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20 pages, 2929 KiB

Open AccessArticle

Multiplex Analysis Platform for Endocrine Disruption Prediction Using Zebrafish

by Sergio Jarque, Jone Ibarra, Maria Rubio-Brotons, Jessica García-Fernández and Javier Terriente

Int. J. Mol. Sci. 2019, 20(7), 1739; https://doi.org/10.3390/ijms20071739 - 8 Apr 2019

Cited by 24 | Viewed by 5049

Abstract

Small fish are an excellent experimental model to screen endocrine-disrupting compounds, but current fish-based assays to detect endocrine disruption have not been standardized yet, meaning that there is not consensus on endpoints and biomarkers to be measured. Moreover, exposure conditions may vary depending [...] Read more.

Small fish are an excellent experimental model to screen endocrine-disrupting compounds, but current fish-based assays to detect endocrine disruption have not been standardized yet, meaning that there is not consensus on endpoints and biomarkers to be measured. Moreover, exposure conditions may vary depending on the species used as the experimental model and the endocrine pathway evaluated. At present, a battery of a wide range of assays is usually needed for the complete assessment of endocrine activities. With the aim of providing a simple, robust, and fast assay to assess endocrine-disrupting potencies for the three major endocrine axes, i.e., estrogens, androgens, and thyroid, we propose the use of a panel of eight gene expression biomarkers in zebrafish larvae. This includes brain aromatase (cyp19a1b) and vitellogenin 1 (vtg1) for estrogens, cytosolic sulfotransferase 2 family 2 (sult2st3) and cytochrome P450 2k22 (cyp2k22) for androgens, and thyroid peroxidase (tpo), transthyretin (ttr), thyroid receptor α (trα), and iodothyronine deiodinase 2 (dio2) for thyroid metabolism. All of them were selected according to their responses after exposure to the natural ligands 17β-estradiol, testosterone, and 3,3′,5-triiodo-L-thyronine (T3), respectively, and subsequently validated using compounds reported as endocrine disruptors in previous studies. Cross-talk effects were also evaluated for all compounds. Full article

(This article belongs to the Special Issue Zebrafish 2.0: A Model for Toxicological Research)

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14 pages, 1696 KiB

Open AccessArticle

Safety Assessment of Compounds after In Vitro Metabolic Conversion Using Zebrafish Eleuthero Embryos

by Arianna Giusti, Xuan-Bac Nguyen, Stanislav Kislyuk, Mélanie Mignot, Cecilia Ranieri, Johan Nicolaï, Marlies Oorts, Xiao Wu, Pieter Annaert, Noémie De Croze, Marc Léonard, Annelii Ny, Deirdre Cabooter and Peter de Witte

Int. J. Mol. Sci. 2019, 20(7), 1712; https://doi.org/10.3390/ijms20071712 - 6 Apr 2019

Cited by 10 | Viewed by 4906

Abstract

Zebrafish-based platforms have recently emerged as a useful tool for toxicity testing as they combine the advantages of in vitro and in vivo methodologies. Nevertheless, the capacity to metabolically convert xenobiotics by zebrafish eleuthero embryos is supposedly low. To circumvent this concern, a [...] Read more.

Zebrafish-based platforms have recently emerged as a useful tool for toxicity testing as they combine the advantages of in vitro and in vivo methodologies. Nevertheless, the capacity to metabolically convert xenobiotics by zebrafish eleuthero embryos is supposedly low. To circumvent this concern, a comprehensive methodology was developed wherein test compounds (i.e., parathion, malathion and chloramphenicol) were first exposed in vitro to rat liver microsomes (RLM) for 1 h at 37 °C. After adding methanol, the mixture was ultrasonicated, placed for 2 h at −20 °C, centrifuged and the supernatant evaporated. The pellet was resuspended in water for the quantification of the metabolic conversion and the detection of the presence of metabolites using ultra high performance liquid chromatography-Ultraviolet-Mass (UHPLC-UV-MS). Next, three days post fertilization (dpf) zebrafish eleuthero embryos were exposed to the metabolic mix diluted in Danieau’s medium for 48 h at 28 °C, followed by a stereomicroscopic examination of the adverse effects induced, if any. The novelty of our method relies in the possibility to quantify the rate of the in vitro metabolism of the parent compound and to co-incubate three dpf larvae and the diluted metabolic mix for 48 h without inducing major toxic effects. The results for parathion show an improved predictivity of the toxic potential of the compound. Full article

(This article belongs to the Special Issue Zebrafish 2.0: A Model for Toxicological Research)

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Figure 1
Mean scores of lethality and sub-lethal toxicity of test compounds unexposed to rat liver microsome (RLM) in zebrafish eleuthero embryos. The bar charts show the results after incubation of three days post fertilization (dpf) zebrafish eleuthero embryos with blank samples that were processed and spiked with different concentrations of (a) parathion, (b) malathion, and (c) chloramphenicol. Control conditions consisted of eleuthero embryos exposed to the RLM extract (without compound spiking) in the medium (indicated as RLM), and eleuthero embryos exposed to the medium without the RLM extract and without spiking (indicated as Med). After 48 h the incubated eleuthero embryos were morphologically screened, and the mean scores calculated, as described in methods. Three independent experiments were performed, the data were pooled and the mean ± SD was calculated. Hence, a total of six eleuthero embryos were processed per concentration, except in the case of Med samples (n = 10) and RLM samples (n = 10). For the statistical analysis, the mean score of RLM was compared with the mean scores of the other samples by using one-way ANOVA with Dunnett’s multiple comparison test. ** p ≤ 0.01, *** p ≤ 0.001. Full article ">Figure 2
Mean scores of lethality and sub-lethal toxicity of test compounds (a) parathion, (b) malathion, and (c) chloramphenicol previously exposed to RLM activated (A) or not (NA) with reduced β-nicotinamide adenine dinucleotide 2′-phosphate (NADPH) and glucose-6-phosphate (G6P) in zebrafish eleuthero embryos. The bar charts show the results after incubation of three dpf zebrafish eleuthero embryos with 4-fold dilutions of reconstituted extracts of processed samples that were analyzed on their content (see <a href="#ijms-20-01712-f003" class="html-fig">Figure 3</a>). Control conditions consisted out of eleuthero embryos exposed to the RLM extract (without compound spiking) in the medium (indicated as RLM), and eleuthero embryos exposed to the medium without the RLM extract and without spiking (indicated as Med). After 48 h the incubated eleuthero embryos were morphologically screened, and the mean scores calculated, as described in methods. Three independent experiments were performed, the data were pooled and the mean ± SD was calculated. Hence, a total of 30 eleuthero embryos were processed per condition. For the statistical analysis, the mean score of RLM was compared with the mean scores of the other samples by using one-way ANOVA with Dunnett’s multiple comparison test. *** p ≤ 0.001. Full article ">Figure 3
Workflow for the determination of sub-lethal toxicity and lethality of test compounds exposed or unexposed to RLM in zebrafish eleuthero embryos. Full article ">Figure 4
Lateral view of untreated five-dpf eleuthero-embryo (a), and of compound-treated eleuthero-embryo with abnormal body shape and non-inflated swim bladder (b), with curved body (c) and with non-inflated swim bladder (d). Full article ">

