 4 µM level of H2O2. We observed accelerated wound closure in DrF cells treated with H2O2. The qPCR results indicated that H2O2 markedly up‐regulated mRNA expression of PCNA and CXCR4. The findings from our study suggest that H2O2 at low levels promotes cell survival, proliferation, migration and wound healing in DrF cells.
... Apart of rainbow trout derived cell lines comet assay has been extensively applied also on cell lines (ZFL and ZF4) derived from small tropical fresh water teleost zebrafish (Danio rerio). ZFL cell line (Fig. 1) is derived from normal adult zebrafish liver and it expresses alanine aminotransferase, aspartate aminotransferase, glucose-6-phosphatase enzyme activities and inducible proteins related to cytochrome P450 1A1 [44] showing the metabolic competency of cells. The cell line has been mainly used for detection of DNA strand breaks induced by model genotoxic compounds such as hydrogen peroxide [45] and benzo(a) pyrene [46][47][48], cytostatic drugs [46][47][48][49] and complex environmental mixtures [48][49][50]. ...
The use of fish models has been proven to be an effective and sensitive tool for the evaluation of genotoxicity of pure compounds and complex mixtures of chemicals in the context of environmental screening of pollutants and hazard assessment in aquatic toxicology. In particular, fish cell lines have been successfully introduced for detection of genotoxic effects and can serve as an alternative to animal testing in preliminary eco-/genotoxicological studies. For this purpose comet assay has been extensively used in fish cell lines for the evaluation of genotoxic potential of chemicals and complex environmental matrices. The most often used fish cell lines in the comet assay are RTG-2, RTgill-W1 and RTL-W1 derived from rainbow trout (Onchorhynchus mykiss) gonads, gills and liver, respectively, and ZFL and ZF4 cells established from zebrafish (Danio rerio) liver and embryos, respectively. The present review gives an overview of the most often-used permanent fish cell lines in genotoxicology and discusses their application in the comet assay.
... The zebrafish liver (ZF-L) cell line was cultured according to the protocols published by Ghosh et al. (1994). Cells were cultured at 26 C in Leibovitz's L15 medium with L-glutamine (SigmaeAldrich) containing 10% fetal bovine serum (FBS, Biochrom, Germany) and 1% (v/v) penicillin/streptomycin solution. ...
Water pollution risks to human health and the environment are emerging as serious concerns in the European Union and worldwide. With the aim to achieve good ecological and chemical status of all European water bodies, the “European Water Framework Directive” (WFD) was enacted. With the framework, bioanalytical techniques have been recognized as an important aspect. However, there are limitations to the application of bioassays directly for water quality assessment. Such approaches often fail to identify pollutants of concern, since the defined priority and monitored pollutants often fail to explain the observed toxicity. In this study, we integrated an effect-based risk assessment with a zebrafish-based investigation strategy to evaluate water sample extracts and fractions collected from the Danube. Four tiered bioassays were implemented, namely RNA-level gene expression assay, protein-level ethoxyresorufin-O-deethylase (EROD) assay, cell-level micronucleus assay and organism-level fish embryo test (FET). The results show that teratogenicity and lethality during embryonic development might be induced by molecular or cellular damages mediated by the aryl hydrocarbon receptor (AhR) -mediated activity, estrogenic activity and genotoxic activity. With the combination of high-throughput fractionation, this effect-based strategy elucidated the major responsible mixtures of each specific toxic response. In particularly, the most toxic mixture in faction F4, covering a log Kow range from 2.83 to 3.42, was composed by 12 chemicals, which were then evaluated as a designed mixture. Our study applied tiered bioassays with zebrafish to avoid interspecies differences and highlights effect-based approaches to address toxic mixtures in water samples. This strategy can be applied for large throughput screenings to support the main toxic compounds identification in water quality assessment.
... A first step would be to make use of zebrafish-derived cell lines and test deiodinase activity following EDC exposure in vitro. For instance, a stable adult zebrafish liver-derived cell line ZF-L is available (Ghosh et al., 1994), as well as other zebrafish embryo-derived cell lines (Choorapoikayil et al., 2013;Lakra et al., 2011). ...
Thyroid hormones (THs) stimulate and coordinate a wide range of processes to ensure normal development, mainly by binding of the most active TH 3,5,3'-triiodothyronine (T3) to nuclear receptors resulting in changes in gene transcription. Local TH action is monitored at three distinct levels by different types of regulators: transmembrane transporters (TH influx and efflux), deiodinases (TH activation and inactivation) and nuclear receptors (TH signalling). Since TH regulators are strongly conserved among vertebrate species, the externally and rapidly developing zebrafish (Danio rerio) has become one of the favourite models to study their role in TH-dependent development. Most regulators are expressed in zebrafish from early stages in development in a dynamic and tissue-specific pattern. Transient or permanent disruption of a given regulator severely perturbs development of multiple organs. These zebrafish deficiency models help to explain why, next to overall hypo-/hyperthyroidism, inactivating mutations in the genes encoding TH regulators such as MCT8 and THRA/B have irreversible adverse effects on human development. Zebrafish are also increasingly used as a high-throughput model to assess the toxicity of various xenobiotics and their impact on development. While adverse effects on TH metabolism and gene expression have been shown, information on direct interaction with TH regulators is scarce, albeit essential to fully understand their mechanism of action. For the future, the combination of novel gene silencing tools, fluorescent reporter lines and (single-cell) transcriptomics holds promise for new zebrafish models to further elucidate the role of each TH regulator in vertebrate development.
... Zebrafish liver cell line ZFL. Zebrafish liver cell line ZFL 26,27 (CVCL_3276) was cultured in a medium consisting of 50% Leibovitz's L-15 (Sigma-Aldrich, Steinheim, Germany), 35% Dulbecco's modified Eagle's medium (Gibco, Paisley, UK), 15% Ham's Nutrient Mixture F-12 (Gibco, Paisley, UK), and phenol red. Additionally, 150 mg/L sodium bicarbonate (Gibco, Paisley, UK), 15 mM HEPES (Gibco, Paisley, UK), 10 µg/mL bovine insulin (Sigma-Aldrich, Steinheim, Germany), 50 ng/mL mouse EGF (Sigma-Aldrich, Steinheim, Germany), and 5% fetal bovine serum (Gibco, Paisley, UK) were supplemented. ...
The nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of cellular defense against oxidative stress and correlated with classical toxicological endpoints. In vitro methods using fish cell lines for the assessment of aquatic toxicity are needed for mechanistic studies and as an alternative to in vivo. We describe an in vitro assay to study oxidative stress using zebrafish cell lines. Transfection efficiency of twelve commercially available transfection reagents were tested in the zebrafish cell lines ZFL, ZF4, and Pac2. The most efficient reagent for each cell line was selected for further experiments. Cells were transiently transfected with an Nrf2-responsive luciferase plasmid. The assay was tested using the oxidative stress inducing chemicals tertbutylhydroquinone, hydrogen peroxide, and sulforaphane. Of the transfected cell lines, ZF4 and ZFL showed higher sensitivity. The latter were used to study potential oxidative stress induced by pesticides (diazinon, deltamethrin, atrazine, metazachlor, terbutylazine, diuron). Besides known inducers, Nrf2 activity was also significantly induced by diazinon, deltametrin, diuron, and metazachlor. Activation of Nrf2 by metazachlor is a novel finding. The described assay could be a valuable tool for research in toxicology to study the stress response of both pure chemicals and environmental water samples.
... Cell culture. The ZFL cell line was purchased from American Type Culture Collection and cultured according to established protocols (27). All media were obtained from Corning, Inc. ...
Background:
Palmitic acid (PA) is the main saturated fatty acid naturally occurring in animal fats and vegetable oils. In recent decades, palm oil, an alternative lipid source containing high amounts of PA, has been widely used to replace fish oil in aquafeed.
Objective:
We investigated the hepatotoxicity of PA in zebrafish and the underlying mechanism.
Methods:
One-month-old zebrafish fed a high-fat diet (HFD) containing 16% soybean oil and 3 PA-incorporated HFDs [4%, 8%, and 12% PA (12PA)] for 2 wk (experiment 1) and 4 wk (experiment 2) were used to evaluate PA-induced liver damage and endoplasmic reticulum (ER) stress. Germ-free (GF) zebrafish fed low-fat, high-fat, or 12PA diets for 5 d were used to study the direct effects of PA on liver damage (experiment 3). GF zebrafish colonized with HFD or 12PA microbiota for 48 h were used to elucidate the indirect effects of PA-altered microbiota on liver damage (experiment 4). Last, GF zebrafish colonized with HFD or 12PA microbiota were used to evaluate the effects of different microbiotas on PA absorption (experiment 5).
