Adult Zebrafish Liver Research Articles

Histopathological changes and transcriptional alterations of three coagulation factors in zebrafish (Danio rerio) following short-term exposure to MC-LR

Microcystin-LR (MC-LR) is the most common hepatotoxic cyanotoxin produced primarily by Microcystis aeruginosa. Although deaths from microcystin toxication have widely been attributed to hypovolemic shock due to hepatic interstitial hemorrhage, so far, information on coagulation factors of MC-LR has been rare. In our present study, the effects of MC-LR on the coagulation factors expression and pathological changes in zebrafish were investigated. Adult zebrafish were injected intraperitoneally with 200μg/kg MC-LR and newly hatched larvae were placed in glass wares containing 200μg MC-LR/L, liver and whole larvae were collected after exposure for 12h, 24h, 48h and 96h, respectively. mRNA levels of coagulation factor IX (FIX), coagulation factor VII (FVII) and coagulation factor VIIi (FVIIi) in liver of adult fish and total zebrafish larvae were analyzed using quantitative real-time PCR. Significant depression of FIX and FVII were observed in adult zebrafish at different time point, and FVIIi were also significantly decreased except for 12h post-injection. In addition, the histopathological changes in adult zebrafish liver were observed, and the severe hepatic injuries were found at 96h. The pathological changes further explained the transcriptional results. The transcriptions of FVII were significantly depressed throughout the entire experiment in zebrafish larvae, and the FIX only significantly depressed after exposure for 48 and 96h, however, FVIIi just was significantly reduced after 12h and 24h stimulation. Whatever the mechanism, the effects of MC-LR on the transcription of these coagulation factors might explain the intrahepatic hemorrhage in liver after exposure to MC-LR, and which would provide new information to elucidate the hepatotoxicity of MC-LR.

Conserved gene regulation during acute inflammation between zebrafish and mammals

Zebrafish (Danio rerio), largely used as a model for studying developmental processes, has also emerged as a valuable system for modelling human inflammatory diseases. However, in a context where even mice have been questioned as a valid model for these analysis, a systematic study evaluating the reproducibility of human and mammalian inflammatory diseases in zebrafish is still lacking. In this report, we characterize the transcriptomic regulation to lipopolysaccharide in adult zebrafish kidney, liver, and muscle tissues using microarrays and demonstrate how the zebrafish genomic responses can effectively reproduce the mammalian inflammatory process induced by acute endotoxin stress. We provide evidence that immune signaling pathways and single gene expression is well conserved throughout evolution and that the zebrafish and mammal acute genomic responses after lipopolysaccharide stimulation are highly correlated despite the differential susceptibility between species to that compound. Therefore, we formally confirm that zebrafish inflammatory models are suited to study the basic mechanisms of inflammation in human inflammatory diseases, with great translational impact potential.

In Vitro Biotransformation of Two Human CYP3A Probe Substrates and Their Inhibition during Early Zebrafish Development.

At present, the zebrafish embryo is increasingly used as an alternative animal model to screen for developmental toxicity after exposure to xenobiotics. Since zebrafish embryos depend on their own drug-metabolizing capacity, knowledge of their intrinsic biotransformation is pivotal in order to correctly interpret the outcome of teratogenicity assays. Therefore, the aim of this in vitro study was to assess the activity of cytochrome P450 (CYP)—a group of drug-metabolizing enzymes—in microsomes from whole zebrafish embryos (ZEM) of 5, 24, 48, 72, 96 and 120 h post-fertilization (hpf) by means of a mammalian CYP substrate, i.e., benzyloxy-methyl-resorufin (BOMR). The same CYP activity assays were performed in adult zebrafish liver microsomes (ZLM) to serve as a reference for the embryos. In addition, activity assays with the human CYP3A4-specific Luciferin isopropyl acetal (Luciferin-IPA) as well as inhibition studies with ketoconazole and CYP3cide were carried out to identify CYP activity in ZLM. In the present study, biotransformation of BOMR was detected at 72 and 96 hpf; however, metabolite formation was low compared with ZLM. Furthermore, Luciferin-IPA was not metabolized by the zebrafish. In conclusion, the capacity of intrinsic biotransformation in zebrafish embryos appears to be lacking during a major part of organogenesis.