17 pages, 2668 KiB

Open AccessArticle

Thyroid Hormone Disruptors Interfere with Molecular Pathways of Eye Development and Function in Zebrafish

by Lisa Baumann, Helmut Segner, Albert Ros, Dries Knapen and Lucia Vergauwen

Int. J. Mol. Sci. 2019, 20(7), 1543; https://doi.org/10.3390/ijms20071543 - 27 Mar 2019

Cited by 33 | Viewed by 5469

Abstract

The effects of thyroid hormone disrupting chemicals (THDCs) on eye development of zebrafish were investigated. We expected THDC exposure to cause transcriptional changes of vision-related genes, which find their phenotypic anchoring in eye malformations and dysfunction, as observed in our previous studies. Zebrafish [...] Read more.

The effects of thyroid hormone disrupting chemicals (THDCs) on eye development of zebrafish were investigated. We expected THDC exposure to cause transcriptional changes of vision-related genes, which find their phenotypic anchoring in eye malformations and dysfunction, as observed in our previous studies. Zebrafish were exposed from 0 to 5 days post fertilization (dpf) to either propylthiouracil (PTU), a thyroid hormone synthesis inhibitor, or tetrabromobisphenol-A (TBBPA), which interacts with thyroid hormone receptors. Full genome microarray analyses of RNA isolated from eye tissue revealed that the number of affected transcripts was substantially higher in PTU- than in TBBPA-treated larvae. However, multiple components of phototransduction (e.g., phosphodiesterase, opsins) were responsive to both THDC exposures. Yet, the response pattern for the gene ontology (GO)-class “sensory perception” differed between treatments, with over 90% down-regulation in PTU-exposed fish, compared to over 80% up-regulation in TBBPA-exposed fish. Additionally, the reversibility of effects after recovery in clean water for three days was investigated. Transcriptional patterns in the eyes were still altered and partly overlapped between 5 and 8 dpf, showing that no full recovery occurred within the time period investigated. However, pathways involved in repair mechanisms were significantly upregulated, which indicates activation of regeneration processes. Full article

(This article belongs to the Special Issue Zebrafish 2.0: A Model for Toxicological Research)

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Number of differentially expressed transcripts in the different treatments and intersects between them. 5 dpf (days post fertilization) = continuous exposure from 0 to 5 dpf; 8 dpf = exposure until 5 dpf + 3 days of recovery. Full article ">Figure 2
Transcriptional changes for eight different transcript lists (two compounds, two time points, up- or down-regulated) with respective enriched GO classes. The bars represent the numbers of up- or down-regulated transcripts for each list with the respective percentage written in it. The total number of differentially expressed transcripts of each list is referred to as “n =” on top of each bar. The upper pie charts show the top three enriched GO classes for the up-regulated transcripts of each list, the lower ones for the down-regulated transcripts (the complete pies are, thus, not representing the high total number of transcripts in each list, which would impede visualization). Full article ">Figure 3
Overview of the percentage of up-regulated (red) or down-regulated (green) transcripts in the 14 different GO classes that were enriched in at least one of the five cluster groups. All treatments were sorted in the same order as for PTU 5 dpf, to enable to visualize the change of response pattern between the time points and compounds. Full article ">Figure 4
Overview of the effects of propylthiouracil (PTU) or tetrabromobisphenol-A (TBBPA) exposure on transcript levels of components of the phototransduction and retinoid recycling pathways. Left: detailed pathways in rod/cone cells and the retinal epithelium; Right: heatmap showing the transcript level changes of components of the pathways. LRAT: lecithin retinol acyltransferase. All other abbreviations are explained in the heatmap on the right. (Figure adapted from Houbrechts [<a href="#B26-ijms-20-01543" class="html-bibr">26</a>]). Full article ">

18 pages, 3090 KiB

Open AccessArticle

Comparison the Effect of Ferutinin and 17β-Estradiol on Bone Mineralization of Developing Zebrafish (Danio rerio) Larvae

by Hoda Zare Mirakabad, Mohammad Farsi, Saeed Malekzadeh Shafaroudi, Abdolreza Bagheri, Mehrdad Iranshahi and Nasrin Moshtaghi

Int. J. Mol. Sci. 2019, 20(6), 1507; https://doi.org/10.3390/ijms20061507 - 26 Mar 2019

Cited by 7 | Viewed by 5410

Abstract

There is an urgent need to develop novel drugs for osteoporosis which occurs due to estrogen deficiency. Phytoestrogens derived from medicinal plants would be the best alternative to chemical drugs with harmful side effects. The main purpose of the present study was to [...] Read more.

There is an urgent need to develop novel drugs for osteoporosis which occurs due to estrogen deficiency. Phytoestrogens derived from medicinal plants would be the best alternative to chemical drugs with harmful side effects. The main purpose of the present study was to investigate the effect of ferutinin compared to 17β-estradiol (E2) on bone mineralization of zebrafish larvae. Regarding the lack of publications, the histology analysis was performed after exposure to E2 to find effective treatment on bone mineralization of developing zebrafish larvae. Then, the larvae were exposed to four concentrations of ferutinin at three time points to assess the mortality, the expression of some related genes and histology of the ceratohyal and hyomandibular of treated larvae. The RT-PCR result of the treatment groups demonstrated the similar expression pattern in the larvae which were exposed to 1.25 μg/mL of ferutinin and 2 µM of E2 at 2 dpf, which confirmed the result of histology analysis. In addition, RT-qPCR of high concentration of ferutinin and E2 demonstrated that bmp2a/b and esr1 were downregulated and upregulated when the larvae were exposed to 5 μg/mL of ferutinin and 10 µM of E2, respectively. Full article

(This article belongs to the Special Issue Zebrafish 2.0: A Model for Toxicological Research)

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21 pages, 2243 KiB

Open AccessArticle

Optimizing the Use of Zebrafish Feeding Trials for the Safety Evaluation of Genetically Modified Crops

by Isabelle J. Gabriëls, Lucia Vergauwen, Marthe De Boevre, Stefan Van Dongen, Ronny Blust, Sarah De Saeger, Mia Eeckhout, Marc De Loose and Dries Knapen

Int. J. Mol. Sci. 2019, 20(6), 1472; https://doi.org/10.3390/ijms20061472 - 23 Mar 2019

Cited by 3 | Viewed by 3888

Abstract

In Europe, the toxicological safety of genetically modified (GM) crops is routinely evaluated using rodent feeding trials, originally designed for testing oral toxicity of chemical compounds. We aimed to develop and optimize methods for advancing the use of zebrafish feeding trials for the [...] Read more.