Results:
In experiment 1, the proportion of PA in the liver linearly increased as its percentage in dietary lipid increased (r2 = 0.83, P < 0.05). In experiment 2, the expression of glucose-regulated protein 78 (Grp78) and C/EBP-homologous protein (Chop) was higher in the 12PA group than in the HFD group (2.2- and 2.7-fold, respectively; P < 0.05). The activity of caspase-12 was increased by 61.1% in the 12PA group compared with the HFD group (P < 0.05). In experiment 3, caspase-12 activity was higher in the 12PA group than in the HFD group (P < 0.05). In experiment 4, GF zebrafish colonized with PA-altered microbiota had higher caspase-12 activity (P < 0.05) than those colonized by HFD microbiota. In experiment 5, PA-altered microbiota promoted PA absorption (P < 0.05) and aggravated ER stress and liver damage in the context of high-PA feeding.
Conclusions:
The PA-altered microbiota indirectly induced ER stress and liver damage in zebrafish. Moreover, the PA microbiota promoted the absorption of PA, leading to enhanced PA overflow into the liver and aggravated hepatotoxicity of PA in zebrafish.
... For this end, a genetically engineered stable cell line of zebrafish origin expressing zfAChE at high amounts along with tdTomato fluorescent protein for easy identification and tracking of transgenic cells was generated. As a basis, the permanent adult zebrafish liver-derived cell line ZF-L (Ghosh et al., 1994) was used. The present communication describes the protocol used for the generation of stably transfected ZF-L cells and the homologous expression of zfAChE. ...
Zebrafish acetylcholinesterase (zfAChE) preparations employed for the evaluation of acetylcholinesterase inhibition are usually extracted from animal tissues, a procedure suffering from both technical and ethical limitations, which may be alleviated using an in vitro expression system for enzyme generation. For this end, a protocol for stable transfection and selection of zebrafish liver (ZF-L) cells using an adapted expression plasmid "ZF-L Exp" was developed. After insertion of zfAChE cDNA, the enzyme was efficiently expressed in transgenic ZF-L cell lines, which were then used as a high yield source of zfAChE activity for acetylcholinesterase (AChE) inhibition assays. An adapted assay protocol was used to demonstrate the effects of carbaryl, dichlorvos and caffeine as model AChE inhibitors towards zfAChE. Dimethyl sulfoxide (DMSO) was also strongly inhibitory towards zfAChE. Finally, we provide data on the stability of zfAChE enzyme preparations. The novel test system provides a promising in vitro test system for the assessment of zfAChE inhibition.
... Cell Viability. Primary hepatocytes were obtained from normal adult Chinese rare minnows using a modified version of the protocol reported by Ghosh et al. 31 Details of the primary cell culture and treatment are in the supplement (Supporting Information (SI) Figure S1). Phe cytotoxicity was assessed using the MTT assay. ...
Phenanthrene (Phe) is one of the most abundant low-molecular-weight polycyclic aromatic hydrocarbons (PAHs). Widespread human and aquatic organism exposure to Phe has been reported, but the toxic effects of Phe and potential mechanisms are unclear. We focused on the chronic hepatotoxicity of Phe in adult Chinese rare minnows (Gobiocypris rarus) and the underlying mechanisms. The chronic effects of exposing Chinese rare minnows to 8.9, 82.3, or 510.0 μg/L Phe for 30 days were examined by histopathological observation, TUNEL assays, caspase activity assays, and gene expression profiles. The liver lesion frequency and hepatocyte apoptosis were increased in Phe-exposed groups. Caspase 9 and caspase 3 enzyme activity in liver tissues was markedly increased. The expression of miR-17/92 cluster members was significantly increased in the 82.3 and 510.0 μg/L groups. Moreover, the response of primary hepatocytes indicated a significant decrease in the mitochondrial membrane potential (MMP) after a 48-h exposure to Phe. Interestingly, miR-18a was significantly decreased in primary hepatocytes in all treatments. Moreover, molecular docking indicated that Phe might have the same binding domain as pri-miR-18a, forming pi-pi and pi-σ interactions with heterogeneous nuclear ribonucleoprotein (hnRNP) A1. Given the above, Phe caused liver lesions and induced hepatocyte apoptosis through the intrinsic apoptosis pathway, and the interaction of Phe with hnRNP A1 contributes to the suppression of miR-18a expression and hepatocyte apoptosis.
... The ZFL cell line (ATCC #CRL-2643), derived from a pool of 10 adult zebrafish livers, has characteristics of liver parenchymal cells and exhibits characteristics consistent with differentiated liver cell function [69]. ZFL cells have been used to investigate toxicological [70,71], pharmacological [72], and innate immune function studies in fish [73]. ...
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.
... 1758) (Chen, 2009). The 613 polygonal epithelial-like morphology of the SCHL cells is a typical in vivo feature of fish hepatocytes, resembling the hepatocyte lines RTL-W1 from O. mykiss, ZFL from zebrafish Danio rerio (Hamilton 1822) and PL from pilchard Sardinops sagax neopilchardus (Jenyns 1842) (Lucila et al., 1993;Ghosh et al., 1994;Williams et al., 2003). The results of the karyotype analysis were in accordance with the report on S. canaliculatus by Shu et al. (2010). ...
... ZFL cells derived from normal liver of adult zebrafish (Ghosh et al., 1994) were obtained from American Type Culture Collection (ATCC number: CRL-2634). They were cultured under humidified air atmosphere at 28°C in a medium composed of 50% Leibovitz L-15, 35% DMEM, and 15% Ham F-12, supplemented with 15 mM HEPES, 0.15 g/L NaHCO 3 , 0.01 mg/mL insulin, 50 ng/mL EGF, 0.1% penicillin/ streptomycin, and 5% heath inactivated FBS. ...
Anticancer drugs enter aquatic environment predominantly via hospital and municipal wastewater effluents where they may, due to their genotoxic potential, cause adverse environmental effects even at very low doses. In this study we evaluated cytotoxic and genotoxic potential of two widely used anticancer drugs, cyclophosphamide (CP) and ifosfamide (IF) as individual compounds and in a complex mixture together with 5-fluorouracil (5-FU) and cisplatin (CDDP) because these four drugs have been frequently detected in an oncological ward effluents. As an experimental model we used zebrafish liver cell (ZFL) line. The cytotoxicity was determined with the MTS assay and genotoxicity with the comet assay and cytokinesis block micronucleus (CBMN) assay that measure the formation of DNA strand breaks and genomic instability, respectively. CP and IF exerted low cytotoxicity towards ZFL cells. Both compounds induced DNA strand breaks and genomic instability, however at relatively high concentrations that are not relevant for the contamination of aquatic environment. The mixture of CP, IF, 5-FU and CDDP was tested at maximal detected concentrations of each drug as determined in the effluents from the oncological ward. The mixture was not cytotoxic and did not induce genomic instability, but it induced significant increase in the formation of DNA strand breaks at concentrations of individual compounds that were several orders of magnitude lower from those that were effective when tested as individual compounds. The results indicate that such mixtures of anticancer drugs may pose a threat to aquatic organisms at environmentally relevant concentrations and contribute to the accumulating evidence that it is not always possible to predict adverse effects of complex mixtures based on the toxicological data for individual compounds.
... The ZFL cell line (ATCC #CRL-2643), derived from a pool of 10 adult zebrafish livers, has characteristics of liver parenchymal cells and exhibits characteristics consistent with differentiated liver cell function [69]. ZFL cells have been used to investigate toxicological [70,71], pharmacological [72], and innate immune function studies in fish [73]. ...
Abstract 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
... The zebrafish (Danio rerio) liver cell line (ZFL) is derived from adult zebrafish (Ghosh et al., 1994) and was obtained from the American Type Culture Collection (ATCC number: CRL-2634). Cells were cultured under a humidified air atmosphere at 28 C in a medium containing 50% Leibovitz L-15 (ATTC), 35% DMEM (Gibco), and 15% Ham F-12 (Gibco), supplemented with 15 mM HEPES (Invitrogen, Paisley, UK), 0.15 g L À1 NaHCO3, 0.01 mg mL À1 insulin, 50 ng mL À1 epidermal growth factor (EGF; Invitrogen, Paisley, UK), 0.1% penicillin/streptomycin, and 5% heat inactivated fetal bovine serum (FBS; ATTC). ...
Anticancer drugs are continuously released into hospital and urban wastewaters, where they, most commonly, undergo conventional treatment in wastewater treatment plants (WWTPs). Wastewaters contain complex mixtures of substances including parent compounds, their metabolites and transformation products (TPs). In this study, samples of hospital effluents and WWTP influents and effluents from Slovenia and Spain were analyzed for twenty-two selected anticancer drugs, their metabolites and transformation products. Acute and chronic toxicity tests were performed on the crustacean Ceriodaphnia dubia, genotoxicity was determined with Tradescantia and Allium cepa micronucleus (MN) assays and in vitro comet assay in zebrafish (Danio rerio) liver cell line (ZFL cells). Sixty of the two hundred-twenty determinations revealed detectable levels of anticancer drug residues. Among the targeted compounds, platinum based were most frequently detected (90%). Furthermore, erlotinib was detected in 80%, cyclophosphamide and tamoxifen in 70% and methotrexate in 60% of the samples. Seven of ten samples were toxic to C. dubia after acute exposure, whereas after chronic exposure all samples reduced reproduction of C. dubia at high sample dilutions. Allium cepa proved insensitive to the potential genotoxicity of the tested samples, while in Tradescantia increased MN frequencies were induced by a hospital effluent and WWTP influents. In ZFL comet assay all but one sample induced a significant increase of DNA strand breaks. Correlations of chemotherapeutics or their TPs were detected for all bioassays except for Allium cepa genotoxicity test, however for each test the highest correlations were found for different substances indicating differential sensitivities of the test organisms.
