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Regulation of Cytochrome P450 Expression in a Novel Liver Cell Line from Zebrafish (Brachydanio rerio)
Abstract
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.
... La fréquence élevée de 13 détection de l'activité spécifique du sous-type zfERβ2 est une découverte importante car ce sous-type d'ER est présent dans les espèces de poissons, mais pas chez les humains (Hawkins et Thomas 2004 L'une des préoccupations majeures en ce qui concerne les essais in vitro sur le gène rapporteur des ER, est liée au contexte de la cellule dans laquelle ils sont réalisés, et qui permet l'expression de co-régulateurs et une machinerie transcriptionnelle pertinents pour cette espèce (Nelson et Habibi 2013). Les essais in vitro au poissons zèbres utilisés dans cette étude ont l'avantage d'utiliser des cellules ZFL qui sont représentatives du contexte des cellules hépatiques de cette espèce modèle (Miranda et al., 1993). Ici, les différences observées entre le poisson zèbre et le dosage humain pour les différentes classes de xénoestrogènes et de matrices environnementales, peuvent refléter les véritables différences inter-espèces qui devraient être prises en compte lors de l'interprétation des données pour l'évaluation des risques pour l'environnement. ...
... Thus, the cell line used for the assays plays a crucial role when interpreting the data. The zebrafish in vitro assays used in this study have the advantage of using ZFL cells that is representative of the liver cell context of this model species (Miranda et al. 1993). Although functional endogenous ER is not detected in this cell system (Cosnefroy et al 2012), it has retained transcriptional machinery and metabolic capacities that make it relevant to the native context. ...
The aim of this PhD thesis was to develop and assess the potential of novel zebrafish (zf)- based in vitro and in vivo reporter gene assays as bio-analytical tools to monitor estrogenic activity and their implementation in effect-directed analysis (EDA) approach to identify fishspecific
estrogenic compounds in complex mixtures. For this purpose, we first characterized the response of the assays towards a panel of (xeno)estrogens, revealing differences in the affinity of zebrafish estrogen receptor (zfER) subtypes to (xeno)-estrogens. Comparison with human cell-based (MELN-hERα) assay further highlighted inter-species differences showing different chemical ranking towards different classes of known ER ligands. Then, application of these tools to different environmental matrices demonstrated for the first time their functionality to detect and quantify estrogenic activity in complex mixtures, highlighting the zfERβ2 assay as the most sensitive among the different in vitro zfER assays. The above in vitro estrogenic activity was also confirmed in vivo at the most contaminated sites by using the EASZY assay, further adding eco-toxicological relevance. Interestingly, we also reported zebrafish-specific activities at several sites that were not active by the human MELN assay, suggesting the occurrence of fish-specific ER ligands. To address this hypothesis, we applied our zebrafish tools in specific higher tier-EDA studies that allowed isolating zebrafish-specific active fractions by multi-step sample fractionation procedures. Chemical analyses of these specific fractions so far identified several candidate compounds, of which few showed higher selectivity towards zfERβ2 than hERα, hence confirming inter-species differences. This work supports recommendations for the integration of these effect-based tools in future water monitoring strategies.
... Primary cell cultures of hepatocytes were obtained from adult zebrafish following a protocol modified from Miranda et al. (1993). Ten to fourteen zebrafish livers were dissected and after washing them in 70% ethanol, they were kept in sterile phosphate buffered saline (PBS, 13 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 135 mM NaCl, 2.5 mM KCl, pH 7.2) plus antibiotic-antimycotic solution (Sigma, St. Louis, USA) till digestion. ...
... In the present work we aimed to determine if known PPARa and PPARc ligands were able to alter the expression of these subtypes in an in vitro model of zebrafish primary hepatocyte culture. Initially trypsin was used for liver tissue dissociation based on the protocol described by Miranda et al. (1993) for establishing a zebrafish liver cell line. The applied method, dissociating cells with trypsin and then adding only FBS to the culture medium, proved to be inefficient for maintaining primary cell cultures longer than 24 h. ...
Peroxisome proliferation is a phenomenon occurring when responsive animals are exposed to certain compounds so-called peroxisome proliferators and is regulated through a nuclear receptor named peroxisome proliferator-activated receptor (PPAR). PPAR family members exhibit a strong binding affinity for both saturated and unsaturated fatty acids. Activators of PPARα include a variety of endogenously present fatty acids, leukotrienes and hydroxyeicosatetraenoic acids (HETEs) and clinically used drugs, such as fibrates. PPARβ activators include fatty acids, prostaglandin A2 (PGA2) and prostacyclin (PGI2). PPARγ is the most selective receptor and, among others, 15-deoxy-Δ12,14 prostaglandin J2 (PGJ2) has been described to be a PPARγ-specific ligand. The aim of the present study was to determine if known PPARα and PPARγ ligands were able to alter the expression of these subtypes in an in vitro model of zebrafish primary hepatocyte culture. With this purpose, a PPARα specific ligand (8S-HETE), a PPARγ specific ligand (PGJ2) and a peroxisome proliferator of the fibrate class (clofibrate) were selected. In addition, the female hormone 17β-estradiol was also used as it is known to interact with PPARs. After cell exposure for 24 h, cells were immunohistochemically stained for both PPARs and immunolabeling was quantified as percentage of positive nuclei and cells. Levels of expression of PPARs were also measured by image analysis as grey level per cell. Expression was induced for both PPARα and PPARγ by clofibrate (at 0.5 mM for PPARα and at 1 and 2 mM for PPARγ), by HETE (1 μM), and by PGJ2 (0.3 and 1 μM for PPARα and 0.3 μM for PPARγ). Expression of PPARγ was also induced at 10 μM by 17β-estradiol. The percentage of PPARα positive nuclei increased significantly at 1 μM HETE and the percentage of PPARγ positive cells decreased at 10 μM 17β-estradiol. As a conclusion, clofibrate, HETE and PGJ2 are able to induce expression of both PPARα and PPARγ in zebrafish primary hepatocyte cultures. Further studies are needed to identify how the expression of different PPAR subtypes is regulated and to elucidate the implication of PPAR subtypes in zebrafish cell functions.
... Primary cell cultures of hepatocytes were obtained from adult zebrafish following a protocol modified from Miranda et al. (1993). Ten to fourteen zebrafish livers were dissected and after washing them in 70% ethanol, they were kept in sterile phosphate buffered saline (PBS, 13 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 135 mM NaCl, 2.5 mM KCl, pH 7.2) plus antibiotic-antimycotic solution (Sigma, St. Louis, USA) till digestion. ...
... In the present work we aimed to determine if known PPARa and PPARc ligands were able to alter the expression of these subtypes in an in vitro model of zebrafish primary hepatocyte culture. Initially trypsin was used for liver tissue dissociation based on the protocol described by Miranda et al. (1993) for establishing a zebrafish liver cell line. The applied method, dissociating cells with trypsin and then adding only FBS to the culture medium, proved to be inefficient for maintaining primary cell cultures longer than 24 h. ...
Peroxisome proliferation is a phenomenon occurring when responsive animals are exposed to certain compounds so-called peroxisome proliferators and is regulated through a nuclear receptor named peroxisome proliferator-activated receptor (PPAR). PPAR family members exhibit a strong binding affinity for both saturated and unsaturated fatty acids. Activators of PPAR alpha include a variety of endogenously present fatty acids, leukotrienes and hydroxyeicosatetraenoic acids (HETEs) and clinically used drugs, such as fibrates. PPAR beta activators include fatty acids, prostaglandin A(2) (PGA(2)) and prostacyclin (PGI(2)). PPAR gamma is the most selective receptor and, among others, 15-deoxy-Delta(12,14) prostaglandin J(2) (PGJ(2)) has been described to be a PPAR gamma-specific ligand. The aim of the present study was to determine if known PPAR alpha and PPAR gamma ligands were able to alter the expression of these subtypes in an in vitro model of zebrafish primary hepatocyte culture. With this purpose, a PPAR alpha specific ligand (8S-HETE), a PPAR gamma specific ligand (PGJ(2)) and a peroxisome proliferator of the fibrate class (clofibrate) were selected. In addition, the female hormone 17 beta-estradiol was also used as it is known to interact with PPARs. After cell exposure for 24 h, cells were immunohistochemically stained for both PPARs and immunolabeling was quantified as percentage of positive nuclei and cells. Levels of expression of PPARs were also measured by image analysis as grey level per cell. Expression was induced for both PPAR alpha and PPAR gamma by clofibrate (at 0.5 mM for PPAR alpha and at 1 and 2 mM for PPAR gamma), by HETE (1 mu M), and by PGJ(2) (0.3 and 1 mu M for PPAR alpha and 0.3 mu M for PPAR gamma). Expression of PPAR gamma was also induced at 10 mu M by 17 beta-estradiol. The percentage of PPAR alpha positive nuclei increased significantly at 1 mu M HETE and the percentage of PPAR gamma positive cells decreased at 10 mu M 17 beta-estradiol. As a conclusion, clofibrate, HETE and PGJ(2) are able to induce expression of both PPAR alpha and PPAR gamma in zebrafish primary hepatocyte cultures. Further studies are needed to identify how the expression of different PPAR subtypes is regulated and to elucidate the implication of PPAR subtypes in zebrafish cell functions. (c) 2005 Elsevier Ltd. All rights reserved.