In Europe, the toxicological safety of genetically modified (GM) crops is routinely evaluated using rodent feeding trials, originally designed for testing oral toxicity of chemical compounds. We aimed to develop and optimize methods for advancing the use of zebrafish feeding trials for the safety evaluation of GM crops, using maize as a case study. In a first step, we evaluated the effect of different maize substitution levels. Our results demonstrate the need for preliminary testing to assess potential feed component-related effects on the overall nutritional balance. Next, since a potential effect of a GM crop should ideally be interpreted relative to the natural response variation (i.e., the range of biological values that is considered normal for a particular endpoint) in order to assess the toxicological relevance, we established natural response variation datasets for various zebrafish endpoints. We applied equivalence testing to calculate threshold equivalence limits (ELs) based on the natural response variation as a method for quantifying the range within which a GM crop and its control are considered equivalent. Finally, our results illustrate that the use of commercial control diets (CCDs) and null segregant (NS) controls (helpful for assessing potential effects of the transformation process) would be valuable additions to GM safety assessment strategies. Full article

(This article belongs to the Special Issue Zebrafish 2.0: A Model for Toxicological Research)

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Figure 1
Effects of increasing dietary maize substitution levels. Range finding feeding trial: Increasing maize substitution levels resulted in (a) a significant decrease in the absolute growth rate (AGR) (n = 3), (b) a significant decrease in the % uptake of carbohydrates from the feed (n = 3) and (c) a significant increase in the hepatosomatic index (HSI) of male fish (n = 3). Artemia supplementation feeding trial: After the introduction of Artemia in the diets, increasing maize substitution levels no longer significantly affected (d) the AGR (n = 4), (e) the % uptake of carbohydrates from the feed (n = 4) or (f) the HSI of male fish (n = 4). Circles represent biological replicates; horizontal lines represent the mean of all replicates; *: significantly different from 0% maize (p < 0.05); #: significant correlation between maize substitution level and the respective parameter (p < 0.05); CCD: commercial control diet; 0% RF: 0% maize substitution values derived from the range finding feeding trial. Full article ">Figure 2
Effects of increasing dietary maize substitution level on liver carbohydrate content. (a) Range finding feeding trial: Increasing maize substitution levels resulted in an increase in the absolute amount of carbohydrates per pooled liver sample in male fish (n = 3) between 0 and 20% maize substitution; (b) Artemia supplementation feeding trial: No differences were observed in the absolute amount of carbohydrates in livers of male fish for the evaluated maize substitution levels (n = 4). *: significantly different from 0% maize (p < 0.05); †: significantly different from 20% maize (p < 0.05); CCD: commercial control diet; 0% RF: 0% maize substitution values derived from the range finding feeding trial. Full article ">Figure 3
Transcriptional effects in zebrafish liver after feeding with 25% of maize substitution. (a) Pie chart summarizing the GO classes affected by feeding with 25% maize. Most differentially transcribed genes (63%) were related to metabolic processes. Details are given for affected pathways related to (b) carbohydrate, lipid and purine/pyrimidine metabolism and (c) amino acid metabolism. Green indicates downregulation and red indicates upregulation relative to 0% maize substitution (false discovery rate: p < 0.05). Dotted arrows represent endocrine regulation, solid arrows indicate pathway conversion steps. Full article ">Figure 4
Equivalence testing based on the distribution-wise equivalence (DWE) criterion. Equivalence (DWE) tested for the contrasts GM vs. null segregant (NS), GM vs. wild type (WT) and NS vs. WT for a selection of endpoints: length, hepatosomatic index, liver carbohydrate and protein contents, gonadosomatic index. Mean equivalence limit scaled differences are presented as a black dot, with a 95% confidence interval. The vertical red lines represent the equivalence limits (ELs) (–1,+1), calculated based on the non-GM reference variation dataset. Full article ">Figure 5
Estimating the natural response variation. Overview of the selected endpoints and corresponding time points measured during the natural response variation feeding trial. Relative condition factor and length were evaluated every 4 weeks, reproduction parameters and quality of the offspring were analyzed at the start of the experiment and at week 12. Hepatosomatic/gonadosomatic index and liver/muscle energy reserves were measured at the end of the feeding trial. W: week. Full article ">

9 pages, 963 KiB

Open AccessArticle

An Integrative Evaluation Method for the Biological Safety of Down and Feather Materials

by Toshikatsu Kawada, Junya Kuroyanagi, Fumiyoshi Okazaki, Mizuki Taniguchi, Hiroko Nakayama, Narumi Suda, Souta Abiko, Satoshi Kaneco, Norihiro Nishimura and Yasuhito Shimada

Int. J. Mol. Sci. 2019, 20(6), 1434; https://doi.org/10.3390/ijms20061434 - 21 Mar 2019

Cited by 6 | Viewed by 4175

Abstract

Background: Down and feather materials have been commonly used and promoted as natural stuffing for warm clothing and bedding. These materials tend to become more allergenic as they become contaminated with microorganisms, in addition to being subjected to several kinds of chemical treatments. [...] Read more.

Background: Down and feather materials have been commonly used and promoted as natural stuffing for warm clothing and bedding. These materials tend to become more allergenic as they become contaminated with microorganisms, in addition to being subjected to several kinds of chemical treatments. The biological or chemical contaminants in these materials pose a major risk to human health, to consumers and manufacturers alike. Here, we report the development of an integrative evaluation method for down and feather materials to assess bacterial contamination and in vivo toxicity. Methods: To assess bacterial contamination, we quantified 16S ribosomal RNA, performed culture tests, and established a conversion formula. To determine in vivo toxicity, we performed a zebrafish embryo toxicity testing (ZFET). Results: Washing the material appropriately decreases the actual number of bacteria in the down and feather samples; in addition, after washing, 16S rRNA sequencing revealed that the bacterial compositions were similar to those in rinse water. The ZFET results showed that even materials with low bacterial contamination showed high toxicity or high teratogenicity, probably because of the presence of unknown chemical additives. Conclusions: We established an integrative evaluation method for down and feather safety, based on bacterial contamination with in vivo toxicity testing. Full article

(This article belongs to the Special Issue Zebrafish 2.0: A Model for Toxicological Research)