... The zebrafish (Danio rerio) liver cell line (ZFL) is derived from adult zebrafish (Ghosh et al., 1994) and was obtained from the American Type Culture Collection (ATCC number: CRL-2634). Cells were cultured under a humidified air atmosphere at 28 C in a medium containing 50% Leibovitz L-15 (ATTC), 35% DMEM (Gibco), and 15% Ham F-12 (Gibco), supplemented with 15 mM HEPES (Invitrogen, Paisley, UK), 0.15 g L À1 NaHCO3, 0.01 mg mL À1 insulin, 50 ng mL À1 epidermal growth factor (EGF; Invitrogen, Paisley, UK), 0.1% penicillin/streptomycin, and 5% heat inactivated fetal bovine serum (FBS; ATTC). ...
... For the study of specific aspects of liver metabolism and physiology, primary liver cell cultures may be partly replaced by established cell lines. Fish cell lines with liver-specific functions were established only recently: PLHC-1 from Poeciliopsis lucida hepatoma [34,39], RTL-W1 from rainbow trout liver [55] and a liver cell line from (Brachy)Danio rerio [27]-all three cell lines express inducible cytochrome P4501A activity. These cell lines provide valuable models to study the relative inducing potency of xenobiotics for cytochrome P4501A [84]. ...
After the original description by Berry and Friend in 1969 of the high yield isolation of rat liver parenchymal cells by collagenase perfusion [1], the use of isolated hepatocytes was soon established as an ideal system for the study of many aspects of hepatic metabolism [2]. In 1976, Birnbaum and colleagues [3] adapted the technique of hepatocyte isolation for studies of hormone-stimulated glycogenolysis in goldfish cells. Since then, isolated fish hepatocytes have been used intensively in fresh suspensions and in primary culture as a powerful, yet simple tool for the investigation of piscine hepatic functions. Cultured hepatocytes have proved to be well suited for applications not only in basic fish physiology, but also in the fields of pharmacology and environmental toxicology [4–7].
... In the past few decades, a number of stable cell lines from zebrafish embryos (ZEM2S) [1] ; (ZF4) [2] and adult caudal fins (AB9 and SJD.1) [3] and liver (ZF-L) [4] have been developed. Cell based assays are important since they can complement findings from in vivo studies. ...
The regenerating caudal fin of an adult zebrafish is a great model system to study gene regulation and function. However, manipulation of essential genes in adult fins can be challenging. In this brief report, we suggest that a fibroblast cell line AB9, isolated from regenerating caudal fins of adult zebrafish, can be used to perform pilot studies for gene regulation. We also provide standardized protocols for morpholino and drug treatment of AB9 cells. Our findings suggest that the AB9 cell line is an easily manipulated system that expresses key regulators of the Cx43 dependent growth and patterning pathway, identified from studies in the regenerating fin. We also provide evidence that expression and function of proteins can be easily manipulated, either through targeted morpholino knockdown or by pharmacological inhibition.
... HepG2 cells were a gift from Dr. Firouz Darroudi (Leiden University Medical Centre, Department of Toxicogenetics, Leiden, The Netherlands). The cells were cultivated at 37°C and 5 % CO 2 in William's medium E containing 15 % FBS, 2 mM L-glutamine and 100 U/mL penicillin/streptomycin. ZFL cell line was derived from normal adult zebrafish (Ghosh et al. 1994) and was obtained from American Type Culture Collection (ATCC number: CRL-2634). Cells were cultured under humidified air atmosphere at 28°C in a medium composed of 50 % Leibovitz L-15, 35 % DMEM and 15 % Ham F-12, supplemented with 15 mM HEPES, 0.15 g/L NaHCO 3 , 0.01 mg/mL insulin, 50 ng/mL EGF, 0.1 % penicillin/streptomycin and 5 % heath inactivated FBS. ...
Due to their increasing use, the residues of anti-neoplastic drugs have become emerging pollutants in aquatic environments. Most of them directly or indirectly interfere with the cell's genome, which classifies them into a group of particularly dangerous compounds. The aim of the present study was to conduct a comparative in vitro toxicological characterisation of three commonly used cytostatics with different mechanisms of action (5-fluorouracil [5-FU], cisplatin [CDDP] and etoposide [ET]) towards zebrafish liver (ZFL) cell line, human hepatoma (HepG2) cells and human peripheral blood lymphocytes (HPBLs). Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and acridine orange/ethidium bromide staining. All three drugs induced time- and dose-dependent decreases in cell viability. The sensitivity of ZFL and HepG2 cells towards the cytotoxicity of 5-FU was comparable (half maximal inhibitory concentration (IC50) 5.3 to 10.4 μg/mL). ZFL cells were more sensitive towards ET- (IC50 0.4 μg/mL) and HepG2 towards CDDP- (IC50 1.4 μg/mL) induced cytotoxicity. Genotoxicity was determined by comet assay and cytokinesis block micronucleus (CBMN) assay. ZFL cells were the most sensitive, and HPBLs were the least sensitive. In ZFL cells, induction of DNA strand breaks was a more sensitive genotoxicity endpoint than micronuclei (MNi) induction; the lowest effective concentration (LOEC) for DNA strand break induction was 0.001 μg/mL for ET, 0.01 μg/mL for 5-FU and 0.1 μg/mL for CDDP. In HepG2 cells, MNi induction was a more sensitive genotoxicity endpoint. The LOEC values were 0.01 μg/mL for ET, 0.1 μg/mL for 5-FU and 1 μg/mL for CDDP. The higher sensitivity of ZFL cells to cytostatic drugs raises the question of the impact of such compounds in aquatic ecosystem. Since little is known on the effect of such drugs on aquatic organisms, our results demonstrate that ZFL cells provide a relevant and sensitive tool to screen genotoxic potential of environmental pollutant in the frame of hazard assessment.
... The human hepatic cell lines used were HepG2 14 and HepaRG, 15 the latter being known for its high biotransformation capabilities. In addition, comparative studies were also performed using human cell lines widely used for screening estrogenic activity, that is, the breast cancer cell lines MELN 16 18 Zebrafish hepatic reporter cell lines (ZELH-zfERα and ZELH-zfERβ2) were cultured exactly as previously described. 5 The MELN 16 and T47D-KBLuc 17 cells derived from human breast cancer MCF-7 and T47D cell lines, respectively, were cultured as previously described. ...
Several human and fish bioassays have been designed to characterize the toxicity and the estrogenic activity of chemicals. However, their biotransformation capability (bio-activation/detoxification processes) is rarely reported, although this can influence the estrogenic potency of test compounds. The fate of two estrogenic chemicals, the UV filter benzophenone-2 (BP2) and the bisphenol A substitute bisphenol S (BPS) was deciphered in eight human and zebrafish in vitro cell models, encompassing hepatic and mammary cellular contexts. BP2 and BPS were metabolized into a variety of gluco- and sulfo-conjugated metabolites. Similar patterns of BP2 and BPS biotransformation were observed among zebrafish models (primary hepatocytes, ZFL and ZELH-zfER cell lines). Interestingly, metabolic patterns in zebrafish models and in the human hepatic cell line HepaRG shared many similarities, while biotransformation rates in cell lines widely used for estrogenicity testing (MELN and T47D-KBLuc) were quantitatively low and qualitatively different. This study provides new data on the comparative metabolism of BP2 and BPS in human and fish cellular models that will help characterize their metabolic capabilities, and underlines the relevance of using in vitro zebrafish-based bioassays when screening for endocrine disrupting chemicals.
... Their detailed studies indicated that these cell lines were valuable for their use as model cell lines for zebrafish research (He et al., 2006). As mentioned, ZFL cells were derived by Collodi et al. from adult zebrafish livers (Collodi et al., 1992;Ghosh et al., 1994). These cells showed the main characteristics of liver parenchymal cells and exhibit properties in culture that were associated with differentiated liver cell function in vivo (Collodi et al., 1992). ...