... Zebrafish liver epithelial cell line (ZLE) was kindly provided by David Barnes and has previously been described (Miranda et al., 1993). The cell line was maintained at 25°C in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum and 1× PSN (penicillin-streptomycin-glutamine, Gibco BRL). ...
... To determine the biological function of zARNT2A in the AhR::ARNT-mediated signal transduction mechanism, a zARNT2A expression vector, pCMV-zARNT2A, was transfected into zebrafish liver epithelial cells (ZLE), and dioxindependent CYP1A mRNA transcription was determined by Northern hybridization analysis. A novel zebrafish cell line, ZLE is sensitive to xenobiotics and responds to TCDD accurately to induce ethoxyresorufin O-deethylase (EROD) activity (Miranda et al., 1993).Figure 2shows that CYP1A mRNA transcription in the ZLE cell line was dose dependent when the concentration of TCDD increased. CYP1A mRNA was not detectable in cultured cells in the absence of TCDD. ...
Aryl hydrocarbon receptor nuclear translocator (ARNT) factors belong to a novel basic-helix-loop-helix–PAS (bHLH-PAS) transcription
factor family that controls a variety of physiological and developmental processes. In a previous study, we obtained a partial
complementary DNA fragment of an ARNT2-like factor from zebrafish embryo, liver, and other tissues by reverse transcription–polymerase
chain reaction. In an effort to characterize the function of this factor, we screened an embryonic complementary DNA library
and obtained a complete cDNA of this ARNT2-like factor, zARNT2A. The deduced protein sequence of zARNT2A encompasses the basic-helix-loop-helix and PAS-A/B motifs and shares highest sequence similarity with the amino-terminal
half of mouse ARNT2 factor. However, it lacks a carboxy-terminal transactivation motif following the PAS-A/B motifs. Transient
expression of zARNT2A in cultured cells resulted in repression of TCDD-dependent CYP1A transcription. Whole-mount in situ hybridization revealed that zARNT2A is expressed in brain and pronephros at prime-5 stages. In adult fish, zARNT2A messenger RNA is transcribed in a wide range of tissues, which indicates that zARNT2A and its corresponding signal transduction mechanisms have important roles in fish development and other physiological aspects.
... Praziquantel Non-hepatotoxic-Anthelminthic Liver necrosis encoding these enzymes (Bresolin et al. 2005;Miranda et al. 1993;Collodi et al. 1994;Trant et al. 2001). ...
The zebrafish as a preclinical drug development model is discussed considering different aspects. 3-Rs Replacement, Reduction and Refinement related to zebrafish for drug discovery are discussed. The strength of zebrafish as a model for preclinical drug development is highlighted. The different aspects of drug development considering the genetics of zebrafish are well covered. Phenotype-based drug development, structure-activity relationship, target-based drug development and toxicities related to zebrafish are elaborated with suitable examples. Organs like heart, liver, intestines, kidney, CNS, haemopoietic system, endocrine system and their drug development-related aspects like pharmacokinetics and drug toxicities in zebrafish are elaborated. Separate topic is dedicated to cancer drug development using zebrafish. Drug resistance related to P-glycoprotein and other related mechanisms is also covered. Practical limitations in use of zebrafish as preclinical drug development models are addressed.KeywordsZebrafishDrug developmentDrug toxicities and zebrafishDrug resistance and zebrafishSAR and zebrafishHeart toxicityLiver toxicityDILICNS and zebrafishCancer and zebrafishLimitations to zebrafish drug research
... The zebrafish liver cell line (ZFL) seemed an appropriate host for transfection of transient reporter vectors of the xenobiotic metabolism pathway, given that former research confirmed the presence of major components of the pathway in the permanent, immortal culture. Reports (Miranda et al. 1993;) disclosed the activity of phase I xenobiotic metabolism enzymes of the cytochrome P450 (CYP) family on the proteomic level. Others (Henry et al. 2001;Evans et al. 2005;Eide et al. 2014) reported basal activity and induction capacity of the zfAhR2, zfAhRR, and zfCyp1A1 genes on the transcriptomic level. ...
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.
... 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.
... Deltamethrin is primarily metabolized by phase I enzymes CYP1A1, CYP1A2, and carboxylesterases 60,61 . The lack of effect in the ZFL cell line may therefore be due to a faster metabolism of the compound within hepatocytes, since functional phase I activity has been reported in vitro 27,62,63 on transcript and protein levels, although also other mechanisms might explain the observed difference in response in ZFL and ZF4. Alterations in antioxidant enzymes, GSH, and LPO levels have also been reported in carp (Cyprinus carpio) after exposure to diazinon 64,65 . ...
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.
... 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. ...
... 13,14 The zebrafish Liver (ZF-L) cell line was established from cells isolated from the liver of D. rerio in 1992 and has been used as a model in several toxicological studies and a promising alternative to the use of animals. 15,16 Established cell cultures are considered to be very effective in some analysis due to their suitability to mechanistic studies. 14 There is a concern, however, whether cell cultures represent reliable models, since they can exhibit altered functions over time, for example, differences in morphology, development, and gene expression. ...
Fish cellular models are commonly used to study the toxic potential of environmentally relevant compounds. Several of these pollutants act on DNA and compromise its integrity. Little is known, however, about the DNA repair ability of these cellular models. Therefore, the aim of this study was to evaluate the DNA base excision repair (BER) of zebrafish Liver (ZF-L) cell line and primary hepatocytes. We performed kinetic studies of the DNA damage levels after exposure to hydrogen peroxide (H2O2, 20 μM for 10 min) using the Comet Assay. Ten minutes after H2O2 treatment, 16% and 50% of the initial damage, measured as comet tail length, were repaired in ZF-L cell line and primary hepatocytes, respectively. Primary hepatocytes repaired 50% of the damages twice as fast as ZF-L cell line and showed DNA damage levels similar to control 40 min after H2O2 treatment. The total recovery time for ZF-L model was of 180 min, which indicates the culture cells have a less efficient BER. In conclusion, both ZF-L cell line and primary hepatocytes exhibit BER activity; however, these cellular models have different repair capacity. In addition, we demonstrated that ZF-L cell line and primary hepatocytes are useful tools for ecotoxicological studies focusing on DNA single-strand breaks and BER.
... Cells at about 90% confluence were used for cryostorage every passage. The cells were collected as mentioned above and resuspended in 1 ml cold DMEM-F12 medium (4 ∘ C) containing 20% FBS and 5% dimethyl sulphoxide (DMSO) (Miranda et al., 1993) and then were dispensed into 2 ml sterile cryovial (Corning), placed into a CoolCell cell-freezing containers (Biocision; www.biocision.com) at −80 ∘ C overnight and finally stored in liquid nitrogen (Wei et al., 2010). To recover the culture from frozen cells, the vial from liquid nitrogen was thawed at 28 ∘ C for 3 min and centrifuged at 112g for 5 min. ...
... His laboratory was the first to purify an inducible form of Cytochrome P450 from fish (Williams and Buhler, 1982) as well as the enzyme responsible for aflatoxin bioactivation (Williams and Buhler, 1983). His laboratory continued to discover new isoforms and orthologs of CYP in a number of species including zebrafish (Miranda et al., 1993;Yin et al., 2008). ...