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Figure 1
qPCR quantification of contaminated bacteria in down and feather samples. (a) Scanning electron microscopic images of unwashed and washed down and feather. Red arrowheads indicate debris. (b) Colony-forming unit (CFU) of down and feather samples. CFU was calculated using standard agar plate protocol. n = 6, error bars indicate SD. (c) qPCR analysis of bacterial numbers in down and feather samples. n = 6, error bars indicate SD. (d) Bacterial proliferation in the down and feather samples. Red signal indicates mCherry-expressing E. coli. Full article ">Figure 2
Compositions of bacteria in contaminated down and feather samples. Phylum (a) and class (b) level of the bacterial composition. n = 3, error bars indicate SD. Bacterial composition of each sample is depicted in <a href="#app1-ijms-20-01434" class="html-app">Figures S2 and S3</a>. (c) Quantification of contaminated bacterial in unwashed and washed samples. n = 3, error bars indicate SD. Full article ">Figure 3
Zebrafish embryo toxicity test (ZFET) of down and feather samples. (a) Scanning electron microscopic images of glue down. Red arrowheads indicate glue chemicals. (b) Representative images of 72 hours-post-fertilization (hpf) zebrafish in ZFET. (c) Survivals and anomalies in ZFET. ** p < 0.01 vs. control. n = 4–6, error bars indicate SD. (d) Quantification of contaminated bacterial in down and feather samples used in ZFET. n = 4, error bars indicate SD. (e) Representative images of macrophage-EGFP zebrafish used in ZFET. Green indicates EGFP-expressed macrophages. Unwashed and glue down immersion water were diluted 5 times to reduce mortality. (f) Quantification of numbers of macrophages in the tail fin. *** p < 0.001 vs. control. n = 8, error bars indicate SD. Full article ">

16 pages, 2368 KiB

Open AccessArticle

A Smart Imaging Workflow for Organ-Specific Screening in a Cystic Kidney Zebrafish Disease Model

by Gunjan Pandey, Jens H. Westhoff, Franz Schaefer and Jochen Gehrig

Int. J. Mol. Sci. 2019, 20(6), 1290; https://doi.org/10.3390/ijms20061290 - 14 Mar 2019

Cited by 16 | Viewed by 6432

Abstract

The zebrafish is being increasingly used in biomedical research and drug discovery to conduct large-scale compound screening. However, there is a lack of accessible methodologies to enable automated imaging and scoring of tissue-specific phenotypes at enhanced resolution. Here, we present the development of [...] Read more.

The zebrafish is being increasingly used in biomedical research and drug discovery to conduct large-scale compound screening. However, there is a lack of accessible methodologies to enable automated imaging and scoring of tissue-specific phenotypes at enhanced resolution. Here, we present the development of an automated imaging pipeline to identify chemical modifiers of glomerular cyst formation in a zebrafish model for human cystic kidney disease. Morpholino-mediated knockdown of intraflagellar transport protein Ift172 in Tg(wt1b:EGFP) embryos was used to induce large glomerular cysts representing a robustly scorable phenotypic readout. Compound-treated embryos were consistently aligned within the cavities of agarose-filled microplates. By interfacing feature detection algorithms with automated microscopy, a smart imaging workflow for detection, centring and zooming in on regions of interests was established, which enabled the automated capturing of standardised higher resolution datasets of pronephric areas. High-content screening datasets were processed and analysed using custom-developed heuristic algorithms implemented in common open-source image analysis software. The workflow enables highly efficient profiling of entire compound libraries and scoring of kidney-specific morphological phenotypes in thousands of zebrafish embryos. The demonstrated toolset covers all the aspects of a complex whole organism screening assay and can be adapted to other organs, specimens or applications. Full article

(This article belongs to the Special Issue Zebrafish 2.0: A Model for Toxicological Research)

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Figure 1
Automated imaging of an ift172-MO-based zebrafish model for human cystic kidney disease. (A–D) Dorsal views on pronephric areas in the Tg(wt1b:EGFP) embryos. Scale bar is 50 µm. (E–H) Bright-field stereo microscope images of the dose–response curve of ift172-MO on 72 hpf zebrafish larvae. Scale bar is 250 µm. (A,E) wild-type, (B,F) 50 µM ift172-MO, (C,G) 100 µM ift172-MO, and (D,H) 500 µM ift172-MO. (I,K) Overlay images of bright-field and fluorescence channels depicting wild-type and cystic pronephric phenotypes acquired with a 10x objective. Scale bar for I is 100 µm and J is 50 µm. (J,L) Enlarged fluorescent 10x views of I and K. (M,N) Montage images illustrating (M) overlay images or (N) pronephric areas of dorsally oriented larvae in a microtiter plate. Full article ">Figure 2
Smart imaging workflow for automated imaging of kidney regions. (A) Workflow chart illustrating the feedback microscopy approach utilised to automatically acquire regions of interest (ROIs). Dotted arrows indicate imaging components of the pre-screen; solid arrows indicate image processing and higher resolution imaging procedures of the smart imaging module. (B) Example of script commands for a single well that are sent to the automated microscope. The script triggers automated centring of the region of interest and high-resolution acquisition. (C) Representative overview image taken with 4x objective followed by (D) centring and higher resolution imaging of the pronephric region with 10x objective. The image shows a maximum projection of the GFP channel overlaid with the bright-field channel. Scale bar in C is 500µm and scale bar in D is 200 µm. Full article ">Figure 3
Quality control and image filtering. (A–D) Pre-processed images used for the quality check step. In both blank and blur detection process, the images acquired with 10x objective were cropped as seen in (A,B,E,F). (C,D) In blur detection, the background noise was reduced using the ‘‘Subtract Background’’ plugin in Fiji followed by variance calculation. Images below 400 variance value were classified as blurry. (G,H) In blank detection, the ‘‘Laplace Filter’’ was used before variance calculation and images above 124 variance value were considered blank. Scale bars in A and C are 50 µm. Full article ">Figure 4
Image categorisation and phenotype detection. Pictorial representation of (A–D) wild-type and (E–H) cystic images acquired with 10x objective and checked by pronephric phenotype detection script. (B,F) Pre-processed images were denoised and thresholded. An optimised bounding box was automatically drawn to ensure the inclusion of kidney tissue. (C,G) Examples of plot profiles for control (C) and cystic kidneys (G). The difference between the resulting profiles were analysed and categorised as either wild-type or cystic kidneys. (D,H) Implementation of confirmatory script to check the previously classified pronephric phenotype. Number of ellipses surrounding the ROI and the ellipse major axis length of the largest ellipse were measured. Images with ellipse values above 299 pixels were classified as wild type. Scale bar in A is 50 µm. Full article ">Figure 5
Automated scoring of pronephric phenotypes. (A–D) Quantification of pronephric tissue and cystic area in the controls (A,B) and cystic kidneys (C,D) acquired with 10x objective. Green depicts GFP-positive pronephric tissue, and red depicts cystic area. Scale bar for A–D is 50 µm. (E,F) Fluorescence and stereo microscope images of dose–response experiments administering rapamycin Co-MO or ift172-MO-injected embryos at 72 hpf. (E,I) The control, (F,J) ift172-MO injected, (G,K) ift172-MO + 25 µM rapamycin-exposed and (H,L) ift172-MO + 50 µM rapamycin-exposed larvae are shown. Scale bar for E–H is 50 µm and for I–L is 250 µm. (M) Montage representing 72 hpf pronephros in a microtiter plate; the first row is ift172-MO injected, last row is wild type and rest of the rows are 25 µM rapamycin-exposed larvae. (N) The corresponding heatmap displays rapamycin-based suppression of kidneys in reference to the cystic (first row) and wild-type (last row) controls. See also <a href="#app1-ijms-20-01290" class="html-app">Figure S3</a>. Full article ">