A novel cell line designated as DRS ( Danio rerio skin) was established and characterized from the skin tissue of wild-type zebrafish, Danio rerio , by the explant technique. The cells thrived well in the Leibovitz’s -15 medium supplemented with 15% FBS and routinely passaged at regular intervals. The DRS cells mainly feature fibroblast-like morphology. The culture conditions of the cells were determined by incubating the cells at varying concentrations of FBS and temperature; the optimum was 15% FBS and 28°C, respectively. Cells were cryopreserved and revived with 70–75% viability at different passage levels. Two extracellular products from bacterial species Aeromonas hydrophila and Edwardsiella tarda were tested and found toxic to the DRS cells. Mitochondrial genes, namely COI and 16S rRNA PCR amplification and partial sequencing authenticated the species of origin of cells. The modal diploid (2n) chromosome number of the cells was 50. The cells were transfected with pmaxGFP plasmid tested positive for green fluorescence at 24–48 h post-transfection, confirming the usefulness of the developed cell line for various in vitro applications.
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 . ”
Graphical abstract
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.
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 251.8 μM in larvae and 5.5 μM 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.
The flavonoid metal-insecticide magnesium-hesperidin complex (MgHP) has recently been considered as a novel insecticide to replace some persistent pesticides. However, it is important to evaluate its action on non-target species, mainly those living in an aquatic environment, as these ecosystems are the final receptors of most chemicals. Reactive oxygen species, antioxidant and oxidative stress biomarkers, genotoxicity as well as cell cycle was evaluated in the liver cell line from zebrafish (Danio rerio; ZF-L) exposed to 0, 0.1, 1, 10, 100 and 1000 ng mL⁻¹ MgHP. MgHP affected cell stability by increasing reactive oxygen species (ROS) in both exposure times (24 and 96 h) at high concentrations. Catalase (CAT) activity decreased after 24 h exposure, and glutathione and metallothionein values increased, avoiding the lipid peroxidation. Genotoxicity increased as MgHP concentration increased, after 24 h exposure, exhibiting nuclear abnormalities; it was recovered after 96 h exposure, evidencing possible stimulation of DNA repair mechanisms. The alteration in the cell cycle (increasing in the Sub-G1 phase and decreasing in the S-phase) was associated with chromosomal instability. In conclusion, the responses of ROS and the antioxidant defense system depended on MgHP concentration and time exposure, while DNA exhibited some instability after 24 h exposure, which was recovered after 96 h.
DNA methylation of enhancers and promoters generally inhibits gene transcription. DNA methylation occurs predominantly at the dinucleotide CpG, a methyl group that is covalently bonded to cytosine. We have previously demonstrated tissue-restricted expression of the uncoupling protein 1 ( Ucp1) in common carp. Here, we characterized the methylation status of the upstream region of the transcriptional initiation site of the carp Ucp1 gene in the liver, brain, kidney, skeletal muscle, and scale. In addition, we explored the direct role of methylation on Ucp1 transcription. Ucp1 expression was higher in the liver than that in other tissues including the kidney, skeletal muscle, and scale. The extent of methylation at nt -2178 and nt -2103 was lower in the liver and kidney than that in the brain, skeletal muscle, and scale. In addition, methylation at the upstream proximal-region of the Ucp1 gene was generally less frequent in the liver compared to that in the other organs. The transcriptional activation assay using the CpG-free luciferase-based reporter suggested that the methylation of the distal and proximal regions of the carp Ucp1 gene did not affect Ucp1 transcription. Unexpectedly, mutation of cytidylic acid to guanylic acid at nt -108 decreased Ucp1 promoter activity. The present results reveal that the status of DNA methylation of the upstream region of the carp Ucp1 gene is different among different tissues, but suggest that the DNA methylation do not directly repress the transcription of Ucp1.
The concern about DNA damage has directed efforts toward evaluating the genotoxic potential of physical and chemical agents. Since the extent of DNA damage is also related to the capacity of the organism in repairing the DNA, the advance of toxicological studies on this area depends on the characterization of the DNA repair mechanisms in the available models. The cellular zebrafish models, for example, replace mammalian cells to answer ecologically relevant questions on aquatic toxicology. So, the aim of the present study was to characterize the nucleotide excision repair (NER) and photoreactivation (PER) in two cellular models of Danio rerio liver, primary hepatocytes and ZF-L (Zebrafish Liver) cell line. We performed kinetic studies of the DNA damage levels after exposure to 6.8 J/m2 UVC using the T4-PDG modified Comet Assay, and determined the expression levels of important genes involved in NER, PER and base excision repair using RT-qPCR. It was observed that both ZF-L cell line and primary hepatocytes exhibit similar NER and PER activity. Primary hepatocytes showed similarities in the gene expression of most of the evaluated repair genes with the original tissue. These results indicate that both primary hepatocytes and ZF-L cells are useful models for toxicological studies aiming to evaluate NER and PER in hepatic cells. Moreover, the similarities in gene expression between the cellular models suggest that the ZF-L cells retain the DNA repair characteristics of the primary hepatocytes and, thus, could serve as replacement to this primary culture, reducing the use of animals in research.
The development of novel agrochemical compounds to reduce the use of pesticides with high ecological impact is urgently needed. A complex of Mg with two flavonoids hesperidin and phenanthroline [Mg(hesp) 2 (phen)], referred to as MgHP, results in high insecticidal activity against urban, agricultural and forest insect pests. In vitro cytotoxicity biomarkers were used to assess the mechanism of action MgHP on fish cells, as this insecticide can reach the aquatic environment and affect its biota. The cytotoxic effects of MgHP were evaluated at different concentrations (0, 0.1, 1, 10, 100 and 1000 ng mL ⁻¹ )in a zebrafish hepatocyte cell line (ZF-L). Twenty-four hours of exposure to high concentrations (10 and 1000 ng mL ⁻¹ )of MgHP affected cell confluence and morphology. Mitochondrial activity and lysosomal retention ability decreased as the MgHP concentration was increased. Cell membrane injury, apoptosis, and necrosis were not induced. These results suggested that toxicity to ZF-L cells was due loss of organellar activity caused by MgHP, which may also include activation of an alternative cell death mechanism. However, after 96 h of exposure, the toxic effects of MgHP may be mitigated, even at high concentrations, enabling cellular population recovery. These data provide important information on the mechanism of action of MgHP on hepatocyte fish cells and stimulate analyses to elucidate the cellular responses to MgHP.
For a wide range of purposes, primary cell cultures and/or cell lines have been prepared from most tissues and organs of a small fraction of the estimated 20,000 species of bony fish. These cell cultures usually have been maintained with mammmalian sera. For many applications their usefulness would be enhanced by a more piscine and defined environment. However, the piscine equivalents of mammalian polypeptide growth and differentiation factors are largely unknown and are unlikely ever to be available commercially. In the future they might be obtained from the medium in which fish cells have been grown. Therefore, by being a potential source of fish polypeptide growth and differentiation factors, a cell line from a fish organ might be utilized as a Rossetta stone to decipher thein vitro proliferation and differentiation of other cells from this or other organs from the same or different species.
Male outbred Sprague-Dawley rats were fed a choline-deficient diet containing 0.10% DL-ethionine (CDE) for 4, 6, 10, 14 or 22 weeks followed by a standard diet for up to 59 weeks. Liver sections were histochemically analyzed for the following parameters: basophilia, glycogen content and the activities of glycogen synthase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphatase (G6PASE), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycerin-3-phosphate dehydrogenase (G3PDH), ‘malic enzyme’ (MDH), alkaline phosphatase (ALKPASE) and γ-glutamyltranspeptidase (GGT). The stop experiments revealed that many of the oval cells proliferating during the first 4–6 weeks may undergo necrotic changes and disappear with time, whereas cholangiofibroses appearing in animals fed CDE for at least 10 weeks are persistent lesions. The sequence of lesions seen in this study, leading from persistent oval cells through cholangiofibroses to cholangiofibromas, strongly suggests that the oval cells are the precursor cells of cholangiocellular tumors. The proliferating oval cells and the hepatic foci consisting of clear and acidophilic or mixed cell populations were always spatially separated and no transitions between oval and parenchymal cells were observed. These results argue against a precursor-product relationship between oval and parenchymal cells. Both proliferating and persistent oval cells, cholangiofibroses and cholangiofibromas showed a strong staining for G6PDH, GAPDH, G3PDH, MDH, ALKPASE and GGT; low PHO, SYN and G6PASE activities were also detected in these lesions. Persistent glycogen-storage foci, which developed in all rats fed CDE for 4–14 weeks followed by a normal lab chow for over a year, had increased PHO, G6PDH, MDH, ALKPASE and GGT activities, while SYN, GAPDH and G3PDH activities remained unaltered and G6PASE activity decreased. Mixed cell foci appearing in animals fed CDE for 22 weeks followed by a normal lab chow for 59 weeks had strongly increased G6PDH, GAPDH, G3PDH, MDH, ALKPASE and GGT activities as well as decreased G6PASE activity. These results indicate that the characteristic metabolic pattern of preneoplastic hepatic foci is independent of the further administration of the carcinogenic diet. The shift from glycogen metabolism to glycolysis and the pentose phosphate pathway occurring during the later stages of CDE-induced hepatocarcinogenesis is an autogenous process apparently directing the disturbed carbohydrate metabolism towards alternative metabolic pathways. A similar metabolic shift also seems to take place during cholangiocarcinogenesis.