... 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. ...
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 early characterization of the ZFL cell line provided by Ghosh et al. (1994) indicated expression of serum albumin and hepatocyte-specific enzymes, as well as two Cyp1a1-like proteins induced by exposure to the known AHR-ligand 2,3,7,8,tetrachlorodibenzodioxin (TCDD). However, Miranda et al. (1993) found no effect on protein expression from exposure to 17estradiol, that expectedly would markedly increase expression of the egg yolk protein vitellogenin (Wallace and Selman, 1985). Nor did they find induced protein expression from exposure to naphthoflavone, a well-known inducer of Cyp1a expression in fish (Goksøyr et al., 1991). ...
The zebrafish (Danio rerio) is a widely used model species in biomedical research. The ZFL cell line, established from zebrafish liver, and freshly isolated primary hepatocytes from zebrafish have been used in several toxicological studies. However, no previous report has compared and characterized these two systems at the level of gene expression. The aim of this study was to evaluate the ZFL cell line in comparison to primary hepatocytes as in vitro models for studying effects of environmental contaminants in zebrafish liver. Using quantitative real-time PCR, the basal level and transcriptional induction potential of key genes involved in toxic responses in the ZFL cell line, primary hepatocytes and whole liver from zebrafish were compared. The study showed that the ZFL cells have lower levels of mRNA of most selected genes compared to zebrafish liver. The induced gene transcription following exposure to ligand was much lower in ZFL cells compared to zebrafish primary hepatocytes at the doses tested. Importantly, oestrogen receptor and vitellogenin genes showed low basal transcription and no induction response in the ZFL cell line. In conclusion, it appears that primary hepatocytes are well suited for studying environmental contaminants including xenoestrogens, but may show large sex-dependent differences in gene transcription. The ZFL cell line shows potential in toxicological studies involving the aryl hydrocarbon receptor pathway. However, low potential for transcriptional induction of genes in general should be expected, especially notable when studying estrogenic responses.
... Primary cell cultures of hepatocytes were obtained from adult zebrafish following a protocol modified from Miranda et al., 1993. Briefly, 10-14 fish were sacrificed under a laminar flow hood by keeping them on ice, sterilized by a quick immersion in 70% alcohol, and the livers were excised and rinsed with phosphate buffered saline (PBS, 13 mM Na2HPO4, 1.5 mM KH2PO4, 135 mM NaCl, 2.5 mM KCl, pH 7.2) plus antibiotic-antimycotic solution (100 U/ml) (Sigma, Milan, Italy), to remove blood cells. ...
... Distinctions between PAH and PHAH also have been seen in vivo, for example in the expression of CYP1A1 mRNA content in livers of scup (Stenotomus chrysops) treated with BNF or 2,3,7,8-tetrachlorodibenzofuran [24,25]. The lack of detectable EROD activity in a liver cell line from zebra fish (Brachydanio rerio) treated with up to 3.6 M BNF for 48 h [26] might also result from metabolism of BNF, although no induction in EROD activity was observed in liver microsomes from zebra fish exposed in vivo to 50 g BNF/L water (approximately 0.2 M) for 48 h [27]. The results stress the importance of determining both the dose-response as well as the time course of response for compounds under investigation. ...
The induction of CYP1A by the polycyclic aromatic hydrocarbon (PAH)-type inducer β-naphthoflavone (BNF) in the Poeciliopsis-lucida hepatocellular carcinoma cell line (PLHC-1), and the effects of the glucocorticoid receptor (GR) agonist dexamethasone (DEX) on this response were examined. Dose-response studies revealed that BNF is three orders of magnitude less potent than the planar halogenated aromatic hydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as an inducer of the CYP1A activity ethoxyresorufin-O-deethylase (EROD), and that the apparent efficacy for the induction by BNF is 50% of that obtained with TCDD. Addition of 10 μM DEX resulted in potentiation of CYP1A induction at all doses of BNF tested. The degree of that potentiation of induction of CYP1A protein levels and EROD activity differed substantially between doses of BNF and at different times of exposure. For example, the maximal degree of potentiation of EROD induction by DEX was 12-fold in PLHC-1 cells treated with 0.1 μM BNF, 19-fold in cells treated with 1 μM BNF, and 8-fold in cells treated with 10 μM BNF. These maximal degrees of potentiation of EROD induction were obtained after 30 h with 0.1 μM BNF, 48 h with 1 μM BNF, and 72 h with 10 μM BNF. These results demonstrate interactions between GR and aryl hydrocarbon receptor pathways that could influence the response of fish to xenobiotic exposure.
... The use of in vitro systems in toxicological studies is rapidly growing in the recent years. ZFL cell-line was isolated in 1992 and was already used in toxicological studies (Miranda et al., 1993; Seok et al., 2007). Cheuk et al. (2008) and Chan et al. (2006) reported data describing the effects of ZFL cell-line exposure to metals, including copper. ...
Copper is an essential metal to aquatic animals, but it can be toxic when in elevated concentrations in water. The objective of the present study was to analyze copper effects in zebrafish hepatocytes (ZFL cell-line). The number of viable cells and copper accumulation were determined in hepatocytes exposed in vitro to different copper concentrations (5-30mgCu/L). Intracellular reactive oxygen species (ROS) formation, total antioxidant capacity against peroxyl radicals, and expression of genes related do DNA repair system were also measured in hepatocytes exposed to 5 and 20mgCu/L. After 24h of exposure, hepatocytes showed an exponential kinetics of copper accumulation. Copper exposure (24 and 48h) significantly reduced hepatocyte number in all concentrations tested, except at the lowest one (5mgCu/L). Exposure to 20mgCu/L for 6, 12 and 24h significantly increased intracellular ROS formation. However, no significant change in total antioxidant capacity was observed. After 12 and 24h of exposure to 20mgCu/L, a significant decrease in expression of p53 and CDKI genes was observed. Conversely, expression of Gadd45alpha, CyclinG1 and Bax genes was significantly induced after 24h of exposure to 20mgCu/L. In hepatocytes exposed to 5mgCu/L, any significant alteration in expression of these genes was observed. In a broad view, most of genes encoding for DNA repair proteins were inhibited after copper exposure, especially in hepatocytes exposed to 20mgCu/L. Taken all together, results obtained suggest that the increased intracellular ROS formation induced by copper exposure would be responsible for the alteration in gene expression pattern observed.
... A cell line, ZSSJ, has been developed from the spleen of adult zebrafish and is one of the few cell lines arising from an internal organ of this species and the first from the spleen. Although approximately 20 zebrafish cell lines have been described, most have been from embryos or external surfaces, such as fins and gills (Bradford et al., 1994;Collodi et al., 1994;Driever & Rangini, 1993;Fan et al., 2004;Gosh & Collodi, 1994;Miranda et al., 1993;Pando et al., 2001;Paw and Zon, 1999;Pepplenbosch et al., 1995;Whitmore et al., 2000, Xing et al., 2008). For the most part, the general properties of ZSSJ were similar to those of the other zebrafish cell lines. ...
A zebrafish spleen cell line, ZSSJ, was developed and its growth arrest by gamma radiation determined and its capacity to stimulate the proliferation of the zebrafish blastula cell line, ZEB2J, measured. ZSSJ was initiated by explant outgrowth, grew adherent with mainly an epithelial-like morphology, and stained strongly for alkaline phosphatase. ZSSJ was not only grown in L-15 with 15% fetal bovine serum at 26 degrees C to 28 degrees degrees C but also grew at room temperature. Cultures of ZSSJ have undergone approximately 40 population doublings, had few cells staining for b-galactosidase activity, which is commonly present in senescent cultures, and many cells with an aneuploid karyotype, which is frequently associated with immortalization. ZSSJ growth was arrested by 30 to 50 Gy of g-irradiation, whereas after 20 Gy, some slight growth was observed. By contrast, growth of the rainbow trout spleen stromal cell line, RTS34st, which has been used as a feeder for zebrafish ES cell cultures, was arrested completely by 20 Gy. In cocultures, nongrowth-arrested ZSSJ stimulated ZEB2J proliferation better than growth-arrested ZSSJ and better than RTS34st. ZSSJ should be useful as a feeder cell line for zebrafish ES cell cultures.