Regulation of vitellogenin (vtg1) and estrogen receptor (er) gene expression in zebrafish (Danio rerio) following the administration of Cd²⁺ and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

We evaluated the individual and joint estrogenic effects of cadmium (Cd(2+)) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the zebrafish liver (ZFL) cell line, zebrafish embryo, larvae and the liver of adult zebrafish. The mRNA expression of vtg1 was inhibited by Cd(2+), but unaffected by TCDD in ZFL cells. Similar changes in the mRNA levels of ERα, ERβ1, ERβ2 and GPER (G protein coupled estrogen receptor) in ZFL cells were also observed. Deletion mutants of vtg1 gene promoters were constructed to investigate transcriptional regulation, and we found that all of the constructs failed to respond to TCDD or Cd(2+). However, after co-transfection with a vtg1 promoter-luciferase construct to the ERα, ERβ1, ERβ2 and GPER expression vectors, decreased luciferase activity was observed in the ERα co-transfection group after treatment with Cd(2+), suggesting that ERα participates in vtg1 transcriptional regulation and is affected by Cd(2+). Differences in the regulation of the mRNA levels of these genes were also observed between different developmental stages and between the livers of male and female zebrafish.

Glucocorticoid activity detected by in vivo zebrafish assay and in vitro glucocorticoid receptor bioassay at environmental relevant concentrations.

Glucocorticoids are pharmaceutical contaminants of emerging concern due to their incomplete removal during wastewater treatment, increased presence in aquatic environment and their biological potency. The zebrafish is a popular model for aquatic toxicology and environmental risk assessment. This study aimed to determine if glucocorticoids at environmental concentrations would perturb expression of selected glucocorticoid-responsive genes in zebrafish and to investigate their potentials as an in vivo zebrafish assay in complementing in vitro glucocorticoid receptor bioassay. The relative expression of eleven glucocorticoid-responsive genes in zebrafish larvae and liver of adult male zebrafish exposed to three representative glucocorticoids (dexamethasone, prednisolone and triamcinolone) was determined. The expression of pepck, baiap2 and pxr was up-regulated in zebrafish larvae and the expression of baiap2, pxr and mmp-2 was up-regulated in adult zebrafish exposed to glucocorticoids at concentrations equivalent to total glucocorticoids reported in environmental samples. The responsiveness of the specific genes were sufficiently robust in zebrafish larvae exposed to a complex environmental sample detected with in vitro glucocorticoid activity equivalent to 478 pM dexamethasone (DEX-EQ) and confirmed to contain low concentration (0.2 ng/L or less) of the targeted glucocorticoids, and possibly other glucocorticoid-active compounds. The findings provided in vivo relevance to the in vitro glucocorticoid activity and suggested that the environmental sample can perturb glucocorticoid-responsive genes in its original, or half the diluted, concentration as may be found in the environment. The study demonstrated the important complementary roles of in vivo zebrafish and in vitro bioassays coupled with analytical chemistry in monitoring environmental glucocorticoid contaminants.

Is CYP3A activity in the liver of adult zebrafish influenced by tricaine methanesulfonate (MS-222)?

Is CYP3A activity in the liver of adult zebrafish influenced by tricaine methanesulfonate (MS-222)?

Anti-oxidative responses of zebrafish (Danio rerio) gill, liver and brain tissues upon acute cold shock

The present study seeks to detect oxidative damage and to compare anti-oxidative responses among liver, gills and brain of adult zebrafish that were cooled from 28°C (control) to 12°C (treatment) for 0–24h. The lipid peroxidation of liver, gill and brain tissues significantly increased at 1h after transfer, but reactive oxygen species in the treatment group increased significantly after 24h as compared to the control. The fish were found to develop a cascading anti-oxidative mechanism beginning with an increase in Cu/Zn-SOD levels, followed by increased CAT and GPx mRNA expressions in the three tissue types. Both smtB and mt2 mRNAs increased in the hepatic and brain tissues following 1h of cold stress, but only smtB exhibited a significant increase in the gills at 1h and 6h after transfer to 12°C. Furthermore, cellular apoptosis in the brain was not evident after cold shock, but liver and gills showed cellular apoptosis at 1–3h, with another peak in the liver at 6h after cold shock. The results suggest that the cold shock induced oxidative stress, and the enzymatic (SOD, GPx and CAT) and non-enzymatic (mt-2 and smt-B) mRNA expressions all play a role in the resulting anti-oxidation within 1–6h of cold shock. A functional comparison showed that the brain had the most powerful antioxidant defense system of the three tissue types since it had the highest smtB mRNA expression and a lower level of cell apoptosis than the liver and gills after exposure to cold stress.