A significant aspect of primary hepatic carcinoma in man is the high positive correlation of hepatocellular carcinoma with infection with hepatitis B virus (HBV)1. Analysis of the relationship between HBV infection and oncogenesis is difficult because natural infection with HBV is limited to man and experimental infection has been achieved only in chimpanzees and gibbons. Furthermore, because HBV has not been successfully propagated in cell culture, basic study of virus-cell interaction of the aetiological agent of one of the most widespread infections of man has been impossible. Recently, however, a cell line (PLC/PRF/5) derived from a human hepatoma biopsy was described which produces the HRV surface antigen (HBsAg) and so provides a tool for the experimental investigation of HBV in viro. We now report the derivation and characterisation of two additional cell lines primary liver carcinomas. In contrast to the PLC/PRF/5 cell line, these cell lines retain the capacity to synthesise many human plasma proteins, including both albumin and alpha-fetoprotein (AFP). One of these lines also produces BHsAg. We also present evidence that HBsAg synthesis and secretion in this cell line are correlated with the growth state of the culture. This finding is in contrast to the continuous HBsAg production found in the PLC/PRF/5 cell line.
Male outbred Sprague-Dawley rats were fed a choline-deficient diet containing 0.10% DL-ethionine for up to 30 weeks. Liver slices from rats killed 4, 6, 10, 14, 22 and 30 weeks after starting the treatment were histochemically analyzed for the following parameters: basophilia, expression of cytokeratin 19 (which in the liver is bile duct epithelial cell-specific), glycogen content and activities of glycogen synthetase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphatase (G6PASE), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycerin-3-phosphate dehydrogenase (G3PDH), 'malic enzyme' (MDH), alkaline phosphatase (ALKPASE) and gamma-glutamyltranspeptidase (GGT). The diet induced necrosis of single parenchymal cells and a massive proliferation of oval cells within 4-6 weeks; thereafter cholangiofibroses, cystic cholangiomas and some cholangiofibromas, but no cholangiocarcinomas, were observed. Oval cells, cholangiofibroses, cystic cholangiomas and cholangiofibromas expressed cytokeratin 19, whereas parenchymal cells, foci of altered hepatocytes and hepatocellular adenomas did not; this observation does not support a precursor-product relationship between oval and parenchymal cells. SYN, PHO, G6PASE, G6PDH, GAPDH, G3PDH, MDH, ALKPASE and GGT activities were detected in oval cells; cholangiofibrotic lesions, cystic cholangiomas and cholangiofibromas stained strongly for GAPDH, G3PDH and MDH. In livers from rats fed the diet for 10 weeks, single hepatocytes storing high amounts of glycogen appeared in the parenchyma. There was no indication of a transition from the oval cell population to hepatocytes storing glycogen in excess. Foci of glycogen-storing cells were scattered all over the lobes after 14 and 22 weeks; they had increased G6PASE, G6PDH, ALKPASE and GGT activities. Mixed cell foci and hepatocellular adenomas developed within 22-30 weeks and exhibited a remarkable decrease of G6PASE activity, a strong increase of G6PDH, GAPDH, G3PDH and MDH activities as well as extremely high ALKPASE and GGT activities. The data support the concept that during hepatocarcinogenesis, a number of sequential changes in the activities of various enzymes involved in carbohydrate metabolism occur and that a correlation between morphology and enzyme pattern in the focal lesions does in fact exist. Furthermore, our results suggest that two different cell lineages are involved in the development of cholangiocellular tumors from oval cells and hepatocellular tumors from hepatocytes.
1. A primary cell culture from rainbow trout (Oncorhynchus mykiss) liver was prepared and evaluated for biotransformation of xenobiotics. 2. The hepatocytes maintained cytochrome P-450 content, as well as their cytochrome P-450-dependent activities, stable for 5-6 days in serum-free medium. Protein and glutathione levels, as well as other enzyme activities important for biotransformation, were close to their fresh cell levels throughout the culture period. 3. The cells were also responsive to cytochrome P-450 inducers. Both beta-naphthoflavone (BNF) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) caused an increase in ethoxyresorufin O-deethylase (EROD) activity, which was dose-dependent over the concentration ranges of 3.6-360 nM and 2.5-100 pM, respectively. The induced activities in BNF exposed cells returned to basal levels within 48 h after replacing the medium with a BNF-free medium. Exposure of cells to TCDD (100 pM) for 48 h induced EROD activity which, in contrast to response of BNF-exposed cells, continued to increase after the medium had been replaced with TCDD-free medium. 4. The results show that trout hepatocytes in primary culture afford a reliable in vitro method for studying the regulation and functions of xenobiotic biotransformation enzymes, and for defining toxic effects of aquatic pollutants in cells.
Four novel nontransformed epithelial cell lines, isolated from fetal or adult mouse liver, were tested: (a) to determine the profile of xenobiotic metabolizing enzymes; (b) to evaluate the inducibility of the polysubstrate (cytochrome P-450-dependent) monooxygenase system by various classes of inducers; and (c) to assess the capacity of the cells to metabolize structurally different procarcinogens. With regard to the phase I pathway, the cells expressed various P-450 (class IA, IA2, IIB, IIE1, IIIA) and flavin adenine dinucleotide-containing monooxygenase-dependent bio-transformation enzyme activities at levels (in lines C2.8 and C6) comparable with those present in murine adult liver preparations. The expression of various P-450s was demonstrated also by immunoprecipitation assays using rabbit polyclonal antibodies. For the phase II pathway, cells expressed substantial levels of glutathione S-transferase, glutathione S-epoxide transferase, and UDP-glucuronosyltransferase. Low expression of epoxide hydrolase was observed. Induction of P-450 function by sodium phenobarbital, beta-naphthoflavone, isosafrole, ethanol, and pregnenolone 16 alpha-carbonitrile, monitored using specific P-450-linked activities, was considerably elevated (over 5-fold in class IIB with the C2.8 and C6 cell lines). The most competent C2.8 and C6 cell lines were able to activate benzo(a)pyrene, cyclophosphamide, dimethylnitrosamine, diethylstilbestrol, and 2-naphthylamine as shown by the significantly increased frequencies of mitotic gene conversion, mitotic crossing-over, and point [reverse] mutation in the diploid D7 strain of Saccharomyces cerevisiae after 4 [cyclophosphamide], 24 [benzo(a)pyrene,2-naphthylamine, dimethylnitrosamine] or 48 [diethylstilbestrol], h of exposure in the presence of 3 x 10(6) cells/flask. The degree of conservation and the inducibility of representative oxidative and postoxidative reactions in the novel epithelial cell lines C2.8 and C6, together with their ability to activate a wide spectrum of procarcinogens, offers a means to study the potential of chemicals for inducing DNA damage in short-term genotoxicity testing. In addition the cells may be suitable for analyzing the metabolic disposition of compounds and the multistage process of carcinogenesis.
Two epithelial cell lines designated LE/2 and LE/6 were established from cells isolated by centrifugal elutriation from the livers of carcinogen-treated rats. Both cell lines exhibit some characteristics of fetal liver cells, such as the expression of the 2.3-kilobase alpha-fetoprotein mRNA, aldolase A, and lactate dehydrogenases 4 and 5. Primary cultures contain gamma-glutamyl transferase-positive cells which do not proliferate in vitro. After the first passage, the LE/2 and LE/6 cell lines are uniformly gamma-glutamyl transferase negative. Neither cell line is transformed as assayed by morphology, anchorage-independent growth, or tumor formation in nude mice. By the 50th passage, LE/6 cells form numerous colonies in soft agar in the presence of epidermal growth factor, while no colonies grow in medium lacking this growth factor. Clonal cell populations derived from five epidermal growth factor-induced soft agar colonies were not tumorigenic in nude mice. This indicates that, although epidermal growth factor-responsive late passage cells had acquired some of the phenotypic properties commonly associated with tumor cells, these cells were not fully transformed. Transformation of LE/6 cells was accomplished by transfection of the rasH oncogene (EJ). Subcutaneous inoculation of rasH (EJ)-transfected LE/6 cells produced tumors at the site of injection with histological features of moderate to well-differentiated trabecular hepatocellular carcinomas. Tumor cell lines derived from the nude mouse tumors are gamma-glutamyl transferase positive and express alpha-fetoprotein mRNA. One clonal cell line expresses both alpha-fetoprotein and albumin mRNA. These results show that nonparenchymal liver epithelial cells transfected with an activated oncogene can give rise to differentiated hepatocellular tumors similar to those induced in livers of rats fed a carcinogenic diet.
Tissues of the fishes are as amenable to the techniques of modern cell culture as mammalian tissues and organs, and yet this vast resource, comprising thousands of vertebrate species, remains largely unexplored. The model systems that have been developed demonstrate the utility of fish cells as sources of special adaptations and exaggerated physiological systems. In this review, we briefly describe several of the successful models along with recent developments in fish cell culture with the hope of stimulating increased interest in the lower vertebrates as useful complements to mammalian cell culture in biomedical research. The topics covered include epithelial ion transport, endocrinological studies, the cellular stress (heat shock) response, thermotolerance, cancer biology, and environmental toxicology.