... To obtain the full-length zebra¢sh AhR cDNA, a poly(dT) primed cDNA library was prepared from zebra¢sh liver cells. The ZF-L cells were used since CYP1A induction has been shown to follow TCDD exposure in this cell line [30,42]. A total of four overlapping clones were obtained after screening approximately 2U10 6 There are several interesting zfAhR2 characteristics worth noting (Fig. 1). ...
The aryl hydrocarbon receptor (AhR) mediates the toxicity of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds in vertebrates. To further establish zebrafish as a vertebrate model to study the molecular mechanism of TCDD toxicity, we have isolated and characterized the cDNA encoding the zebrafish aryl hydrocarbon receptor (zfAhR2). Analysis of the deduced protein sequence revealed the 1027 amino acid protein is approximately 200 amino acids longer than previously isolated receptors. zfAhR2 is homologous to previously cloned PAS proteins within the basic helix-loop-helix and PAS domains. The C-terminal domain of zfAhR2 diverges from the mammalian AhR at position 420, and does not contain a Q-rich domain. zfAhR2 mRNA is first detected by Northern blot analysis at 24 h post fertilization, and expression increases throughout early development. Treatment of zebrafish embryos and zebrafish liver cells with graded doses of TCDD results in a dose-dependent increase in zfAhR2 mRNA. The time course for zfAhR2 and cytochrome P4501A mRNA induction by TCDD are similar. In vitro produced zfAhR2 protein dimerizes with the rainbow trout aryl hydrocarbon receptor nuclear translocator (rtARNTb) and binds dioxin response elements derived from the rainbow trout CYP1A gene. Finally, transient coexpression of zfAhR2 and rtARNTb in COS-7 cells results in a TCDD dose-related increase in transcription driven by the rainbow trout CYP1A promoter and enhancer.
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.
Fish cell cultures from fish are used in studies on the fate and effects of xenobiotics at the cellular level. So far, special attention has been paid to primary cultures of hepatocytes. This cell type has been particularly useful for studies on biotransformation of xenobiotics. Among biotransformation enzymes, a number of studies have been devoted to cytochrome P4501A due to its inducibility by ecotoxicologically relevant pollutants. Biomarkers of DNA damage, i.e., DNA adducts and single strand breaks, have also been investigated. Furthermore, primary cultures of fish hepatocytes appear as promising models for studies on the effects of xenobiotics on critical steps of fish reproductive physiology. All the data are discussed in an ecotoxicological perspective.
Zebrafish: Methods for Assessing Drug Safety and Toxicity offers a practical guide for using zebrafish as a tool for toxicology studies. Consolidating key protocols and approaches to help researchers navigate the important and evolving field of zebrafish models for toxicity screening, this new title describes the methods for using the zebrafish as a model organism to assess compound-induced toxicity on all major organs. Individual chapters that concentrate on assays for each organ system are included and various analytical tools including microscopy, microplate readers, high content imaging systems, ECG, blood pressure monitors, high speed video and motion detectors are described.
This study investigated the inductive response of cytochrome P4501A (CYP1A) in the zebrafish (Danio rerio) following exposure to Aroclor 1254, β-naphthoflavone (βNF), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and then investigated TCDD modulation of aflatoxin B1(AFB1) metabolism and hepatic AFB1–DNA adduction. Aroclor 1254 fed at 500 ppm for 1 to 9 days or intraperitoneal (ip) injection of 75–200 mg Aroclor 1254/kg body weight failed to induce CYP1A protein or associated 7-ethoxyresorufin-O-deethylase (EROD) activity. By contrast, dietary βNF at 500 ppm for 3 or 7 days induced CYP1A protein and EROD activity approximately threefold above controls. A single ip injection of 150 mg/kg βNF showed maximal induction of CYP1A protein and EROD activity near 24 hr, both of which decreased to control levels during the next 6 days. Single ip administration of 25, 50, 100, or 150 mg βNF/kg body weight provided dose-responsive increases in CYP1A and EROD activity. Dietary exposure to 0.75 ppm TCDD for 3 days also significantly induced CYP1A and EROD. The effect of TCDD on the metabolism of [3H]AFB1in zebrafish was then investigated. The major [3H]AFB1metabolites excreted in water over 24 hr in the control group were aflatoxicol, aflatoxicol-glucuronide, and parent AFB1. By contrast, the predominant metabolites in the TCDD-pretreated group were aflatoxicol-M1-glucuronide, aflatoxicol, aflatoxin M1plus aflatoxicol-M1(unresolved), aflatoxicol-glucuronide, and parent AFB1. Surprisingly, hepatic AFB1–DNA adduction was approximately fourfold higher in the TCDD treated group than in controls. This significant difference could not be explained by increased capacity for bioactivation of AFB1as measured by anin vitroAFB1-exo-8,9-epoxide trapping assay. However, it was demonstrated that both control and induced zebrafish have high capacity to bioactivate aflatoxin M1to a reactive intermediate, such that secondary bioactivation of this genotoxic intermediate may be responsible for the increased DNA binding.
Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatocytes isolated from immature rainbow trout (Oncorhynchus mykiss) by collagenase perfusion were investigated with respect to induction of cytochrome P450 1A (CYP1A) enzyme activities and protein contents as well as DNA damage. Exposure of primary rainbow trout hepatocytes to TCDD resulted in increased CYP1A contents, as determined by immunoblotting, enhanced activities of 7-ethoxyresorufin-O-deethylase (EROD) and increased DNA damage as determined by the comet assay. By means of electron microscopy, no symptoms of cytotoxicity could be observed except for slight increases of lysosomal components and the smooth endoplasmic reticulum. Whereas CYP1A contents constantly increased over the duration of the entire experiment, EROD activities remained constant from day 3 of exposure to 1 nM TCDD; maximum induction of CYP1A activities was reached with 0.1 nM TCDD after 5 days. DNA damage increased in a time- and dose-dependent fashion until day 3. After 5 days, DNA damage was less pronounced, and the number of damaged nuclei declined in all TCDD concentrations. Since TCDD has been shown to not directly react with DNA, metabolism of TCDD or TCDD-induced changes in other metabolic pathways are suspected to result in the production of DNA-reactive (endogenous) substances.
This chapter presents a description of the methodology applied to zebrafish. With the general approaches of animal cell culture, the chapter provides an overview of the basic equipment, techniques, and concepts. Zebrafish embryo cell cultures can be initiated using techniques adapted from mammalian cell culture techniques, although several methodological aspects require approaches somewhat unique to fish species. The culture techniques that have benefitted other model systems can thus be applied to zebrafish developmental biology including genomic manipulation and cell selection in vitro. The chapter also discusses the application of the techniques to other interesting models, such as the Japanese pufferfish, Fugu. In principle, the approaches for zebrafish can be applied to any fish species. The Japanese pufferfish possesses a highly compact genome, and has gained interest among those involved in the sequencing and mapping of vertebrate genomes. Cell cultures from these fish provide a biological complement to the genomic libraries derived to study the molecular biology of Fugu.
An enzyme-linked immunosorbent assay (ELISA) for measuring cytochrome P4501A (CYP1A) expression in vitro in fish hepatoma cells is described. Cells were cultured as monolayers in 96-microwell cell culture plates and exposed to polychlorinated biphenyl (PCB) congeners 77, 105, 153, and 169; 3-methylcholanthrene (3-MC), and β-naphthoflavone (BNF) for 3 d. Relative CYP1A protein content, CYP1A enzymatic activity, and total protein content were determined directly within the wells. At low concentrations of PCB 77, PCB 169, and 3-MC, the ethoxyresorufin-O-deethylase (EROD) activity was induced, but it was inhibited at high concentrations of these compounds. However, CYP1A protein content measured in an ELISA performed with intact cells increased monotonically in response to the concentration. No CYP1A induction was observed for PCB 105 and PCB 153. Because comparison between EROD activity and CYP1A amount gives information about the catalytic efficiency of CYP1A in the cells, this noncompetitive, solid-phase ELISA is recommended as a complementary method to the EROD assay. This novel ELISA method may be an accurate in vitro technique for a rapid and sensitive screening of CYP1A-inducible compounds.
