Hexabromocyclododecane-induced developmental toxicity and apoptosis in zebrafish embryos

Hexabromocyclododecane (HBCD) is widely used as a brominated flame retardant, and has been detected in the aquatic environment, wild animals, and humans. However, details of the environmental health risk of HBCD are not well known. In this study, zebrafish embryos were used to assess the developmental toxicity of the chemical. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to various concentrations of HBCD (0, 0.05, 0.1, 0.5, and 1.0 mg L-1) until 96 h. Exposure to 0.1, 0.5, and 1.0 mg L-1 HBCD significantly increased the malformation rate and reduced survival in the 0.5 and 1.0 mg L-1 HBCD exposure groups. Acridine orange (AO) staining showed that HBCD exposure resulted in cell apoptosis. Reactive oxygen species (ROS) was significantly induced at exposures of 0.1, 0.5, and 1.0 mg L-1 HBCD. To test the apoptotic pathway, several genes related to cell apoptosis, such as p53, Puma, Apaf-1, caspase-9, and caspase-3, were examined using real-time PCR. The expression patterns of these genes were up-regulated to some extent. Two anti-apoptotic genes, Mdm2 (antagonist of p53) and Bcl-2 (inhibitor of Bax), were down-regulated, and the activity of capspase-9 and caspase-3 was significantly increased. The overall results demonstrate that waterborne HBCD is able to produce oxidative stress and induce apoptosis through the involvement of caspases in zebrafish embryos. The results also indicate that zebrafish embryos can serve as a reliable model for the developmental toxicity of HBCD. (C) 2009 Elsevier B.V. All rights reserved.

Developmental toxicity and alteration of gene expression in zebrafish embryos exposed to PFOS

Perfluorooetanesulfonate (PFOS) is a persistent organic pollutant, the potential toxicity of which is causing great concern. In the present study, we employed zebrafish embryos to investigate the developmental toxicity of this compound. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to 0.1, 0.5, 1, 3 and 5 mg/L PFOS. Hatching was delayed and hatching rates as well as larval survivorship, were significantly reduced after the embryos were exposed to 1, 3 and 5 mg/L PFOS until 132 hpf. The fry displayed gross developmental malformations, including epiboly deformities, hypopigmentation, yolk sac edema, tail and heart malformations and spinal curvature upon exposure to PFOS concentrations of I mg/L or greater. Growth (body length) was significantly reduced in the 3 and 5 mg/L PFOS-treated groups. To test whether developmental malformation was mediated via apoptosis, flow cytometry analysis of DNA content, acridine orange staining and TUNEL assay was used. These techniques indicated that more apoptotic cells were present in the PFOS-treated embryos than in the control embryos. Certain genes related to cell apoptosis, p53 and Bax, were both significantly up-regulated upon exposure to all the concentrations tested. In addition, we investigated the effects of PFOS on marker genes related to early thyroid development (hhex and pax8) and genes regulating the balance of androgens and estrogens (cyp19a and cyp19b). For thyroid development, the expression of hhex was significantly up-regulated at all concentrations tested, whereas pax8 expression was significantly up-regulated only upon exposure to lower concentrations of PFOS (0.1, 0.5, 1 mg/L). The expression of cyp19a and of cyp19b was significantly down-regulated at all exposure concentrations. The overall results indicated that zebrafish embryos constitute a reliable model for testing the developmental toxicity of PFOS, and the gene expression patterns in the embryos were able to reveal some potential mechanisms of developmental toxicity. (C) 2008 Elsevier Inc. All rights reserved.

Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase

Double-stranded RNA (dsRNA) has been shown to be a useful tool for silencing genes in zebrafish (Danio rerio), while the blocking specificity of dsRNA is still of major concern for application. It was reported that siRNA (small interfering RNA) prepared by endoribonuclease digestion (esiRNA) could efficiently silence endogenous gene expression in mammalian embryos. To test whether esiRNA could work in zebrafish, we utilized Escherichia coli RNaseIII to digest dsRNA of zebrafish no tail (ntl), a mesoderm determinant in zebrafish and found that esi-ntl could lead to developmental defects, however, the effective dose was so close to the toxic dose that esi-ntl often led to non-specific developmental defects. Consequently, we utilized SP6 RNA polymerase to produce si-ntl, siRNA designed against ntl, by in vitro transcription. By injecting in vitro synthesized si-ntl into zebrafish zygotes, we obtained specific phenocopies of reported mutants of ntl. We achieved up to a 59%no tail phenotype when the injection concentration was as high as 4 mu g/mu L. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization analysis showed that si-ntl could largely and specifically reduce mRNA levels of the ntl gene. As a result, our data indicate that esiRNA is unable to cause specific developmental defects in zebrafish, while siRNA should be an alternative for downregulation of specific gene expression in zebrafish in cases where RNAi techniques are applied to zebrafish reverse genetics.

Knockdown and overexpression of Unc-45b result in defective myofibril organization in skeletal muscles of zebrafish embryos

We thank John Stubblefield for editing, Junling Li for the assistance in the Western blot analysis. This research was supported by a training grant from National Institutes of Health (#T32 AR07592) and a research grant MB-8713-08 from United States - Israel Binational Agriculture Research and Development Fund.

Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos

Thymidylate synthase (TS), which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU) and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 mu M 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 mu M ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5'-UTR of TS mRNA, which corresponded to nt 13-32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.

Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos

Thymidylate synthase (TS), which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU) and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 mu M 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 mu M ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5'-UTR of TS mRNA, which corresponded to nt 13-32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.