The localization of albumin was investigated in rat liver, fixed by perfusion, with peroxidase-labeled monospecific antibodies against rat serum albumin purified by affinity chromatography. By light microscopy, albumin is present uniformly in all parenchymal cells with no difference in the intensity of reaction in the different parts of hepatic lobules. By electron microscopy, albumin is localized in the entire secretory apparatus including the rough and smooth endoplasmic reticulum, Golgi complex, and secretory vacuoles. In the rough endoplasmic reticulum, focal negative segments are interposed between positive regions. In the Golgi region, albumin is found both in stacked cisternae and at the trans aspect in the portion called GERL (Golgi-endoplasmic reticulum--lysosome). Whereas albumin and lipoprotein particles are separated in terminal dilatations of the endoplasmic reticulum and in the cisternae on the cis face of the Golgi apparatus, they are usually intermixed in vacuoles of the trans face. Similarly, most secretion vacuoles below the sinusoidal lining contain albumin and lipoprotein particles together, although a few are also found with only one secretory product. These observations suggest that, after synthesis in the rough endoplasmic reticulum, albumin is segregated into smooth transitional elements and transported to the Golgi region where it is packaged together with other secretory products such as lipoproteins. These secretion vacuoles move up the sinusoidal surface, where they are discharged. The possible involvement of GERL in the proteolytic cleavage of proalbumin to albumin is considered.
A cell line, RTL-W1, has been developed from the normal liver of an adult rainbow trout by proteolytic dissociation of liver fragments. RTL-W1 can be grown routinely in the basal medium, L-15, supplemented with 5% fetal bovine serum. In this medium, the cells have been passaged approximately 100 times over an 8-year period. The cells do not form colonies or grow in soft agar. The cultures are heteroploid. The cell shape was predominantly polygonal or epithelial-like, but as cultures became confluent, bipolar or fibroblast-like cells appeared. Among the prominent ultrastructural features of RTL-W1 were distended endoplasmic reticulum and desmosomes. Benzo[a]pyrene was cytotoxic to RTL-W1. Activity for the enzyme, 7-ethoxyresorufin O-deethylase (EROD), which is a measure of the cytochrome P4501A1 protein, increased dramatically in RTL-W1 upon their exposure to increasing concentrations of either beta-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). With these properties, RTL-W1 should be useful for studying the expression of the cytochrome P450 enzymes and as a tool for assessing the toxic potency of environmental contaminants.
A permanent cell line has been established from an aflatoxin-induced hepatoma of rainbow trout. The cell line is now 17 years old and has been carried through 133 serial passages. Chromosome analysis indicates the cells are heteroploid and show a bimodal distribution with modal numbers of 54 and 60. Cells are routinely cultured at 18°C and are easily stored in liquid nitrogen.They are susceptible to both the viruses of infectious hematopoietic necrosis and infectious pancreatic necrosis. It is not known if these cells have undergone malignant transformation. However, numerous attempts to cultivate normal liver cells have failed. This culture may represent the first salmonid cell line derived from tumorous tissue.
An in vitro system, the H4IIE rat hepatoma cell bioassay, was characterized for use in assessing the overall toxic potency of PCBs, PCDDs, and PCDFs in extracts from environmental samples. The in vitro bioassay of cytochrome P450IA1 catalytic activity in the H4IIE cells in response to planar halogenated hydrocarbons (PHHs) was repeatable over time and standards were reproducible among laboratories when dosing conditions were similar. Three common extraction/cleanup procedures tested had no adverse affect on the response of the cells and biogenic interferences were not encountered. The results of the bioassay can complement chemical residue analysis and direct the need for such analysis, as well as aid in the interpretation of biological effects data from environmental studies.
IntroductionSpecific Assays of Microsomal EnyzmesInvestigations of Phospholipid-Protein InteractionsPreparation of MicrosomesInterfering Enzymes and Substrate Forms
A human hepatocellular carcinoma (HCC) cell line (KYN-1) has been established from a resected HCC of a 58-yr-old Japanese,
male patient with HCC. Original resected HCC was moderately differentiated and proliferated in a solid pattern with vague
trabecular structure in part. This cell line has been maintained for 10 mo. through 50 passages. Morphological features of
KYN-1 cells demonstrated one or more large, round-to-oval nuceli with prominent nucleoli and eosinophilic polygonal-to-spindle
abundant cytoplasm. In addition, some of these cells contained mucicarmin-positive materials in the cytoplasm. The cells exhibited
a typical epithelial feature with pavementlike cell arrangement, and lacked contact inhibition. The doubling times of the
cells grown in a serum-containing and a serum-fre, medium were about 31 h and 10 to 11 d, respectively. Functonally, KYN-1
cells produced albumin, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), ferritin, β2-microglobulin (BMG), and α1-anti-trypsin (AAT). Positive reactions for albumin, AFP, CEA, and ferritin were identified in the cells by immunohistochemical
techniques. Chromosome study revealed the chromosome number in a range from 61 to 74 without mode. The tumorigenicity of KYN-1
cells was identified by the tumor formation after subcutaneous inoculation of the cells into nude mice. The developed tumor
showed compact growth of the tumor cells with gland formations containing mucicarmin-positive materials. Features of adenocarcinoma
were identified by electron microscopy. The tumor cells were also identified to contain albumin, AFP, CEA, ferritin, and AAT
by immunohistochemical techniques. AFP, CEA, and BMG were detected in the sera of nude mice. Thus, KYN-1 cells represented
the morphologic features of adenocarcinoma, retaining some characteristics of original HCC. These findings suggest that KYN-1
is a new human HCC cell line with transformation to adenocarcinoma, which will provide useful information to clarify the histogenesis
of combined hepatocellular and cholangiocellular carcinoma.
Rainbow trout hepatocytes in primary monolayer culture maintained several important cell functions and enzyme activities involved in xenobiotic metabolism. Furthermore, cytochrome P-450-dependent 7-ethoxyresorufin O-deethylase (EROD) activity and the amount of the major polycyclic aromatic hydrocarbons (PAH)-inducible form of cytochrome P-450 (measured by ELISA using antibody towards cod P-450c) increased after treatment of cells with β-naphthoflavone (BNF) or 2.3.7.8-tetrachlorodibenzo-p-dioxin (TCDD). Cultured trout hepatocytes seems to be a promising in vitro model which may serve as a screening test for toxic constituents in industrial effluents and could serve as an interesting system for studies on regulation of xenobiotic metabolism by exogenous and endogenous factors.
Primary cultures of rainbow trout hepatocytes were used to study the expression of CYP1A1 mRNA, its protein product (P4501A1), and catalytic activities (7-ethoxyresorufin-O-deethylase, EROD) during a 96-h period after exposure of cells to β-naphthoflavone (BNF) or 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD). Hepatocytes were isolated from immature rainbow trout by a two-step perfusion method and incubated at 10 °C. Cells were exposed to the inducers for 48 h after 24 h of preculturing. The EROD activity of BNF-treated hepatocytes was higher than that of the control hepatocytes 12 h after addition of the inducer. Activities peaked after 48 h and had declined to control levels after 72 h. EROD activity in TCDD-treated cells was significantly elevated after 12 h, but in contrast to BNF-exposure, activities continued to increase during the experimental period. The content of P4501A1 protein, measured with an indirect ELISA technique (using anti-codP4501A1 IgG), increased linearly during the first 12 h and remained constant thereafter. In TCDD-exposed cells the immunochemically determined P4501A1 levels changed in parallel with EROD activity. CYP1A1 mRNA levels, determined by Northern blot and slot-blot analyses (using the trout P4501A1 cDNA pSg15 probe), were hardly detectable in control cells. In BNF- and TCDD-treated cells a 2.8-kb mRNA band was detected by the probe 6 h before the protein and catalytic activities became detectable. The elevated levels of CYP1A1 mRNA were sustained more effectively by TCDD than by BNF. In addition, a second mRNA band at 1.9 kb was seen. The results suggest that transcriptional activation is probably the prime factor even though post-transcriptional events may be involved in the regulation of P4501A1 induction by PAHs in rainbow trout hepatocytes.