Characterization of glutathione-S-transferases in zebrafish (Danio rerio)

Glutathione-S-transferases (GSTs) are one of the key enzymes that mediate phase II of cellular detoxification. The aim of our study was a comprehensive characterization of GSTs in zebrafish (Danio rerio) as an important vertebrate model species frequently used in environmental research. A detailed phylogenetic analysis of GST superfamily revealed 27 zebrafish gst genes. Further insights into the orthology relationships between human and zebrafish GSTs/Gsts were obtained by the conserved synteny analysis. Expression of gst genes in six tissues (liver, kidney, gills, intestine, brain and gonads) of adult male and female zebrafish was determined using qRT-PCR. Functional characterization was performed on 9 cytosolic Gst enzymes after overexpression in E. coli and subsequent protein purification. Enzyme kinetics was measured for GSH and a series of model substrates. Our data revealed ubiquitously high expression of gstp, gstm (except in liver), gstr1, mgst3a and mgst3b, high expression of gsto2 in gills and ovaries, gsta in intestine and testes, gstt1a in liver, and gstz1 in liver, kidney and brain. All zebrafish Gsts catalyzed the conjugation of GSH to model GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and monochlorobimane (MCB), apart from Gsto2 and Gstz1 that catalyzed GSH conjugation to dehydroascorbate (DHA) and dichloroacetic acid (DCA), respectively. Affinity toward CDNB varied from 0.28mM (Gstp2) to 3.69mM (Gstm3), while affinity toward MCB was in the range of 5μM (Gstt1a) to 250μM (Gstp1). Affinity toward GSH varied from 0.27mM (Gstz1) to 4.45mM (Gstt1a). Turnover number for CDNB varied from 5.25s−1 (Gstt1a) to 112s−1 (Gstp2). Only Gst Pi enzymes utilized ethacrynic acid (ETA). We suggest that Gstp1, Gstp2, Gstt1a, Gstz1, Gstr1, Mgst3a and Mgst3b have important role in the biotransformation of xenobiotics, while Gst Alpha, Mu, Pi, Zeta and Rho classes are involved in the crucial physiological processes. In summary, this study provides the first comprehensive analysis of GST superfamily in zebrafish, presents new insight into distinct functions of individual Gsts, and offers methodological protocols that can be used for further verification of interaction of environmental contaminants with fish Gsts.

Haploinsufficiency of Def activates p53-dependent TGFβ signalling and causes scar formation after partial hepatectomy.

The metazoan liver exhibits a remarkable capacity to regenerate lost liver mass without leaving a scar following partial hepatectomy (PH). Whilst previous studies have identified components of several different signaling pathways that are essential for activation of hepatocyte proliferation during liver regeneration, the mechanisms that enable such regeneration to occur without accompanying scar formation remain poorly understood. Here we use the adult zebrafish liver, which can regenerate within two weeks following PH, as a new genetic model to address this important question. We focus on the role of Digestive-organ-expansion-factor (Def), a nucleolar protein which has recently been shown to complex with calpain3 (Capn3) to mediate p53 degradation specifically in the nucleolus, in liver regeneration. Firstly, we show that Def expression is up-regulated in the wild-type liver following amputation, and that the defhi429/+ heteroozygous mutant (def+/−) suffers from haploinsufficiency of Def in the liver. We then show that the expression of pro-inflammatory cytokines is up-regulated in the def+/− liver, which leads to distortion of the migration and the clearance of leukocytes after PH. Transforming growth factor β (TGFβ) signalling is thus activated in the wound epidermis in def+/− due to a prolonged inflammatory response, which leads to fibrosis at the amputation site. Fibrotic scar formation in def+/− is blocked by the over-expression of Def, by the loss-of-function of p53, and by treatment with anti-inflammation drug dexamethasone or TGFβ-signalling inhibitor SB431542. We finally show that the Def- p53 pathway suppresses fibrotic scar formation, at least in part, through the regulation of the expression of the pro-inflammatory factor, high-mobility group box 1. We conclude that the novel Def- p53 nucleolar pathway functions specifically to prevent a scar formation at the amputation site in a normal amputated liver.