Zebrafish (Brachydanio rerio) were evaluated as a small fish model for environmental carcinogenesis monitoring. Criteria used for evaluation included: (1) sensitivity to dose response exposures of 6 known carcinogens by 4 routes of exposure, (2) histopathologic evaluation of the resulting lesions and comparison with responses in other species, (3) response to promoters of neoplasia, (4) ability to conduct carcinogen metabolism studies to understand mechanisms of action, (5) determining the role of oncogenes in zebrafish carcinogenesis, and (6) success in developing transgenics that would increase the species' usefulness as a carcinogen monitoring model. Findings were (1) zebrafish did respond to known carcinogens with variable but overall acceptable sensitivity, (2) the histopathology of their responses was similar to that observed in other small fish species, (3) they did not respond well in limited promotional studies, (4) metabolism studies were very difficult due to small fish and organ size and results did not always support observed tumor responses, (5) ras and p53 genes were sequenced but lack of grossly observable tumors prevented the determination of mutations in these genes, and (6) although progress was made, the production of transgenic fish was not achieved. Although none of these results would disqualify zebrafish as environmental monitors, their strict requirement for tropical or subtropical water temperatures would make them completely unsuitable for temperate water use.
The heterodimeric complex of Ah receptor (AHR) Ah receptor nuclear translocator (ARNT) is an ubiquitous transcription factor which mediates the expression of vertebrate xenobiotic-response genes, such as CYP1A1 and CYP1A2. AHR also performs key functions in murine tissue differentiation. Both AHR and ARNT factors share conserved function domains with PAS domain families, such as Drosophila CNS-developing modulator, SIM, and biological rhythm factor, PER. By using RT-PCR technique, we have obtained partial cDNA fragments of zebrafish AHR and ARNT from fish tissues. We found that both ahr and arnt genes are active during fish embryogenesis. The mRNAs of AHR and ARNT are also transcribed in oocytes as maternal mRNA. The deduced amino acid sequences derived from the amplified cDNA fragments share significant homology with the respective mammalian AHR and ARNT PAS domain sequences. The xenobiotic, 2,3,7,8-TCDD, strongly induces zebrafishCYP1A expression during embryogenesis. Apparently, both AHR and ARNT factors are present in fish embryos in forms that can accurately respond to the proper ligands to induce CYP1A. We speculate these factors play similar functional roles in fish development as they do during developemnt of murine embryos.
Zebrafish (Danio rerio) has been extensively studied and well described for environmental toxicity studies. Molecular biology and genetics have recently been used to elucidate the underlying mechanisms of toxicity in zebrafish and to predict effects in mammals. The versatile zebrafish is now incorporated in many areas of toxicological programs for assessing human risk and for preclinical drug discovery and screening.
This chapter explains the utility of zebrafish as a model for toxicological research. Zebrafish (Danio rerio) have long been the genetic model of choice for vertebrate developmental biologists, as it provides several advantages for investigating organ and tissue development. Zebrafish have become a powerful model organism for investigating the molecular and cellular mechanisms by which environmental chemicals disrupt normal developmental processes. The utility of zebrafish as a laboratory model organism also makes it an excellent system for studying processes in juvenile and adult organisms, including reproductive development, carcinogenesis, aging, and the influence of environmental chemicals on these processes. The advent of modern genetic tools and genome sequencing projects has elevated zebrafish as a suitable model to effectively study human disease and pathophysiology, ushering in a new era of comparative biology and medicine. The chapter reviews the mechanistic toxicology and advantages of the zebrafish model system.
Quantification of levels of cytochrome P4501A (CYP1A) gene expression in sentinel species of fishes has been proposed as a
management tool to evaluate contamination of aquatic systems. Based on preliminary studies, we hypothesized that differences
in CYP1A mRNA inducibility among individuals, populations, or species might lead to spurious conclusions when using this approach
in environmental monitoring programs. To address this possibility, we quantitated and compared CYP1A mRNA induction levels
in four species of common Atlantic Coast estuarine fish: smooth flounder, hogchoker, striped bass, and Atlantic tomcod, which
were treated with model chemicals (beta naphthoflavone (β-NF), or benzo[a]pyrene at 10 ppm) known to induce CYP1A mRNA, or
were exposed to contaminated environments. Species-specific CYP1A DNA probes were generated from PCR (polymerase chain reaction)
amplification of genomic DNA using conserved oligonucleotide primers, and, along with cloned rainbow trout and Atlantic tomcod
CYP1A cDNA probes were used to quantify CYP1A mRNA levels in northern blot analyses. Successful PCR amplification of CYP1A
hybridizable DNA fragments was observed for all four species. Results from northern blot analyses showed large differences
in CYP1A mRNA induction among species; only Atlantic tomcod exhibited significant induction of CYP1A mRNA for both chemically
treated (97-fold) and environmentally exposed fish (34-fold). Significant, although lower, levels of induction were observed
in β-NF treated (14-fold) smooth flounder, but not in environmentally exposed smooth flounder. Only low levels (not significant)
of CYP1A gene induction were detected in hogchokers and striped bass. We conclude that CYP1A mRNA inducibility differed significantly
among fish taxa perhaps due to differences in regulation of gene expression, suggesting that careful selection of sentinel
species should be exercised prior to the use of CYP1A mRNA induction in environmental monitoring programs. However, the significance
of differences in CYP1A mRNA inducibility in relation to higher level biological endpoints has yet to be determined.
ARNT factors are a cluster of bHLH-PAS factors that heterodimerize with other specific bHLH-PAS factors to mediate a wide range of biological responses. Previously, we obtained a truncated form of ARNT2-like factor, ARNT2A, from zebrafish, which encompasses the basic-helix-loop-helix and PAS A/B domains, but lacks a transactivation domain at its carboxyl end. Herein, we report another truncated ARNT2-like factor, ARNT2X, in zebrafish, which differs from ARNT2A at its N-terminal region. In cultured ZLE cells, transiently expressed ARNT2X and ARNT2A inhibited 2,3,7,8-TCDD-activated cyp1a1 transcription with different efficiencies. In the developing embryo, arnt2X mRNA was consistently expressed in the retinal and neural tube regions until the hatching stages, but it exhibited a more specific pattern at larval stages, including expression in the brain, eyes, hypothalamus, pharyngeal skeleton, heart, liver, pronephros duct, pectoral fin, and epithelial cells of the swim bladder. In contrast, arnt2A transcription diminished after hatching. Microinjecting a recombinant arnt2X-expression vector into fertilized eggs before cleavage stages caused severe defects in brain, eyes, pectoral fin, heart, and gut development. This suggests that the ARNT-mediated signal transduction pathways play important roles in fish tissue development.
As the zebrafish, Danio rerio, has been increasingly used as an animal model for biomedical research, we aimed to establish zebrafish cell line models for inflammation and cancer studies in this thesis. Several zebrafish cell lines were characterized and their genetic and physiological properties were compared. We also developed a set of tool methods to investigate cellular signaling events in zebrafish cell lines. Our case studies illustrated that zebrafish cell lines are as reliable models as the widely used mammalian cell cultures. Taking advantage of the transparency of zebrafish embryos and cell implantation protocols, zebrafish cell lines can serve as a bridge platform between in vitro, in silico, ex vivo and in vivo studies in order to enhance our understanding of molecular mechanisms underlying disease progression.
Isolation and functional characterization of a dioxin-inducible CYP1A regulatory region from zebrafish (Danio rerio)
To investigate the role of detoxification-related liver genes in amnesic shellfish poisoning toxin metabolism, red sea bream Pagrus major were exposed to domoic acid (DA, 2mugg(-1) wet weight) for 24h. Hepatic mRNA expression levels of AHR, ARNT, CYP1 and GSTs were determined by semi-quantitative RT-PCR. The cytosolic factors aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) mRNA levels of DA exposure group were substantially enhanced by 113.3% and 90.9%, respectively. Consistent with this result, the phase I xenobiotic metabolizing enzyme (XME) cytochrome P-450 1A (CYP1A) was significantly induced. In contrast, the transcriptions of three major phase II XME glutathione S-transferases as well as heat shock protein 70 were not significantly affected by DA exposure. These results suggest a possible role of CYP1A after DA exposure in the toxin metabolism of marine fish, possibly through the AHR/ARNT signaling pathway.