Cultured fish cells can be used in a variety of cytotoxicity and genotoxicity assays for the preliminary testing of environmental chemical hazards that may be hazardous to the aquatic biota. Such assays can also be used to evaluate synergistic and antagonistic interactions between combinations of test agents and to establish structure-activity relationships for series of related chemicals. A range of fish cell lines are available for use in such assays and a variety of endpoints may be used. To detect toxicants that require bioactivation the chosen cell line must have significant P-450 activity, or a metabolizing component must be incorporated into the assay. Fish cells in culture respond to the same chemical mutagens and clastogens that are genotoxic to mammalian cells in culture. However, since fish cells in culture are eurythermic, they represent a unique system for studying temperature as a parameter in mediating the genotoxicity and the cytotoxicity of a test agent.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
The zebrafish is a popular model for studies of vertebrate development and toxicology. However, in vitro approaches with this organism have not been fully exploited because cell culture systems have been unavailable. We developed methods for the culture of cells from blastula-stage diploid and haploid zebrafish embryos, as well as cells from the caudal and pelvic fin, gill, liver, and viscera of adult fish. The haploid embryo-derived cells differentiated in culture to a pigmented phenotype and expressed, upon exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin, a protein that was immunologically and functionally similar to rainbow trout cytochrome P450IA1. Zebrafish cultures were grown in a complex basal nutrient medium supplemented with insulin, trout embryo extract, and low concentrations of trout and fetal bovine serum; they could not be maintained in conventional culture medium containing a high concentration of mammalian serum. Using calcium phosphate-mediated transfection, a plasmid constructed for use in mammalian cells was introduced into zebrafish embryo cell cultures and expressed in a stable manner. These results indicated that the transfection procedures utilized in mammalian systems can also be applied to zebrafish cell cultures, providing a means for in vitro alteration of the genotype and phenotype of the cells.
The aim of the work presented in this paper was to compare toxic threshold concentrations of three substances obtained from growth test in rainbow trout (Salmo gairdneri) with data from early life-stages in zebrafish. The growth test was conducted over a period of 7 wk in case of 4-chloroaniline and 4 wk in case of 3,4-dichloroaniline and diazinon. The data from the experiment in zebrafish originate from life-cycle studies; here, only the results obtained within the first 6 wk of development after fertilization are considered. These time limits have been set, as in the FRG a growth test in rainbow trout extending over 4 wk and an early life-stage test in zebrafish extending over 6 wk are being discussed for the Chemical Act.
Ah receptor was identified and characterized in cytosol and nuclear extracts from the rainbow trout hepatoma cell line RTH-149. The cytosolic receptor was detectable with both halogenated ([3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)) and non-halogenated ([3H]3-methylcholanthrene and [3H]benzo[a]pyrene) aromatic hydrocarbons and sedimented at approximately 9 S after velocity sedimentation on sucrose gradients. The apparent binding affinity (kd) of cytosolic Ah receptor was always less than 1 nM as derived from Scatchard or Woolf plot analyses. The same analyses indicated a concentration of Ah receptor in the RTH-149 cells of approximately 20 fmol/mg cytosolic protein or approximately 4400 receptor sites per cell. Thus, this trout hepatoma cell line has a low concentration of high-affinity binding sites in comparison to Ah receptor concentrations in cytosol obtained from rodent tissues. Incubation of whole cells with the radioligand [3H]TCDD resulted in transformation of the cytosolic Ah receptor to a nuclear binding form which could be detected as a specifically labeled peak sedimenting at approximately 6 S on sucrose gradients. Aryl hydrocarbon hydroxylase was induced after exposure of RTH-149 cells to TCDD or benz[a]anthracene for 24 hr in culture. These data demonstrate the existence of the Ah receptor in a cell line derived from a nonmammalian species and provide an additional step toward understanding the mechanisms by which fish respond to specific aquatic contaminants.
A recently described chicken liver cell line, LMH, was characterized to evaluate responsiveness to estrogen. Expression of the endogenous apolipoprotein (apo) II gene was induced by 17 beta-estradiol when LMH cells were cultured with chicken serum. The response was low and yielded apoll mRNA at only 0.3% of the level seen in estrogenized rooster liver. Higher levels of apoll mRNA were achieved when LMH cells were transiently transfected with an expression plasmid for estrogen receptor. A transfected apoll gene was strongly expressed only when cotransfected with receptor. Expression of the endogenous vitellogenin (VTG) II gene was not detected. However, when cotransfected with a receptor expression plasmid, VTG II reporter plasmids were expressed in LMH cells in response to 17 beta-estradiol. These results suggest that estrogen responsiveness of LMH cells is limited by the availability of functional receptor. Low levels of estrogen receptor mRNA were detected in LMH cells, and receptor binding sites and mRNA were greatly increased following transient transfection with a receptor expression plasmid. Using this transient transfection protocol, several VTG II reporter plasmids were compared in LMH cells and chick embryo fibroblasts. A plasmid containing VTG II estrogen response elements linked to a heterologous promoter was regulated by estrogen in both cell types. In contrast, reporter plasmids containing the VTG II promoter were regulated by estrogen in LMH cells but were not expressed at all in chick embryo fibroblasts. These results suggest that regulation of the VTG II gene involves cell type-specific elements in addition to estrogen response elements.(ABSTRACT TRUNCATED AT 250 WORDS)
An extract of 21-day rainbow trout embryos stimulated growth of several piscine cell lines in the absence of added serum. Established lines from trout (RTG-2 and STE-137), salmon (CHSE-214), carp (EPC), and goldfish (CAR) and early-passage cells initiated from trout embryos grew in serum-free medium containing the embryo extract. In addition the extract was sufficient for maintaining long-term cultures of CHSE-214 cells for several months through a minimum of 20 passages (approximately 50 population doublings) in the absence of serum. Optimal response was achieved with 100 micrograms of extract protein per ml, but a significant growth-promoting effect was observed with as little as 2.5 micrograms/ml. The activity was nondialyzable, protease-sensitive, and stable in 200 mM acetic acid. The level of mitogenic response induced by the extract could not be duplicated with purified mammalian growth factors added individually or in combination, and the extract did not stimulate DNA synthesis in quiescent mouse fibroblasts. These results suggest that trout embryo extract may contain a novel growth-promoting activity for fish cells.
This study describes a long-term test over three generations, using zebrafish (Brachydanio rerio) as the test species and concentrations of 1, 0.2, and 0.04 mg/L 4-chloroaniline (CA) as a model substance. The effect of the compound on the ecologically important parameters reproduction and growth was the focus of interest. Reduction in egg release by fish raised under CA was the most sensitive parameter in the test. Compared to the toxic threshold concentration for growth (0.4 mg/L), egg release was affected by a ten-fold lower concentration (0.04 mg/L). This study demonstrates that a long-term test is still the most appropriate method to assess the chronic toxicity of a substance on fish. A chronic toxicity test is proposed which comprises two generations, with the zebrafish as test species.
A human hepatocellular carcinoma (HCC) cell line (KYN-1) has been established from a resected HCC of a 58-yr-old Japanese, male patient with HCC. Original resected HCC was moderately differentiated and proliferated in a solid pattern with vague trabecular structure in part. This cell line has been maintained for 10 mo. through 50 passages. Morphological features of KYN-1 cells demonstrated one or more large, round-to-oval nuclei with prominent nucleoli and eosinophilic polygonal-to-spindle abundant cytoplasm. In addition, some of these cells contained mucicarmin-positive materials in the cytoplasm. The cells exhibited a typical epithelial feature with pavementlike cell arrangement, and lacked contact inhibition. The doubling times of the cells grown in a serum-containing and a serum-free medium were about 31 h and 10 to 11 d, respectively. Functionally, KYN-1 cells produced albumin, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), ferritin, beta 2-microglobulin (BMG), and alpha 1-anti-trypsin (AAT). Positive reactions for albumin, AFP, CEA, and ferritin were identified in the cells by immunohistochemical techniques. Chromosome study revealed the chromosome number in a range from 61 to 74 without mode. The tumorigenicity of KYN-1 cells was identified by the tumor formation after subcutaneous inoculation of the cells into nude mice. The developed tumor showed compact growth of the tumor cells with gland formations containing mucicarmin-positive materials. Features of adenocarcinoma were identified by electron microscopy. The tumor cells were also identified to contain albumin, AFP, CEA, ferritin, and AAT by immunohistochemical techniques. AFP, CEA, and BMG were detected in the sera of nude mice. Thus, KYN-1 cells represented the morphologic features of adenocarcinoma, retaining some characteristics of original HCC. These findings suggest that KYN-1 is a new human HCC cell line with transformation to adenocarcinoma, which will provide useful information to clarify the histogenesis of combined hepatocellular and cholangiocellular carcinoma.
Fish represent the largest and most diverse group of vertebrates. Their evolutionary position relative to other vertebrates
and their ability to adapt to a wide variety of environments make them ideal for studying both organismic and molecular evolution.
A number of other characteristics make them excellent experimental models for studies in embryology, neurobiology, endocrinology,
environmental biology, and other areas. In fact, they have played a critical role in the development of several of these disciplines.
Research techniques that enable scientists to make isogenic lines in a single generation, create and maintain mutants, culture
cells, and transfer cloned genes into embryos signal an increasing role for fish as experimental models.