Short-term treatment of adult male zebrafish (Danio Rerio) with 17α-ethinyl estradiol affects the transcription of genes involved in development and male sex differentiation

The synthetic estrogen 17α-ethinyl estradiol (EE2) disturbs reproduction and causes gonadal malformation in fish. Effects on the transcription of genes involved in gonad development and function that could serve as sensitive biomarkers of reproductive effects in the field is, however, not well known. We have studied mRNA expression in testes and liver of adult zebrafish (Danio rerio) males treated with 0, 5 or 25ng/L EE2for 14days. qPCR analysis showed that the mRNA expression of four genes linked to zebrafish male sex determination and differentiation, Anti-Mullerian Hormone, Double sex and mab-related protein, Sry-related HMG box-9a and Nuclear receptor subfamily 5 group number 1b were significantly decreased by 25ng/L, but not 5ng/L EE2 compared with the levels in untreated fish. The decreased transcription was correlated with a previously shown spawning failure in these males (Reyhanian et al., 2011. Aquat Toxicol 105, 41–48), suggesting that decreased mRNA expression of genes regulating male sexual function could be involved in the functional sterility. The mRNA level of Cytochrome P-45019a, involved in female reproductive development, was unaffected by hormone treatment. The transcription of the female-specific Vitellogenin was significantly induced in testes. While testicular Androgen Receptor and the Estrogen Receptor-alpha mRNA levels were unchanged, Estrogen receptor-beta was significantly decreased by 25ng/L EE2. Hepatic Estrogen Receptor-alpha mRNA was significantly increased by both exposure concentrations, while Estrogen Receptor-beta transcription was unaltered. The decreased transcription of male-predominant genes supports a demasculinization of testes by EE2 and might reflect reproductive disturbances in the environment.

Tissue-, sex- and development-specific transcription profiles of eight UDP-glucuronosyltransferase genes in zebrafish (Danio rerio) and their regulation by activator of aryl hydrocarbon receptor

UDP-Glucuronosyltransferases (Ugts) are phase II biotransformation enzymes that glucuronidate numerous endogenous and xenobiotic substrates. Based on the reported zebrafish Ugt gene repertoire, primers for the Ugt1a and Ugt1b family and for individual Ugt5a1, Ugt5a3, Ugt5a4, Ugt5a5, Ugt5c2 and Ugt5c3 were designed and applied in RT-qPCR analyses. Transcriptional expression profiles of these Ugts were analyzed in intestine, liver, gonad and brain of female and male adult zebrafish and at different embryonic developmental stages. We found tissue-, sex- and developmental-specific expression patterns for all isoforms. Throughout all tissues, the most abundant Ugts were Ugt1a, Ugt1b, Ugt5a1 and Ugt5a3. Expression during embryonic development was assessed between 24 and 120 hpf. Ugts showed a development-dependent expression. The pattern of Ugt1a, Ugt1b, Ugt5a1, Ugt5a3 and Ugt5a4 were similar with highest expression at 24 hpf followed by a decrease and rebound increase up to 120 hpf. To analyze for transcriptional regulation of Ugts by the arylhydrocarbon receptor (ahr2), zebrafish eleuthero-embryos were exposed to 5, 25 and 50μg/L benzo(a)pyrene (BaP), a model ahr2 regulator for cyp1a. Besides transcriptional induction of ahr2 and cyp1a, BaP produced a significant induction of Ugt1a, Ugt5a1, Ugt5a3 and Ugt5a5 as well as a down-regulation of Ugt1b. These data demonstrate the link between ahr2 signalling and transcriptional expression of Ugt genes. This is the first study showing transcriptional expression of eight different Ugts in tissues and during embryonic development and offers new perspectives on the involvement of Ugts in fish xenobiotic metabolism.

Arsenic exposure alters expression of cell cycle and lipid metabolism genes in the liver of adult zebrafish (Danio rerio)

Adult zebrafish (Danio rerio) were used to investigate mRNA expression in the liver following 7-day and 21-day exposures to 0, 10, 50, or 500ppb sodium arsenite. Arsenic exposure has been linked to several human disorders including cancers and cardiovascular and metabolic diseases. Quantitative PCR was employed to determine the mRNA expression of genes involved in cell cycle regulation [cyclin E1 (ccne1), WEE1 A kinase (wee1)], DNA damage repair [breast cancer 2 (brca2)] and lipid transport and metabolism [carnitine O-octanoyltransferase (crot), fatty acid binding protein-3 (fabp3) and 3-hydroxy-3-methylglutaryl-CoA synthase 1 (hmgcs1)]. Results from the 7-day exposure showed sex- and dose-specific changes in expression of wee1, brca2, crot and hmgcs1. No significant differences from controls were observed in fish exposed for 21 days. Expression of all genes, except ccne1, was significantly different between the 7- and 21-day exposures. The results presented here correlate with prior findings from our lab and others, and offer further insight into potential mechanisms of low-dose arsenic exposure.