Effects of environmental factors on the toxicity of pesticides to zebrafish embryos

During the last century mean global temperatures have been increasing. According to the predictions, the temperature change is expected to exceed 1.5ºC in this century and the warming is likely to continue. Freshwater ecosystems are among the most sensitive mainly due to changes in the hydrologic cycle and consequently changes in several physico-chemical parameters (e.g. pH, dissolved oxygen). Alterations in environmental parameters of freshwater systems are likely to affect distribution, morphology, physiology and richness of a wide range of species leading to important changes in ecosystem biodiversity and function. Moreover, they can also work as co-stressors in environments where organisms have already to cope with chemical contamination (such as pesticides), increasing the environmental risk due to potential interactions. Therefore, the objective of this work was to evaluate the effects of climate change related environmental parameters on the toxicity of pesticides to zebrafish embryos. The following environmental factors were studied: pH (3.0-12.0), dissolved oxygen level (0-8 mg/L) and UV radiation (0-500 mW/m2). The pesticides studied were the carbamate insecticide carbaryl and the benzimidazole fungicide carbendazim. Stressors were firstly tested separately in order to derive concentration- or intensity-response curves to further study the effects of binary combinations (environmental factors x pesticides) by applying mixture models. Characterization of zebrafish embryos response to environmental stress revealed that pH effects were fully established after 24 h of exposure and survival was only affected at pH values below 5 and above 10. Low oxygen levels also affected embryos development at concentrations below 4 mg/L (delay, heart rate decrease and edema), and at concentrations below 0.5 mg/L the survival was drastically reduced. Continuous exposure to UV radiation showed a strong time-dependent impact on embryos survival leading to 100% of mortality after 72 hours of exposure. The toxicity of pesticides carbaryl and carbendazim was characterized at several levels of biological organization including developmental, biochemical and behavioural allowing a mechanistic understanding of the effects and highlighting the usefulness of behavioural responses (locomotion) as a sensitive endpoint in ecotoxicology. Once the individual concentration response relationship of each stressor was established, a combined toxicity study was conducted to evaluate the effects of pH on the toxicity of carbaryl. We have shown that pH can modify the toxicity of the pesticide carbaryl. The conceptual model concentration addition allowed a precise prediction of the toxicity of the jointeffects of acid pH and carbaryl. Nevertheless, for alkaline condition both concepts failed in predicting the effects. Deviations to the model were however easy to explain as high pH values favour the hydrolysis of carbaryl with the consequent formation of the more toxic degradation product 1- naphtol. Although in the present study such explanatory process was easy to establish, for many other combinations the “interactive” nature is not so evident. In the context of the climate change few scenarios predict such increase in the pH of aquatic systems, however this was a first approach focused in the lethal effects only. In a second tier assessment effects at sublethal level would be sought and it is expectable that more subtle pH changes (more realistic in terms of climate changes scenarios) may have an effect at physiological and biochemical levels with possible long term consequences for the population fitness.

Metabolic profile analysis of zebrafish embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Lipid abundance in zebrafish embryos is regulated by complementary actions of the endocannabinoid system and retinoic acid pathway

The endocannabinoid system (ECS) and retinoic acid (RA) signaling have been associated with influencing lipid metabolism. We hypothesized that modulation of these pathways could modify lipid abundance in developing vertebrates and that these pathways could have a combinatorial effect on lipid levels. Zebrafish embryos were exposed to chemical treatments altering the activity of the ECS and RA pathway. Embryos were stained with the neutral lipid dye Oil-Red-O (ORO) and underwent whole-mount in situ hybridization. Mouse 3T3-L1 fibroblasts were differentiated under exposure to RA modulating chemicals and subsequently stained with ORO and analyzed for gene expression by qRT-PCR. ECS activation and RA exposure increased lipid abundance and the expression of lipoprotein lipase. Additionally, RA treatment increased expression of CCAAT/enhancer binding protein alpha. Both ECS receptors and RA receptor subtypes were separately involved in modulating lipid abundance. Finally, increased ECS or RA activity ameliorated the reduced lipid abundance caused by peroxisome proliferator-activated receptor gamma (PPARγ) inhibition. Therefore, the ECS and RA pathway influence lipid abundance in zebrafish embryos and have an additive effect when treated simultaneously. Furthermore, we demonstrated that these pathways act downstream or independently of PPARγ to influence lipid levels. Our study shows for the first time that the RA and ECS pathways have additive function in lipid abundance during vertebrate development.

Toxicity assessment of TiO2 nanoparticles in zebrafish embryos under different exposure conditions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Protein Profiles in Zebrafish (Danio rerio) Embryos Exposed to Perfluorooctane Sulfonate

Perfluorooctane sulfonate (PFOS) is widely distributed and persistent in the environment and in wildlife, and it has the potential for developmental toxicity. However, the molecular mechanisms that lead to these toxic effects are not well known. In the present study, proteomic analysis has been performed to investigate the proteins that are differentially expressed in zebrafish embryos exposed to 0.5 mg/l PFOS until 192 h postfertilization. Two-dimensional electrophoresis coupled with mass spectrometry was employed to detect and identify the protein profiles. The analysis revealed that 69 proteins showed altered expression in the treatment group compared to the control group with either increase or decrease in expression levels (more than twofold difference). Of the 69 spots corresponding to the proteins with altered expression, 38 were selected and subjected to matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (TOF/TOF) analysis; 18 proteins were identified in this analysis. These proteins can be categorized into diverse functional classes such as detoxification, energy metabolism, lipid transport/steroid metabolic process, cell structure, signal transduction, and apoptosis. Overall, proteomic analysis using zebrafish embryos serves as an in vivo model in environmental risk assessment and provides insight into the molecular events in PFOS-induced developmental toxicity.