The outstanding characteristic of most vertebrate eggs is their large size. These specialized cells have grown so huge that many can be appreciated without instrumental aids as part of our manifest environment—from the amphibian eggs that grace rural ponds to the omelets and caviar that complement our cuisine. The egg proper of the domestic chicken averages 32 mm in diameter (Romanoff, 1943), for instance, but even this size is exceeded by a number of living vertebrates, such as the coelacanth, with eggs measuring 85–90 mm (Anthony and Millot, 1972). The chicken egg acquires about 99% of its size during the 6 days before laying (Romanoff and Romanoff, 1949), when approximately 1 g protein is accumulated per day (Cutting and Roth, 1973). Thus, pronounced growth occurs in these single cells over a relatively short period of time.
Adult rat parenchymal hepatocytes in primary culture can be induced to enter into DNA synthesis and mitosis. The optimal conditions for hepatocyte replication are low plating density (less than 10,000 cells/sq cm) and 50% serum from two-thirds partially hepatectomized rats (48 hr after hepatectomy). Approximately 80% of the hepatocytes enter the cell cycle, and most of these cells go through mitosis. The replicating hepatocytes remain positive for glucose-6-phosphatase and negative for gamma-glutamyl transpeptidase, and they accumulate fat, in analogy to regenerating liver. Most of the replicating hepatocytes enter into multiple consecutive rounds of DNA synthesis. Dose-response studies between control animal serum and hepatocyte labeling index indicate that in unoperated animals the serum contains substances stimulatory as well as inhibitory for hepatic growth, with the inhibitory effect prevailing at high concentrations. After partial hepatectomy, the inhibitory activity disappears whereas the hepatopoietin activity reaches almost 90% of maximal biological effectiveness at 25% serum concentration. Addition of hormones to the system shows that the hepatopoietin activity is not identical to epidermal growth factor, platelet-derived growth factor, thyroxine, glucagon, or hydrocortisone. Norepinephrine abolishes the difference between control and hepatectomized serum but does not restore hepatopoietin activity when added to heat-inactivated serum. The results show that this system of replicating hepatocytes can be used to investigate the trophic factors that control growth of normal and neoplastic hepatocytes.
Systematic design of replacement chemicals with reduced toxicities will require knowledge of mechanisms of action of parent compounds, especially in species which occupy the environment of most likely exposure. For aquatic systems, the rainbow trout has proven a valuable model for studying mechanisms of carcinogenicity. By comparison, small aquarium species show great potential as in situ field monitors of aquatic contamination by toxic chemicals but are less developed for mechanism studies. Fish species, especially rainbow trout, have also proven useful alternatives to traditional rodent models for comparative studies on mechanisms of action of nonaquatic carcinogens. These kinds of comparative studies form an essential basis for extrapolation of animal studies to man. Carcinogenicity testing of individual compounds and their replacements can provide only limited information on the expected impact of such chemicals on natural populations, since these populations are unavoidably exposed to potent modulators of the carcinogenic response. Hence any program which aims at redesign of commercial chemicals with reduced toxicities must have as a prior aim the full understanding of the mechanisms of joint carcinogen-inhibitor-promotor interactions. Because of their high sensitivity, low cost per individual, and low background tumor incidences, fish models such as the rainbow trout may be the only vertebrate models in which it is economically practical to initiate such complex studies.
The toxicity and carcinogenicity of number of chemicals, for which there are toxicity and/or carcinogenicity data in mouse and rat, were tested in medaka, a small aquarium fish (Oryzias latipes). The water-soluble chemicals were dissolved in the water and the water-insoluble chemicals were incorporated into the diet. Observation for 24 wk revealed induction of liver cell carcinoma by 6 chemicals; aflatoxins B1 and G1, sterigmatocystin, ortho-aminoazotoluene, methylazoxymethanol acetate and N-nitrosodiethylamine. In medaka fed PCB or DDT only preneoplastic liver cell nodules were observed. No evident carcinogenicity was found in medaka treated with 2-fluorenylamine, 2-fluorenylacetamide, DL-ethionine, luteoskyrin, ochratoxin A, phenobarbital, 1-(2-tetrahydrofuryl)-5-fluorouracil, sodium benzoate, 2,3,4,5,6-penta-O-acetyl-D-gluconyl isothiocyanate, citrinin and fusarenon X, respectively, although toxic lesions or hyperplastic changes were demonstrated histologically. These results confirm the practical utility and rapidity of the use of medaka for screening carcinogenicity and toxicity of chemicals, since neoplastic or toxic morphological changes develop rapidly following administration of relatively small quantities of test materials.
Homozygous diploid zebra fish have been produced on a large scale by the application of simple physical treatments. Clones of homozygous fish have been produced from individual homozygotes. These clones and associated genetic methods will facilitate genetic analyses of this vertebrate.
Oval cells are liver epithelial cells that proliferate during hepatocarcinogenesis and chemically induced severe liver injury. It has been suggested that these cells represent hepatic stem cells which might play an important role in the histogenesis of cholangiocellular as well as hepatocellular carcinomas. In order to test this hypothesis highly purified oval cell preparations and propagable oval cell lines are needed. In the present study the isolation, biochemical characterization, and long-term culture of oval cells from rats fed a choline-deficient/DL-ethionine-supplemented diet for 6, 14, or 22 weeks are described. The freshly isolated oval cells were gamma-glutamyltranspeptidase-positive, cytokeratin 7-, 8-, 18-, and 19-positive, albumin-positive, peroxidase-negative, and alpha-fetoprotein-negative and expressed lactate dehydrogenase isoenzymes 1-5. In addition, low but clearly measurable glucose-6-phosphatase and high gamma-glutamyltranspeptidase and alkaline phosphatase activities (when compared to activities in untreated liver parenchymal cells) were measured in oval cells. Three oval cell lines, OC/CDE 6, OC/CDE 14, and OC/CDE 22, were established. They contained small and large epithelial cells replicating to form uniform monolayers with a cobblestone appearance; furthermore, a very low number of mononucleated giant cells were also present in the three cell lines. OC/CDE 6, OC/CDE 14, and OC/CDE 22 cells were gamma-glutamyltranspeptidase-negative, were transiently albumin-positive, maintained the glucose-6-phosphatase activity levels measured in freshly isolated oval cells, and expressed lactate dehydrogenase isoenzymes 2-5. After exposure of the cultured oval cells to dimethyl sulfoxide or sodium butyrate, 35-40% of the cells reexpressed albumin, and glucose-6-phosphatase activity was enhanced; in addition, sodium butyrate strongly increased gamma-glutamyltranspeptidase and alkaline phosphatase activities. In conclusion, oval cells express phenotypic markers of liver parenchymal as well as bile duct epithelial cells and possess a certain intrinsic plasticity. In order to test if the oval cells indeed represent an intermediate step in the differentiation of certain cells within the bile duct and ductular epithelial cell compartment to parenchymal cells, the three cell lines described herein will be transformed in vitro and their potential to give rise to cholangiocellular and/or hepatocellular carcinomas will be verified in vivo.
The expression and induction of cytochrome P450 by 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) and beta-naphthoflavone (BNF) in a new liver cell line from adult zebrafish (Brachydanio rerio) were studied. Subcellular fractions from control, BNF- or TCDD-treated cells did not show detectable bands in immunoblots probed with antibodies to the constitutive forms of trout P450 (LMC1, LMC2, LMC3, LMC4, and LMC5), suggesting that either zebrafish liver cells lack P450s closely related to those constitutively expressed in trout or that the concentrations of the orthologous P450s were too low to be detected. However, upon exposure to TCDD, the cells expressed a major immunoreactive 54-kDa protein and a minor 50-kDa protein recognized by antibodies to rainbow trout P4501A1. These immunoreactive proteins were observed in microsomal and mitochondrial fractions of TCDD-treated cells but were not detected in cell cultures treated with dimethyl sulfoxide (DMSO) (vehicle control) or BNF. The activities of ethoxyresorufin O-deethylase (EROD) and 7,12-dimethylbenzanthracene (DMBA) hydroxylase were markedly increased by TCDD but not by BNF in this cell line. EROD activity was more sensitive than DMBA hydroxylase activity of TCDD-treated liver cells to diagnostic inhibitors such as alpha-naphthoflavone and anti-trout P4501A1 IgG. The TCDD-treated cells converted DMBA to various metabolites, one of which is the putative proximate carcinogen, DMBA-3,4-diol. These results suggest that TCDD, but not BNF, induces one or possibly two forms of P450 immunochemically and functionally related to trout P4501A1, in cultured zebrafish liver cells.
Techniques for the characterization of UDP-glucuronyl transferase, glucose-6-phosphatase and other tightly bound microsomal enzymes Address for correspondence
- D Zaldm
- Vessey
- Da
Zaldm D, Vessey DA. Techniques for the characterization of UDP-glucuronyl transferase, glucose-6-phosphatase and other tightly bound microsomal enzymes. In: Glock D, ed. Methods of biochemicaI analysis, Vol.21. New York: Wiley; 1973. Address for correspondence: Paul Collodi, Ph.D., Department of Animal Sciences, Purdue University, West Lafayette, IN 47907, U.S.A.
Metabolic significance of transamination
- Cooper Ajl Meister
Cooper AJL, Meister A. Metabolic significance of transamination.