Effects of chronic dietary selenomethionine exposure on repeat swimming performance, aerobic metabolism and methionine catabolism in adult zebrafish (Danio rerio)

In a previous study we reported impaired swimming performance and greater stored energy in adult zebrafish (Danio rerio) after chronic dietary exposure to selenomethionine (SeMet). The goal of the present study was to further investigate effects of chronic exposure to dietary SeMet on repeat swimming performance, oxygen consumption (MO2), metabolic capacities (standard metabolic rate [SMR], active metabolic rate [AMR], factorial aerobic scope [F-AS] and cost of transport [COT]) and gene expression of energy metabolism and methionine catabolism enzymes in adult zebrafish. Fish were fed SeMet at measured concentrations of 1.3, 3.4, 9.8 or 27.5μgSe/g dry mass (d.m.) for 90d. At the end of the exposure period, fish from each treatment group were divided into three subgroups: (a) no swim, (b) swim, and (c) repeat swim. Fish from the no swim group were euthanized immediately at 90d and whole body triglycerides, glycogen and lactate, and gene expression of energy metabolism and methionine catabolism enzymes were determined. Individual fish from the swim group were placed in a swim tunnel respirometer and swimming performance was assessed by determining the critical swimming speed (Ucrit). After both Ucrit and MO2 analyses, fish were euthanized and whole body energy stores and lactate were determined. Similarly, individual fish from the repeat swim group were subjected to two Ucrit tests (Ucrit-1 and Ucrit-2) performed with a 60min recovery period between tests, followed by determination of energy stores and lactate. Impaired swim performance was observed in fish fed SeMet at concentrations greater than 3μg Se/g in the diet. However, within each dietary Se treatment group, no significant differences between single and repeat Ucrits were observed. Oxygen consumption, SMR and COT were significantly greater, and F-AS was significantly lesser, in fish fed SeMet. Whole body triglycerides were proportional to the concentration of SeMet in the diet. While swimming resulted in lesser concentrations of glycogen in the body, exposure to SeMet in the diet had no significant effect on glycogen content. Exposure to SeMet significantly down-regulated mRNA abundance of protein tyrosine phosphatase 1B (PTP 1B) in muscle, and β-hydroxyacyl coenzyme A dehydrogenase (HOAD), sterol regulatory element binding protein 1 (SREBP 1) and methionine adenosyltransferase 1 alpha (MAT 1A) in liver of adult zebrafish. Overall the results of this study suggest chronic exposure of adult zebrafish to SeMet in the diet can cause both cellular and organismal effects that could affect fitness and survivability of fish.

Quercetin 3-O-methyl ether protects FL83B cells from copper induced oxidative stress through the PI3K/Akt and MAPK/Erk pathway

Quercetin is a bioflavonoid that exhibits several biological functions in vitro and in vivo. Quercetin 3-O-methyl ether (Q3) is a natural product reported to have pharmaceutical activities, including antioxidative and anticancer activities. However, little is known about the mechanism by which it protects cells from oxidative stress. This study was designed to investigate the mechanisms by which Q3 protects against Cu2+-induced cytotoxicity. Exposure to Cu2+ resulted in the death of mouse liver FL83B cells, characterized by apparent apoptotic features, including DNA fragmentation and increased nuclear condensation. Q3 markedly suppressed Cu2+-induced apoptosis and mitochondrial dysfunction, characterized by reduced mitochondrial membrane potential, caspase-3 activation, and PARP cleavage, in Cu2+-exposed cells. The involvement of PI3K, Akt, Erk, FOXO3A, and Mn-superoxide dismutase (MnSOD) was shown to be critical to the survival of Q3-treated FL83B cells. The liver of both larval and adult zebrafish showed severe damage after exposure to Cu2+ at a concentration of 5μM. Hepatic damage induced by Cu2+ was reduced by cotreatment with Q3. Survival of Cu2+-exposed larval zebrafish was significantly increased by cotreatment with 15μM Q3. Our results indicated that Cu2+-induced apoptosis in FL83B cells occurred via the generation of ROS, upregulation and phosphorylation of Erk, overexpression of 14-3-3, inactivation of Akt, and the downregulation of FOXO3A and MnSOD. Hence, these results also demonstrated that Q3 plays a protective role against oxidative damage in zebrafish liver and remarked the potential of Q3 to be used as an antioxidant for hepatocytes.