30 pages, 5551 KiB

Open AccessArticle

From mRNA Expression of Drug Disposition Genes to In Vivo Assessment of CYP-Mediated Biotransformation during Zebrafish Embryonic and Larval Development

by Evy Verbueken, Chloé Bars, Jonathan S. Ball, Jelena Periz-Stanacev, Waleed F. A. Marei, Anna Tochwin, Isabelle J. Gabriëls, Ellen D. G. Michiels, Evelyn Stinckens, Lucia Vergauwen, Dries Knapen, Chris J. Van Ginneken and Steven J. Van Cruchten

Int. J. Mol. Sci. 2018, 19(12), 3976; https://doi.org/10.3390/ijms19123976 - 10 Dec 2018

Cited by 28 | Viewed by 4828

Abstract

The zebrafish (Danio rerio) embryo is currently explored as an alternative for developmental toxicity testing. As maternal metabolism is lacking in this model, knowledge of the disposition of xenobiotics during zebrafish organogenesis is pivotal in order to correctly interpret the outcome [...] Read more.

The zebrafish (Danio rerio) embryo is currently explored as an alternative for developmental toxicity testing. As maternal metabolism is lacking in this model, knowledge of the disposition of xenobiotics during zebrafish organogenesis is pivotal in order to correctly interpret the outcome of teratogenicity assays. Therefore, the aim of this study was to assess cytochrome P450 (CYP) activity in zebrafish embryos and larvae until 14 d post-fertilization (dpf) by using a non-specific CYP substrate, i.e., benzyloxy-methyl-resorufin (BOMR) and a CYP1-specific substrate, i.e., 7-ethoxyresorufin (ER). Moreover, the constitutive mRNA expression of CYP1A, CYP1B1, CYP1C1, CYP1C2, CYP2K6, CYP3A65, CYP3C1, phase II enzymes uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) and sulfotransferase 1st1 (SULT1ST1), and an ATP-binding cassette (ABC) drug transporter, i.e., abcb4, was assessed during zebrafish development until 32 dpf by means of quantitative PCR (qPCR). The present study showed that trancripts and/or the activity of these proteins involved in disposition of xenobiotics are generally low to undetectable before 72 h post-fertilization (hpf), which has to be taken into account in teratogenicity testing. Full capacity appears to be reached by the end of organogenesis (i.e., 120 hpf), although CYP1—except CYP1A—and SULT1ST1 were shown to be already mature in early embryonic development. Full article

(This article belongs to the Special Issue Zebrafish 2.0: A Model for Toxicological Research)

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1619 KiB

Open AccessArticle

Effects of Hydroxylated Polybrominated Diphenyl Ethers in Developing Zebrafish Are Indicative of Disruption of Oxidative Phosphorylation

by Jessica Legradi, Marinda Van Pomeren, Anna-Karin Dahlberg and Juliette Legler

Int. J. Mol. Sci. 2017, 18(5), 970; https://doi.org/10.3390/ijms18050970 - 3 May 2017

Cited by 16 | Viewed by 5167

Abstract

Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) have been detected in humans and wildlife. Using in vitro models, we recently showed that OH-PBDEs disrupt oxidative phosphorylation (OXPHOS), an essential process in energy metabolism. The goal of the current study was to determine the in vivo [...] Read more.

Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) have been detected in humans and wildlife. Using in vitro models, we recently showed that OH-PBDEs disrupt oxidative phosphorylation (OXPHOS), an essential process in energy metabolism. The goal of the current study was to determine the in vivo effects of OH-PBDE reported in marine wildlife. To this end, we exposed zebrafish larvae to 17 OH-PBDEs from fertilisation to 6 days of age, and determined developmental toxicity as well as OXPHOS disruption potential with a newly developed assay of oxygen consumption in living embryos. We show here that all OH-PBDEs tested, both individually and as mixtures, resulted in a concentration-dependant delay in development in zebrafish embryos. The most potent substances were 6-OH-BDE47 and 6′-OH-BDE49 (No-Effect-Concentration: 0.1 and 0.05 µM). The first 24 h of development were the most sensitive, resulting in significant and irreversible developmental delay. All substances increased oxygen consumption, an effect indicative of OXPHOS disruption. Our results suggest that the induced developmental delay may be caused by disruption of OXPHOS. Though further studies are needed, our findings suggest that the environmental concentrations of some OH-PBDEs found in Baltic Sea wildlife in the Baltic Sea may be of toxicological concern. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Full article ">Figure 1
Developmental delay in zebrafish embryos exposed to 2′-OH-6′-Cl-BDE68. Images of 48 hpf old embryos exposed to different concentrations (upper row). The delayed embryos look like control embryos at 11 and 20 hpf (lower row). Magnification was 2×. Full article ">Figure 2
Irreversible developmental delay in zebrafish embryos exposed to hydroxylated polybrominated diphenyl ethers. Development after exposure of zebrafish from 0 to 24 h (blue bars). The red bar presents the development from 24 to 48 hpf after the medium was replaced with clean medium. Concentrations are the LOECs (1 dpf) from <a href="#ijms-18-00970-t001" class="html-table">Table 1</a>. The error bars are the standard deviation over three replicates. Full article ">Figure 3
Representative graph showing that hydroxylated polybrominated diphenyl ethers (OH-PBDEs) lead to increased oxygen consumption. Amount of oxygen per well (µM) during the first 24 h of development. Solvent control (DMSO), positive control (Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) 0.5 µM) and six different OH-PBDEs at different exposure concentrations (Lowest Observed Effect Concentration (LOECs) for oxygen consumption in <a href="#ijms-18-00970-t001" class="html-table">Table 1</a>). The error bars present the standard deviation over 12 wells (n = 12). Full article ">Figure 4
Effects on growth of zebrafish embryos exposed to a mixture of OH-PBDEs for 24 h. The concentrations of the test compounds in the mixture were modelled to represent levels found in Baltic blue mussels, and were tested as concentrated mixtures (10X and 100X) or diluted mixtures (10× and 100×). The error bars show the standard deviation over three replicates. Full article ">

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Open AccessArticle

Altered Adipogenesis in Zebrafish Larvae Following High Fat Diet and Chemical Exposure Is Visualised by Stimulated Raman Scattering Microscopy

by Marjo J. Den Broeder, Miriam J. B. Moester, Jorke H. Kamstra, Peter H. Cenijn, Valentina Davidoiu, Leonie M. Kamminga, Freek Ariese, Johannes F. De Boer and Juliette Legler

Int. J. Mol. Sci. 2017, 18(4), 894; https://doi.org/10.3390/ijms18040894 - 24 Apr 2017

Cited by 55 | Viewed by 9614

Abstract

Early life stage exposure to environmental chemicals may play a role in obesity by altering adipogenesis; however, robust in vivo methods to quantify these effects are lacking. The goal of this study was to analyze the effects of developmental exposure to chemicals on [...] Read more.