Immunoblots (Western blot) of mullet liver microsomes revealed the presence of multiple forms of P-450 that appear to be structurally related to rainbow trout CYP1A1 and CYP2K1 and to P-450 LMC1 and LMC4, but not to LMC5. 3-Methylcholanthrene but not beta-naphthoflavone induced a major 58 kDa liver protein and a minor 56 kDa protein in mullet that both cross-reacted with anti-trout CYP1A1 IgG. The levels of immunodetectable P-450s and the activities of microsomal lauric acid hydroxylase, DMBA hydroxylase and progesterone 6 beta-hydroxylase were several times lower in mullet liver than in rainbow trout liver; however, progesterone 16 alpha-hydroxylase and progesterone 20 alpha-hydroxylase activities were 4-fold and 6-fold higher, respectively, in mullet than in trout liver.
1. Induction of zebrafish P450 by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was studied in liver tissue, primary liver cell culture and multipassage cell culture derived from zebrafish haploid and diploid embryos and liver. 2. TCDD induced two hepatic proteins (54 and 50 kDa) in vivo which were recognized by anti-trout P4501A1 IgG. The 54-kDa protein was induced by TCDD in primary and multipassage hepatocyte cultures and in haploid and diploid embryo-derived cells. The proteins in liver homogenates were not induced by aqueous exposure of zebrafish to beta-naphthoflavone (BNF). 3. Homogenates of zebrafish liver, cultured hepatocytes and embryo-derived cells also exhibited increased ethoxyresorufin O-deethylase (EROD) and 7-12-dimethyl-benz[a]anthracene (DMBA) hydroxylase activity following TCDD exposure.
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.
The nuclease P1 modification of the 32P-postlabeling technique was used to study the biological activity of 7H-dibenzo[c,g]carbazole (DBC) and some of its derivatives, including N-methyldibenzo[c,g]carbazole (N-MeDBC), 5,9-dimethyldibenzo[c,g]carbazole (5,9-diMeDBC), 5,9,N-trimethyldibenzo[c,g]carbazole (5,9,N-triMeDBC), 6-methoxydibenzo[c,g]carbazole (6-McODBC), N-acetyldibenzo[c,g]carbazole (N-AcDBC), N-hydroxymethyldibenzo[c,g]carbazole (N-HMeDBC) in primary mouse embryo cells. A very good correlation was found between carcinogenic specificity in vivo of these N-heterocyclic aromatic hydrocarbons and their DNA-adduction in vitro. Primary mouse embryo cells were able to metabolize and detect tissue-specific sarcomagens N-MeDBC and 6-MeODBC as well as derivatives with both sarcomagenic and hepatocarcinogenic activity, DBC, N-AcDBC, and N-HMeDBC. The strong specific hepatocarcinogen 5,9-diMeDBC in vivo, did not induce any DNA-adducts in the embryo cells, which suggests that the enzymatic composition of the target tissue probably is the determining factor in the organ specificity of this derivative. 5,9,N-triMeDBC, derivative without any carcinogenic activity in vivo, did not induce any DNA-adducts in primary mouse embryo cells. Pretreatment of cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) apparently stimulated DNA-adduct formation in the cells exposed to DBC, 6-MeODBC, and N-MeDBC. No or a very slight effect of TCDD on DNA-adduct formation was found in cells exposed to N-HMeDBC and N-AcDBC. Preliminary results have shown that TCDD slightly induced cytochrome P4501A1-linked ethoxyresorufin O-deethylase (EROD) activity in primary mouse embryo cells. These data suggest the role of cytochrome P4501A1 in the metabolism of DBC derivatives with sarcomagenic activity.
As is the case with mammals, an ever-increasing number of cytochromes P450 (CYPs) are being characterized from fish. The focus of work on fish CYPs has been primarily on environmental induction of CYP1A by pollutants such as the polycyclic aromatic hydrocarbons, polychlorinated biphenyls, dioxins and dibenzofurans. This response has been the basis for a sensitive biomonitoring tool of ecosystem health for a number of years. Studies have documented a correlation between CYP1A induction, pollutant levels and tumor incidence, especially in bottom-dwelling species. The rainbow trout has been utilized as a tumor model to document the role of CYP1A modulation in the inhibition or promotion of cancer. Fish are also very responsive to the class of chemicals known as xenoestrogens. Recent evidence is presented documenting the modulation of CYPs by xenoestrogens and their potential role as modulators of the tumor response. In this paper, we summarize the current knowledge concerning the occurrence of CYPs in fish and focus on the role of CYP1A induction in environmental monitoring of various genotoxic carcinogens and in the modulation of cancer in the trout model. Finally, the important class of aquatic pollutants known as xenoestrogens have now been shown to modulate CYP levels perhaps leading to alterations in tumor response or other adverse effects.
In aquatic toxicology, cytotoxicity tests using continuous fish cell lines have been suggested as a tool for (1) screening or toxicity ranking of anthropogenic chemicals, compound mixtures and environmental samples, (2) establishment of structure-activity relationships, and (3) replacement or supplementation of in vivo animal tests. Due to the small sample volumes necessary for cytotoxicity tests, they appear to be particularly suited for use in chemical fractionation studies. The present contribution reviews the existing literature on cytotoxicity studies with fish cells and considers the influence of cell line and cytotoxicity endpoint selection on the test results. Furthermore, in vitro/in vivo correlations between fish cell lines and intact fish are discussed.
During recent years, fish cell lines have been increasingly used for purposes beyond their meanwhile established role for cytotoxicity measurements. They have been successfully introduced for detection of genotoxic effects, and cell lines are now applied for investigations on toxic mechanisms and on biomarkers such as cytochrome P4501A. The development of recombinant fish cell lines may further support their role as a bioanalytical tool in environmental diagnostics.
Organophosphate (OP) anticholinesterases were found to modulate metabolic activities of human neuroblastoma cells and hepatocytes, which was detectable by the Cytosensor microphysiometer. The nerve gas ethyl-S-2-diisopropylaminoethyl methylphosphorothiolate (VX), at 10 microM, produced significant reduction in cell metabolism within 2 min, as measured by changes in the acidification rate of the medium. The reduction was dose- and time-dependent and irreversible after 4 h of exposure. Two alkaline degradation products of VX produced no cytotoxicity. Exposure for 24 h to 3 microM VX caused 36% and 94% irreversible loss of metabolism in hepatocytes and neuroblastoma cells, respectively. The insecticides parathion and chlorpyrifos stimulated hepatocyte metabolism but inhibited neuroblastoma cells. Their oxons were more active. Exposure of neuroblastoma cells for 4 h to VX, parathion, paraoxon, diisopropylfluorophosphate or chlorpyrifos gave an LC50 of 65, 775, 640, 340, or 672 microM, respectively, whereas 24 h gave an LC50 of 0.7, 3.7, 2.5, 29, and 31 microM, respectively. Preincubation of hepatocytes with phenobarbital enhanced their response to parathion and VX due to metabolic bioactivation. Atropine partially blocked the effects of VX and paraoxon on both cell types, which suggests the involvement of a muscarinic receptor as the target for cytotoxicity. There was no correlation between OP in vivo neurotoxicity and in vitro cytotoxicity. It is suggested that the former results from their cholinesterase inhibition, while the latter results from action on different targets and requires much higher concentrations.
To evaluate the rpsL transgenic zebrafish (Brachydanio rerio) mutation assay, we treated the embryos with benzo[a]pyrene (B[a]P) (10 microg/ml) or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) (300 microg/ml) for 16h and determined the mutation spectra. These treatments were previously reported to induce mutant frequencies that were 4.3 and 2.4 times the control value, respectively. In the B[a]P-treated group, half of the mutations were single base substitutions, 74% of which occurred at G:C base pairs. Among G:C base pair substitutions, G:C to T:A and G: C to C:G transversions were predominant, suggesting that B[a]P induced mutations in zebrafish embryos by mechanisms previously described in mammalian tissues. In the MeIQx-treated group, about 60% of the mutations were deletions. Some specific mutations were found, but the compound primarily amplified the background mutation level; improvement in the conditions of treatment may be required for elucidating MeIQx-mutagenesis in this system. This study showed that transgenic zebrafish may be a useful tool for detecting mutagens in aquatic environments and for elucidating mutagenic mechanisms.