Comprehensive profiling of zebrafish hepatic proximal promoter CpG island methylation and its modification during chemical carcinogenesis

BackgroundDNA methylation is an epigenetic mechanism associated with regulation of gene expression and it is modulated during chemical carcinogenesis. The zebrafish is increasingly employed as a human disease model; however there is a lack of information on DNA methylation in zebrafish and during fish tumorigenesis.ResultsA novel CpG island tiling array containing 44,000 probes, in combination with immunoprecipitation of methylated DNA, was used to achieve the first comprehensive methylation profiling of normal adult zebrafish liver. DNA methylation alterations were detected in zebrafish liver tumors induced by the environmental carcinogen 7, 12-dimethylbenz(a)anthracene. Genes significantly hypomethylated in tumors were associated particularly with proliferation, glycolysis, transcription, cell cycle, apoptosis, growth and metastasis. Hypermethylated genes included those associated with anti-angiogenesis and cellular adhesion. Of 49 genes that were altered in expression within tumors, and which also had appropriate CpG islands and were co-represented on the tiling array, approximately 45% showed significant changes in both gene expression and methylation.ConclusionThe functional pathways containing differentially methylated genes in zebrafish hepatocellular carcinoma have also been reported to be aberrantly methylated during tumorigenesis in humans. These findings increase the confidence in the use of zebrafish as a model for human cancer in addition to providing the first comprehensive mapping of DNA methylation in the normal adult zebrafish liver.

Proteomics Profiling of Single Organs from Individual Adult Zebrafish

The model organism zebrafish (Danio rerio) is extensively utilized in studies of developmental biology but is also being investigated in the context of a growing list of human age-related diseases. To facilitate such studies, we here present protein expression patterns of adult zebrafish organs, including blood, brain, fin, heart, intestine, liver, and skeletal muscle. Protein extracts were prepared from the different organs of two zebrafish and analyzed using liquid chromatography coupled to high-resolution tandem mass spectrometry. Zebrafish tissue was digested directly after minimal fractionation and cleaned up (the shotgun approach). Proteins were identified using Mascot software. In total, 1394 proteins were identified of which 644 were nonredundant. Of these, 373 demonstrated an organ-specific expression pattern and 57 had not been shown on protein level before. These data emphasize the need for increased research at the protein level to facilitate the selection of candidate proteins for targeted quantification and to refine systematic genetic network analysis in vertebrate development, biology, and disease.

DNA methylation levels in the 5′ flanking region of the vitellogenin I gene in liver and brain of adult zebrafish (Danio rerio)—Sex and tissue differences and effects of 17α-ethinylestradiol exposure

Vitellogenin is produced in the liver of sexually mature female fish in response to endogenous estrogens. Exogenous estrogens also induce synthesis of vitellogenin in the liver of male and juvenile fish and vitellogenin is a frequently used biomarker for estrogen exposure. The epigenetic state, e.g. histone acetylation and DNA methylation, in the region of a gene or in its 5′ flanking region influences the gene expression. DNA methylation positions in multicellular eukaryotes are mostly found on cytosine bases located 5′ to guanine, i.e. in CpG sites. Here, we have for the first time analyzed the DNA methylation levels of three CpG sites located in the 5′ flanking region of the vitellogenin I gene in liver and brain from adult zebrafish (Danio rerio) utilizing Pyrosequencing technology. This sequencing technique allows determination of methylation levels of multiple individual CpG sites. Our purpose was to assess any differences in methylation levels related to sex, tissue and exposure to estrogen. Out of the seven vitellogenin genes identified in the zebrafish, vitellogenin I is the most highly expressed during vitellogenesis. We found that the methylation levels of all three CpG sites were higher in male liver than in female liver. In brain, which does not express vitellogenin, females and males showed similar, high methylation levels in the analyzed CpG positions. Exposure of adult zebrafish to 17α-ethinylestradiol (100ng/L) for 14 days decreased the methylation levels in the 5′ flanking region of vitellogenin I in the liver in both females and males. These results suggest that induced expression of vitellogenin in fish following exposure to estrogens might involve alterations in DNA methylation.