Early life stage exposure to environmental chemicals may play a role in obesity by altering adipogenesis; however, robust in vivo methods to quantify these effects are lacking. The goal of this study was to analyze the effects of developmental exposure to chemicals on adipogenesis in the zebrafish (Danio rerio). We used label-free Stimulated Raman Scattering (SRS) microscopy for the first time to image zebrafish adipogenesis at 15 days post fertilization (dpf) and compared standard feed conditions (StF) to a high fat diet (HFD) or high glucose diet (HGD). We also exposed zebrafish embryos to a non-toxic concentration of tributyltin (TBT, 1 nM) or Tris(1,3-dichloroisopropyl)phosphate (TDCiPP, 0.5 µM) from 0–6 dpf and reared larvae to 15 dpf under StF. Potential molecular mechanisms of altered adipogenesis were examined by qPCR. Diet-dependent modulation of adipogenesis was observed, with HFD resulting in a threefold increase in larvae with adipocytes, compared to StF and HGD. Developmental exposure to TBT but not TDCiPP significantly increased adipocyte differentiation. The expression of adipogenic genes such as pparda, lxr and lepa was altered in response to HFD or chemicals. This study shows that SRS microscopy can be successfully applied to zebrafish to visualize and quantify adipogenesis, and is a powerful approach for identifying obesogenic chemicals in vivo. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Open AccessArticle

ZeGlobalTox: An Innovative Approach to Address Organ Drug Toxicity Using Zebrafish

by Carles Cornet, Simone Calzolari, Rafael Miñana-Prieto, Sylvia Dyballa, Els Van Doornmalen, Helma Rutjes, Thierry Savy, Davide D’Amico and Javier Terriente

Int. J. Mol. Sci. 2017, 18(4), 864; https://doi.org/10.3390/ijms18040864 - 19 Apr 2017

Cited by 76 | Viewed by 11031

Abstract

Toxicity is one of the major attrition causes during the drug development process. In that line, cardio-, neuro-, and hepatotoxicities are among the main reasons behind the retirement of drugs in clinical phases and post market withdrawal. Zebrafish exploitation in high-throughput drug screening [...] Read more.

Toxicity is one of the major attrition causes during the drug development process. In that line, cardio-, neuro-, and hepatotoxicities are among the main reasons behind the retirement of drugs in clinical phases and post market withdrawal. Zebrafish exploitation in high-throughput drug screening is becoming an important tool to assess the toxicity and efficacy of novel drugs. This animal model has, from early developmental stages, fully functional organs from a physiological point of view. Thus, drug-induced organ-toxicity can be detected in larval stages, allowing a high predictive power on possible human drug-induced liabilities. Hence, zebrafish can bridge the gap between preclinical in vitro safety assays and rodent models in a fast and cost-effective manner. ZeGlobalTox is an innovative assay that sequentially integrates in vivo cardio-, neuro-, and hepatotoxicity assessment in the same animal, thus impacting strongly in the 3Rs principles. It Reduces, by up to a third, the number of animals required to assess toxicity in those organs. It Refines the drug toxicity evaluation through novel physiological parameters. Finally, it might allow the Replacement of classical species, such as rodents and larger mammals, thanks to its high predictivity (Specificity: 89%, Sensitivity: 68% and Accuracy: 78%). Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Complete ZeGlobalTox experimental setup. (A) Acute Toxicity experimental pipeline; (B) ZeGlobalTox experimental pipeline. Drugs are added from 96 hpf. Cardiotoxicity is evaluated at 100 hpf, neurotoxicity at 120 hpf, and hepatotoxicity at 132 hpf. Abbreviations: NOEC (no observed effect concentration). Full article ">Figure 2
96 hpf mortality concentration response curve (red line), compared to DEAB (Diethylaminobenzaldehyde)(blue line), for (A) (±)-Epinephrine hydrochloride; (B) Ciprofloxacin; (C) Cisapride; (D) d-(+)-glucose; (E) Digoxigenin; (F) Docetaxel; (G) Dofetilide; (H) Finasteride; (I) Flupirtine; (J) Fusidic Acid; (K) Isoniazid; (L) l-Cysteine; (M) l-Glutamine; (N) Methyldopa; (O) NaCl; (P) Pindolol; (Q) Riluzole; (R) Suramin; (S) Trifluoperazine hydrochloride; and (T) Vincristine. Full article ">Figure 3
Cardiotoxicity evaluation results. (A) Scheme of the experimental procedure; (B) Bar graphs showing heart beat frequency in beats per minute (bpm); (C) QT corrected interval (QTc); (D) Ejection fraction (EJF); (E) and longest cardiac arrest of 100 h old zebrafish larvae. Asterisks indicate statistical significance after a One-way ANOVA: * p < 0.05; ** p < 0.01; *** p < 0.001. Black bar: negative control. Red bar: positive control. n = 16 but for DMSO n = 46. Full article ">Figure 4
Locomotion results. (A) Scheme of the experimental procedure; (B) Bar graphs showing total distance moved corrected to the DMSO group. Asterisks indicate statistical significance after a One-way ANOVA * p < 0.05; ** p < 0.01; *** p < 0.001. Black bar: negative control. Red bar: positive control. Experiment performed once with 16 larvae per condition. n = 43 for the DMSO. Full article ">Figure 5
Hepatotoxicity results (A) Scheme of the experimental procedure (B). Bar graphs showing average liver area in mm. (C) Bar graphs showing the percentage of larvae presenting steatosis or yolk lipid accumulation after oil red O stain (D–F) Representative oil red O whole mount staining images of (D) DMSO, (E) EtOH and (F) APAP; black arrows point at non-affected liver (D), liver with steatosis (E), and yolk lipid retention (F), respectively. Asterisks indicate statistical significance after One-way ANOVA (liver area) or Fisher’s exact test (steatosis and yolk lipid retention): * p < 0.05; ** p < 0.01; *** p < 0.001. Black bar: negative control (B). Red bar: positive control (B). n = 20 but for DMSO n = 45. Full article ">Figure 6
ZeCardio β software user pipeline. (A) Video acquisition of larvae incubated with the candidate drug; (B) Video import into the software; (C) User drawn line along the heart axis; (D–H) GUI (Graphical User Interface) display of (D) Chamber kymographs; (E) atrial and ventricular BPM (Beats Per Minute) values; (F) Distribution plot over time of atrial and ventricular BPM; (G) QTc interval and EJF (Ejection Fraction) values and (H) Cardiac arrest events; (I) Output values are presented in .csv format. Kymographs and measurements are displayed in green or blue for ventricle or atrium respectively. Full article ">Figure 7
Lipid droplets on a zebrafish liver stained with Oil Red O. Steatosis is considered when three or more droplets are seen within the liver area. Full article ">