Fish are known to have two distinct classes of aryl hydrocarbon receptors, and their roles in mediating xenobiotic toxicity remain unclear. In this study, we have identified and characterized a cDNA tentatively named zebrafish AHR1 (zfAHR1). Analysis of the deduced amino acid sequence reveals that the protein is distinct from zfAHR2 and is more closely related to the mammalian aryl hydrocarbon receptor (AHR). zfAHR1 and zfAHR2 share 40% amino acid identity overall and 58% in the N-terminal half. The zfAHR1 gene maps to linkage group 16 in a region that shares conserved synteny with human chromosome 7 containing the human AHR, suggesting that the zfAHR1 is the ortholog of the human AHR. zfAHR2 maps to a separate linkage group (LG22). Both zfAHR mRNAs are expressed in early development, but they are differentially expressed in adult tissues. zfAHR2 can dimerize with zfARNT2b and binds with specificity to dioxin-responsive elements (DREs). Under identical conditions, zfAHR1/zfARNT2b/DRE complexes are formed; however, the interactions are considerably weaker. In COS-7 cells expressing zfARNT2b and zfAHR2, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure leads to a significant induction of dioxin-responsive reporter genes. In identical experiments, TCDD exposure fails to induce the reporter gene in zfAHR1-expressing cells. Ligand-binding experiments suggested that the differential zfAHR activities are attributable to differences in TCDD binding because only zfAHR2 exhibits high-affinity binding to [(3)H]TCDD or beta-naphthoflavone. Finally, using chimeric zfAHR1/zfAHR2 constructs, the lack of TCDD-mediated transcriptional activity was localized to the ligand-binding and C-terminal domains of zfAHR1.
The zebrafish (Danio rerio) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. The zebrafish genome will be completely sequenced within the next 1-2 years. Together with the substantial historical database regarding basic developmental biology, toxicology, and gene transfer, the rich foundation of molecular genetic and genomic data makes zebrafish a powerful model system for clarifying mechanisms in toxicity. In contrast to the highly advanced knowledge base on molecular developmental genetics in zebrafish, our database regarding infectious and noninfectious diseases and pathologic lesions in zebrafish lags far behind the information available on most other domestic mammalian and avian species, particularly rodents. Currently, minimal data are available regarding spontaneous neoplasm rates or spontaneous aging lesions in any of the commonly used wild-type or mutant lines of zebrafish. Therefore, to fully utilize the potential of zebrafish as an animal model for understanding human development, disease, and toxicology we must greatly advance our knowledge on zebrafish diseases and pathology.
Metallothionein-1 (MT-1) cDNA clones were isolated from a common carp (Cyprinus carpio) uninduced hepatopancreas cDNA library. Northern blot assay using the common carp (cc) MT-1 cDNA as a probe showed high fold induction of ccMT mRNA levels in the intestine and kidney following exposure to Cd2+ and Zn2+. Using polymerase chain reaction (PCR), primers designed from the cDNA sequences allowed the isolation of ccMT-1 gene fragments including the 5'-flanking region. The 600 bp 5'-flanking region of ccMT-1 gene carries four putative metal regulatory regions, one AP1, two SP1, one c-Jun site, and a TATA box. The 5'-flanking region of the ccMT-1 gene obtained was a functional promoter responding to the administration of various metal ions as well as hydrogen peroxide (H2O2) and lipopolysaccharide (LPS). When tested in primary cultures of cc hepatocytes, Zn2+ had the highest fold (20 times) induction of the 600 bp cloned ccMT-1 gene promoter, followed by Cu2+, Hg2+, Ni2+ and Pb2+ (4-5-fold inductions); H2O2 and LPS had a 6-7-fold induction. In conclusion, the ccMT-1 is a constitutively expressed MT and its gene promoter is inducible by various metal ions and chemical agents.
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The recently identified AHR repressor (AHRR) forms a negative feedback loop with the AHR. We investigated AHRR structure, function, evolution, and regulation in zebrafish, a powerful model in developmental biology and toxicology. We identified and cloned two distinct AHRR cDNAs that encode predicted proteins of 550 (AHRR1) and 573 (AHRR2) amino acids. The ahrr1 and ahrr2 genes map to zebrafish chromosomes 24 and 2, respectively, both of which share conserved synteny with human chromosome 5, the location of human AHRR. Mapping and phylogenetic analysis show that AHRR1 and AHRR2 are co-orthologs of the mammalian AHRR. In transient transfection assays, AHRR1 and AHRR2 repressed constitutive and TCDD-inducible transactivation by AHR2. Expression of both AHRR mRNAs was induced in ZF-L cells by AHR agonists but not by non-agonists. TCDD induced AHRR1 and AHRR2 expression in a dose-dependent manner in ZF-L cells, with EC50 values similar to those for induction of CYP1A. Both AHRRs were expressed and induced by TCDD in zebrafish embryos. Thus, zebrafish possess duplicate AHR-regulated AHRR paralogs that act in a negative feedback loop to repress the AHR signaling pathway.
A simple and efficient procedure was employed for the electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to sheets of pure, unmodified nitrocellulose. Immobilized proteins could then be radiographically visualized in situ by reaction with specific antibody and the subsequent binding of radioiodinated Staphylococcus protein A to the immune complexes. The detection of murine leukemia virus antigens in complex cellular lysates was used to demonstrate the efficacy of this technique.
Antibodies prepared against the major β-naphthoflavone (BNF)-inducible cytochrome P450 (P450) forms from three species of fish (rainbow trout, Atlantic cod, and scup) well separated in teleost phylogeny, were used to investigate the immunochemical relatedness of liver microsomal P450 in different species of BNF-treated fish and rat. Rabbit polyclonal IgG against all three P450s and mouse monoclonal antibodies prepared against scup P450E were employed in this study. Liver microsomes were prepared from BNF-treated specimens of hagfish, herring, rainbow trout, cod, scup, perch, plaice and rat. With Western blotting it was shown that the various antibodies cross-reacted with a protein band in liver microsomes in the P450-region of each of the BNF-treated fish species. The apparent molecular weight of the cross-reacting proteins showed differences within the range 54,000-59,000 daltons. The effects of the different antibodies on the microsomal BNF-inducible 7-ethoxyresorufin O-deethylase (EROD) activity gave inhibition patterns that reflected to a certain extent the phylogenetic relationship of the species investigated. In rat microsomes a protein band of relative molecular mass similar to rat P450c (Mr=54,000) was recognized by all antibodies. In addition, a second band of lower molecular mass was strongly recognized by anti-cod P450c antibodies, and faintly stained with anti-rainbow trout P450LM4b IgG and anti-scup P450E MAb 1-12-3. This band could correspond to rat P450d, the isosafrole-inducible rat isoenzyme. Considering the long separate evolutionary history of some of these fishes (50-200 million years), the results demonstrate that certain antigenic epitopes in the BNF-inducible P450 isoenzymes have been strongly conserved during the evolution of fish species. These conserved epitopes seem however not to be directly involved in the measured EROD activities. Furthermore, the results suggest that the BNF-inducible P450s in fish contain regions with structural similarity to the homologous counterpart that has evolved through gene duplication into a P450 family in mammals containing at least two gene products (the P450IA gene family).
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.
Messenger RNA and genomic DNA from Atlantic tomcod (Microgadus tomcod) from the cancer prone Hudson River population and two rivers in Maine were screened for size polymorphisms in cytochrome P-450IA. In northern blot analysis, we found that approximately 10% (16 of 176) of Hudson tomcod exhibited a polymorphism in P-450IA hybridizable mRNA not seen in any tomcod from Maine. All tomcod displayed a 3.0 kb P-450IA mRNA while variant individuals exhibited an additional 2.2 kb P-450IA mRNA band. Southern blot analysis on these same fish confirmed the genetic basis of this polymorphism. All individuals exhibited a single P-450IA hybridizable DNA fragment while tomcod with the variant genotype also displayed a second smaller (< 1 kb) DNA fragment. The results of multiple restriction enzyme digests on these DNA's suggest that this polymorphism represents a second smaller P-450IA allele in these fish. This is the first demonstration of multiple forms of the cytochrome P-450IA gene in any species of fish.