Induction of oxidative stress and apoptosis by silver nanoparticles in the liver of adult zebrafish

Silver nanoparticles (AgNPs) may induce deleterious effects in aquatic life on environmental release. The hepatotoxicity of AgNPs was assessed in the liver of adult zebrafish, with the aim of studying the roles of oxidative damage and apoptosis. Zebrafish were exposed to an AgNP solution in which free Ag + ions were absent at the time of treatment. However, the metal-sensitive metallothionein 2 (MT2) mRNA was induced in the liver tissues of AgNP-treated zebrafish, suggesting that Ag + ions were released from AgNPs after treatment. It is also possible that MT2 mRNA was induced in the liver tissues by AgNP-generated free radicals. A number of cellular alterations including disruption of hepatic cell cords and apoptotic changes were observed in histological analysis of the liver tissues. The levels of malondialdehyde, a byproduct of cellular lipid peroxidation, and total glutathione were increased in the tissues after treatment with AgNPs. The mRNA levels of the oxyradical-scavenging enzymes catalase and glutathione peroxidase 1a were reduced in the tissues. AgNP treatment induced DNA damage, as demonstrated by analysis with the double-strand break marker γ-H2AX and the expression of p53 protein in liver tissues. In addition, the p53-related pro-apoptotic genes Bax, Noxa, and p21 were upregulated after treatment with AgNPs. These data suggest that oxidative stress and apoptosis are associated with AgNP toxicity in the liver of adult zebrafish.

NTPDase family in zebrafish: Nucleotide hydrolysis, molecular identification and gene expression profiles in brain, liver and heart

The nucleoside triphosphate diphosphohydrolase (NTPDase) family cleaves tri- and diphosphonucleosides to monophosphonucleosides and is responsible for terminating purinergic transmission. Since the NTPDase family in zebrafish is poorly understood, here we evaluated the nucleotide hydrolysis in three tissues of adult zebrafish (brain, liver, and heart), confirmed the presence of distinct NTPDase members by a phylogenetic analysis and verified their relative gene expression profiles in the respective tissues. A different profile of ATP and ADP hydrolysis in the brain, liver, and heart as a function of time and protein concentration was observed. Sodium azide (20mM), ARL 67156 (300 microM) and Suramin (300 microM) differently altered the nucleotide hydrolysis in zebrafish tissues, suggesting the contribution of distinct NTPDase activities. Homology-based searches identified the presence of NTPDase1-6 and NTPDase8 orthologs and the phylogeny also grouped three NTPDase2 and two NTPDase5 paralogs. The deduced amino acid sequences share the apyrase conserved regions, conserved cysteine residues, putative N-glycosylation, phosphorylation, N-acetylation sites, and different numbers of transmembrane domains. RT-PCR experiments revealed the existence of a distinct relative entpd1-6 and entpd8 expression profile in brain, liver, and heart. Taken together, these results indicate that several NTPDase members might contribute to a tight regulation of nucleotide hydrolysis in zebrafish tissues.

Induction of estrogen-responsive gene transcription in the embryo, larval, juvenile and adult life stages of zebrafish as biomarkers of short-term exposure to endocrine disrupting chemicals

Real-time quantitative RT-PCR was adopted to investigate the transcription of the estrogen-responsive genes in different developmental stages of zebrafish exposed to endocrine disrupting chemicals (EDCs). The lowest observed effect concentrations (LOECs) of 17β-estradiol for inducing vtg1 transcription were 0.25, 0.5, 0.25 and ≤ 0.1 μg/L for embryo, larvae, juvenile and adult male zebrafish, respectively, while the LOECs of nonylphenol for induction of vtgs transcription were 50 μg/L in embryo and 100 μg/L in larvae and adult stages. The mRNA levels of the two vtgs were low in both the embryo and larvae stages, even at the highest 17β-estradiol or nonylphenol exposure concentrations, while the mRNA levels in liver of adult zebrafish of the two vtgs were 10 2 or even 10 4 times higher than those of the control groups at the corresponding nonylphenol or 17β-estradiol exposure concentrations. Similarity, the ERα and ERβ mRNA levels in juvenile and adult zebrafish livers were also higher. Results suggest that in the early developmental stages of zebrafish might be more sensitive (low LOECs) to the presence of EDCs such as nonylphenol, but juvenile and adult zebrafish have a more effective (high induction levels). The use of zebrafish juveniles, larvae and embryos offers an alternative stage to detect EDCs.