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Open AccessArticle

Morphofunctional Alterations in Zebrafish (Danio rerio) Gills after Exposure to Mercury Chloride

by Rachele Macirella and Elvira Brunelli

Int. J. Mol. Sci. 2017, 18(4), 824; https://doi.org/10.3390/ijms18040824 - 13 Apr 2017

Cited by 50 | Viewed by 8812

Abstract

Mercury (Hg) is a global pollutant that may exert its toxic effects on living organisms and is found in both aquatic and terrestrial ecosystems in three chemical forms; elemental, organic, and inorganic. The inorganic form (iHg) tends to predominantly accumulate in aquatic environments. [...] Read more.

Mercury (Hg) is a global pollutant that may exert its toxic effects on living organisms and is found in both aquatic and terrestrial ecosystems in three chemical forms; elemental, organic, and inorganic. The inorganic form (iHg) tends to predominantly accumulate in aquatic environments. The gill apparatus is a very dynamic organ that plays a fundamental role in gas exchange, osmoregulation, acid-base regulation, detoxification, and excretion, and the gills are the primary route of waterborne iHg entrance in fish. In the present work we investigated the morphofunctional and ultrastructural effects in Danio rerio gills after 96 h exposure to two low HgCl₂ concentrations (7.7 and 38.5 µg/L). Our results clearly demonstrated that a short-term exposure to low concentrations of mercury chloride resulted in gill morphology alterations and in the modifications of both Na^+/K⁺-ATPase and metallothioneins (MTs) expression pattern. The main morphological effects recorded in this work were represented by hyperplasia and ectopia of chloride cells (CCs), lamellar fusion, increased mucous secretion, alteration of pavement cells (PVCs), detachment of the secondary epithelium, pillar cell degeneration, degeneration, and apoptosis. Trough immunohistochemistry and real-time PCR analysis also showed a dose-related modulation of Na⁺/K⁺-ATPase and MTs. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Open AccessArticle

Zebrafish as an Alternative Vertebrate Model for Investigating Developmental Toxicity—The Triadimefon Example

by Maria Zoupa and Kyriaki Machera

Int. J. Mol. Sci. 2017, 18(4), 817; https://doi.org/10.3390/ijms18040817 - 12 Apr 2017

Cited by 66 | Viewed by 9142

Abstract

Triadimefon is a widely used triazole fungicide known to cause severe developmental defects in several model organisms and in humans. The present study evaluated in detail the developmental effects seen in zebrafish embryos exposed to triadimefon, confirmed and expanded upon previous phenotypic findings [...] Read more.

Triadimefon is a widely used triazole fungicide known to cause severe developmental defects in several model organisms and in humans. The present study evaluated in detail the developmental effects seen in zebrafish embryos exposed to triadimefon, confirmed and expanded upon previous phenotypic findings and compared them to those observed in other traditional animal models. In order to do this, we exposed embryos to 2 and 4 µg/mL triadimefon and evaluated growth until 120 h post-fertilization (hpf) through gross morphology examination. Our analysis revealed significant developmental defects at the highest tested concentration including somite deformities, severe craniofacial defects, a cleft phenotype along the three primary neural divisions, a rigorously hypoplastic or even absent mandible and a hypoplastic morphology of the pharyngeal arches. Interestingly, massive pericardial edemas, abnormal shaped hearts, brachycardia and inhibited or absent blood circulation were also observed. Our results revealed that the presented zebrafish phenotypes are comparable to those seen in other organism models and those derived from human observations as a result of triadimefon exposure. We therefore demonstrated that zebrafish provide an excellent system for study of compounds with toxic significance and can be used as an alternative model for developmental toxicity studies to predict effects in mammals. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Open AccessArticle

Triazole Fungicides Inhibit Zebrafish Hatching by Blocking the Secretory Function of Hatching Gland Cells

by Javiera F. De la Paz, Natalia Beiza, Susana Paredes-Zúñiga, Misque S. Hoare and Miguel L. Allende

Int. J. Mol. Sci. 2017, 18(4), 710; https://doi.org/10.3390/ijms18040710 - 4 Apr 2017

Cited by 70 | Viewed by 7193

Abstract

In animals, hatching represents the transition point from a developing embryo to a free-living individual, the larva. This process is finely regulated by many endogenous and environmental factors and has been shown to be sensitive to a variety of chemical agents. It is [...] Read more.