7,12-Dimethylbenz[a]anthracene and its 3,4-, 5,6-, 8,9- and 10,11-dihydrodiols have been tested for mutagenicity towards . TA100 in the presence of rat-liver post-mitochondrial supernatants from Aroclor-treated rats. At non-toxic concentrations, the non-K-region 3,4-dihydrodiol was six-fold more active than the parent hydrocarbon. At these concentrations, the 8,9-dihydrodiol showed some mutagenic activity, but the 5,6- and 10,11-dihydrodiols were inactive.
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.
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.
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.
Purification of cytochrome P450 from liver microsomes of untreated juvenile male rainbow trout yielded five fractions designated LMC1 to LMC5. All fractions, except LMC4 and LMC5, appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed minimum molecular weights of 50,000 (LMC1), 54,000 (LMC2), 56,000 (LMC3), 58,000 (LMC4), and 59,000 (LMC5). Specific contents ranged from 2.8 (LMC3) to 14.9 (LMC5) nmol heme/mg protein. The catalytic activity of LMC1, LMC2, and LMC5 toward various substrates was examined. LMC2 exhibited the highest estradiol 2-hydroxylase activity and progesterone 16 alpha-hydroxylase activity. LMC2 also was most active in the metabolic activation of aflatoxin B1 (AFB1). In contrast, LMC5 was most active in catalyzing the 6 beta- and 16 beta-hydroxylation of testosterone and the 6 beta-hydroxylation of progesterone. LMC1 showed the highest lauric acid hydroxylase activity. The three isozymes tested had low activity (for LMC2 and LMC5) or no activity (for LMC1) toward benzphetamine or benzo[a]pyrene. Polyclonal antibodies to all five isozymes were raised in rabbits and the antibodies were used to examine the contribution of the P450s to microsomal enzyme activities. The results of microsomal enzyme inhibition studies with polyclonal antibodies showed that anti-LMC2 IgG significantly inhibited the oxidative metabolism of testosterone, lauric acid, AFB1, and benzphetamine. Anti-LMC5 IgG inhibited the oxidation of progesterone, estradiol, benzo[a]pyrene, and benzphetamine. Anti-LMC1 IgG slightly inhibited the microsomal hydroxylation of lauric acid. Anti-LMC3 and anti-LMC4 IgG did not inhibit any of the measured microsomal enzyme activities. These findings suggest that individual constitutive isozymes of trout cytochrome P450 have well-defined contributions to the microsomal metabolism of steroids, fatty acids, and xenobiotics.
The metabolic activation of the carcinogens benzo[a]pyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA) was examined in cell lines derived from bluegill fry (BF-2), rainbow trout (RTG-2) and brown bullhead (BB). All three cell lines metabolized BP (0.5 microgram/ml medium) almost completely to water-soluble metabolites within 120 h, but the maximum amount of BP bound to DNA ranged from only 5 pmol/mg DNA in the BF-2 cells to 17 in the BB cells and 44 in the RTG-2 cells. The major BP-DNA adduct in the BB and BF-2 cells was that formed by reaction of (+)-anti-BP-7,8-diol-9,10 epoxide [(+)anti-BPDE] with deoxyguanosine. This adduct was also present in the RTG-2 cell DNA, but there were larger amounts of unidentified polar BP-DNA adducts. Exposure of the cells to [3H]BP-7,8-diol, a metabolic precursor of (+)anti-BPDE, resulted in binding of 1.5, 12 and 35 pmol BP per mg DNA in the BF-2, BB and RTG-2 cells, respectively. More than 90% of the BP-7,8-diol added to the BF-2 cultures was recovered as a glucuronic acid conjugate, but the RTG-2 cells formed more glutathione conjugates than glucuronide conjugates. The BB cells formed both types of conjugates at a slower rate for more than 75% of the 7,8-diol was recovered unchanged after 24 h. The three cell lines differed in the proportion of a 0.1 microgram/ml dose of DMBA metabolized in 48 h: the values ranged from 47% in the BF-2 cells to 78% in the BB cells and 97% in the RTG-2 cells. The amount of DMBA bound to DNA ranged from 4.7 to 8.6 pmol/mg DNA in the three cell lines: DMBA-3,4-diol-1,2-epoxide (DMBADE) adducts were present in the BB cell DNA, but no significant amounts of DMBADE-DNA adducts were detected in the RTG-2 or BF-2 cell DNA. These results demonstrate that fish cell cultures can activate BP to an ultimate carcinogenic metabolite, (+)anti-BPDE, but the level of binding of this metabolite to DNA is much lower than that which occurs in rodent embryo cell cultures. In BF-2 cell cultures formation of BP-7,8-diol-glucuronide effectively prevents the activation of this diol to (+)anti-BPDE. A substantial proportion of the BP-7,8-diol is also metabolized to glucuronide and glutathione conjugates in BB and RTG-2 cells. DMBA also binds to DNA at very low levels in these fish cell cultures. Thus effective conjugation of diols and their metabolites by fish cell lines appears to greatly reduce metabolic activation of hydrocarbons through the bay-region diol epoxide pathway that predominates in mammalian cell cultures.
Hepatocytes were isolated from adult male and female rats and maintained in monolayer culture for up to 24 hr. The degree of preservation of representative phase I and phase II xenobiotic biotransformation enzymes was studied in these cells immediately after isolation, after attachment in culture, and after 24 hr in culture. Regarding phase I pathways, hepatocytes during 24 hr lost 50% of cytochrome P-450, but maintained high mixed function oxidase activities; 75% of aryl hydrocarbon hydroxylase and 65% of benzphetamine demethylase activities were preserved in hepatocytes from males, whereas in hepatocytes from females 70 and 50% of these activities, respectively, were maintained. Of phase II pathways, glutathione transferase activity after 24 hr, tested toward 1,2-dichloro-4-nitrobenzene as substrate, was diminished in male hepatocytes to 20% of the initial liver activity and in female cells, to 35%, whereas the activity tested toward 1-chloro-2,4-dinitrobenzene as substrate was stable. UDP-glucuronosyltransferase activities, tested toward p-nitrophenol and phenolphthalein as substrates, were slightly increased during 24 hr of culture of hepatocytes to levels higher than in liver before perfusion. The level of UDP-glucuronic acid, the endogenous substrate for the enzyme, was reduced after isolation to only 6% of the initial liver value, and then increased during culture to a level approximately 60% of normal. Thus, the changes in xenobiotic biotransformation enzymes and associated constituents in cultured hepatocytes were not uniform, although biotransformation capability remained reasonably intact.
We have purified five isozymes of liver microsomal (LM) P-450 from beta-naphthoflavone-fed rainbow trout. Four forms (LM3, LM1, LM4a and LMx) were resolved on DEAE-Sepharose. Chromatography on hydroxylapatite further resolved LMx into two components, LM2 and LM4b. This latter form, obtained in highest yield (5%), had an apparent minimum molecular weight (Mr), as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), of 58,000, a specific content of 11.9 nmoles/mg, a lambda max in the carbon monoxide-ligated, reduced difference spectrum of 447.0 nm, and was active towards benzo[a]pyrene in a reconstituted system. A second form, LM4a, obtained in a final yield of 2%, had a specific content of 10.3 and was indistinguishable from Lm4b by Mr, lambda max, or activity towards benzo[a]pyrene. Form LM2 (2% yield) had a specific content of 10.8, a Mr of 54,000, a lambda max of 449.5 nm, and was not effective in reconstitution of benzo[a]pyrene-hydroxylase. In addition, two other forms with lower specific contents were obtained, LM1 and LM3. Neither LM1 nor LM3 was active towards benzo[a]pyrene. The properties of LM2, LM4a and LM4b were further examined with the aid of antibodies prepared from rabbits. Antibodies to LM4a and LM4b each cross-reacted with the other antigen and formed lines of identity on Ouchterlony plates, and both IgGs exhibited some cross-reaction to P-448 from rat. Neither antibody cross-reacted with trout LM2, and LM2-IgG did not cross-react with any other purified P-450. Benzo[a]pyrene-hydroxylase, catalyzed by either LM4a or LM4b, was inhibited by LM4b-IgG but not by LM4a-IgG, suggesting that these antibodies recognize different antigenic sites. Further comparison of LM4a and LM4b by amino acid composition, peptide mapping, kinetic properties, sensitivity to alpha-naphthoflavone, and regioselectivity towards benzo[a]pyrene-dihydrodiol formation indicates that these forms are highly similar in structure and function.