In animals, hatching represents the transition point from a developing embryo to a free-living individual, the larva. This process is finely regulated by many endogenous and environmental factors and has been shown to be sensitive to a variety of chemical agents. It is commonly evaluated in bioassays in order to establish the effects of different agents on early development and reproductive capabilities in fish and other aquatic animals. In fish, the breakdown of the chorion is achieved by the secretion of choriolysin by hatching gland cells (HGCs) into the perivitelline space (PVS), coupled with spontaneous movements of the developing larva. In this work, we used zebrafish to assay the effects of a family of widely used agrochemicals—triazoles Triadimefon (FON), Triadimenol (NOL) and free triazole (1,2,4-T)—on hatching success. We found a strong inhibition of hatching by triazole exposure which was correlated with morphological changes and a reduction in the secretory function of the HGCs. As a consequence, the release of choriolytic enzymes by HGCs was reduced. We also found that HGC secretion reduction after exposure to FON can be rescued by co-incubation with a dopamine D2 receptor antagonist but not by antagonists of the D1-like receptors. This suggests a specific pathway through which this family of fungicides may be impairing a critical event in the fish life cycle. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Figure 1
Hatching inhibition in zebrafish after triazole exposure. (A) The percentage of hatching success at 4 dpf of larvae exposed to different concentrations of triazoles during embryogenesis is shown. LC5096h, hatching IC5096h, and IC5096h/10 were used as the high, medium, and low exposure concentrations respectively; (B) The percentage of dead larvae under each condition at 8 dpf was quantified. Control, unexposed animals. With chorion, high Triadimefon (FON) exposure and animals left in the chorion. Dechorionated, high FON exposure in which chorion was manually removed. Half of the animals die by the eighth day if left in the chorion, but if they are dechorionated at day 2, lethality decreases to about 10%. Exact concentration details are presented in <a href="#app1-ijms-18-00710" class="html-app">Table S1</a>; (A,B) Kruskal-Wallis, Dunn’s multiple comparisons test (statistical significance is compared with respective controls * p < 0.05, ** p < 0.01, *** p < 0.001). Full article ">Figure 2
Number and morphology measurements of hatching gland cells (HGCs) in zebrafish embryos exposed to triazoles. (A,B) Representative images of the pericardial area including the HGCs, labeled with GFP in cldnB:mGFP fish; (A) control, and (B) representative Triadimefon (FON) and Triadimenol (NOL)-treated 2 dpf embryo; (C) There was no difference in the number of HGCs between treated and control embryos at 1 or 2 dpf; (D) There was also no difference in cell size at 2 dpf; (E) Circularity index measurements were made on HGCs and were compared between treated embryos and controls. The shape of HGCs in treated animals with FON and NOL is more circular compared to untreated ones. Kruskal-Wallis, Dunn’s multiple comparisons test (statistical significance is compared with controls **** p < 0.0001). Scale bar: 50 μm. Full article ">Figure 3
Triazoles prevent secretion of the HGC granules. HGCs with granular content of representative 50–60 hpf (A) control, (B) FON, (C) NOL, and (D) 1,2,4-T-treated animals; (E,F) The number of HGCs containing granules in embryos exposed to a high concentration of triazoles for 48 h starting at blastula (E), or 1 dpf (F) stages, was counted and averaged. Scale bar: 20 µm. Kruskal-Wallis, Dunn’s multiple comparisons test (statistical significance is compared with controls * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Full article ">Figure 4
Triadimefon increases locomotor activity (LMA) in 3 dpf larvae. Larvae incubated with the medium FON concentration show a significant increase in LMA when they are compared with non-treated animals. The hyperactivity appears after 3 h of incubation and persists until at least seven hours. Data are presented as average ± SEM from 32 to 40 larvae per condition from four independent experiments. Comparisons were performed using two-way ANOVA, with Sidak’s post-test (statistical significance is compared with controls * p < 0.05, ** p < 0.01, *** p < 0.001). Full article ">Figure 5
HGC granule secretion is inhibited by FON but rescued by a D2 dopamine receptor antagonist. The graph shows quantification of HGCs with granular content in 44 and 70 hpf larvae under the treatments indicated (E3, control medium; FON, FON high (36 mg/L); incubation was carried out with FON alone, the D2 receptor antagonist Spiperone (Spi), and the D1 receptor antagonist SCH23390 (SCH), or combinations of FON and the antagonists. Granule secretion (naturally occurring arround 48 hpf) is inhibited in fish treated with FON. Coincubation with Spi restores secretion to values indistinguishable from those observed in control conditions; in contrast, SCH coincubation has no significant difference with FON incubation alone. Kruskal-Wallis, Dunn’s multiple comparisons test (statistical significance is compared with respective controls *** p < 0.001; ns, not significant). Full article ">Figure 6
Triadimefon induces the expression of prolactin after 3 h of treatment in 42 hpf embryos. Pfaffl calculated fold-change in embryos incubated with FON at 26 and 16 mg/L relative to non-treated animals. FON 16 mg/L affects prolactin (prl) expression (A) but does not induce a significant change in tyrosine hydroxylase (th1) mRNA levels (B). Unpaired t-test with Welch’s correction was performed for statistical analysis. * p < 0.05; ns, not significant. Full article ">

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Open AccessArticle

Methylmercury Induced Neurotoxicity and the Influence of Selenium in the Brains of Adult Zebrafish (Danio rerio)

by Josef Daniel Rasinger, Anne-Katrine Lundebye, Samuel James Penglase, Ståle Ellingsen and Heidi Amlund

Int. J. Mol. Sci. 2017, 18(4), 725; https://doi.org/10.3390/ijms18040725 - 29 Mar 2017

Cited by 36 | Viewed by 7464

Abstract

The neurotoxicity of methylmercury (MeHg) is well characterised, and the ameliorating effects of selenium have been described. However, little is known about the molecular mechanisms behind this contaminant-nutrient interaction. We investigated the influence of selenium (as selenomethionine, SeMet) and MeHg on mercury accumulation [...] Read more.

The neurotoxicity of methylmercury (MeHg) is well characterised, and the ameliorating effects of selenium have been described. However, little is known about the molecular mechanisms behind this contaminant-nutrient interaction. We investigated the influence of selenium (as selenomethionine, SeMet) and MeHg on mercury accumulation and protein expression in the brain of adult zebrafish (Danio rerio). Fish were fed diets containing elevated levels of MeHg and/or SeMet in a 2 × 2 full factorial design for eight weeks. Mercury concentrations were highest in the brain tissue of MeHg-exposed fish compared to the controls, whereas lower levels of mercury were found in the brain of zebrafish fed both MeHg and SeMet compared with the fish fed MeHg alone. The expression levels of proteins associated with gap junction signalling, oxidative phosphorylation, and mitochondrial dysfunction were significantly (p < 0.05) altered in the brain of zebrafish after exposure to MeHg and SeMet alone or in combination. Analysis of upstream regulators indicated that these changes were linked to the mammalian target of rapamycin (mTOR) pathways, which were activated by MeHg and inhibited by SeMet, possibly through a reactive oxygen species mediated differential activation of RICTOR, the rapamycin-insensitive binding partner of mTOR. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Figure 1
Heatmaps of significantly regulated (p < 0.05, 2-way ANOVA) proteins in the brain of zebrafish (Danio rerio). Zebrafish were fed control, methylmercury (10 µg Hg/g; MeHg), selenomethionine (5 µg Se/g; SeMet), or MeHg and SeMet (10 µg Hg/g + 5 µg Se/g) supplemented diets. After eight weeks of exposure, the brains (n = 12) were sampled and subjected to quantitative intact proteomics analysis. Differential analysis (2-way ANOVA) and hierarchical clustering (Pearson correlation) were performed using the Qlucore omics-explorer. (A,B) depict significantly differentially expressed proteins after exposure to MeHg and SeMet, respectively (p < 0.05, 2-way ANOVA); (C) Depicts the proteins displaying significant MeHg-SeMet interaction effects. The numbers in plots describe the unique difference in gel protein spot identifiers. Protein spots successfully identified by LC-MS/MS are denoted with an asterisk (*). See <a href="#app1-ijms-18-00725" class="html-app">Table S1</a> for further details on individual proteins. Full article ">Figure 2
Preparative two-dimensional (2D) gel showing the picked and successfully identified significantly regulated (p < 0.05, 2-way ANOVA) proteins in brain of zebrafish (Danio rerio) after exposure to methylmercury (10 µg Hg/g; MeHg), selenomethionine (5 µg Se/g; SeMet) or MeHg and SeMet (10 µg Hg/g + 5 µg Se/g) supplemented diets. The numbers in the plot describe the unique difference in gel protein spot identifiers. The location of the spots relative to the x axis of the plot reflects the approximate isoelectric points of the protein spots. See <a href="#app1-ijms-18-00725" class="html-app">Table S1</a> for further details on individual proteins. Full article ">Figure 3
Heatmaps of significantly regulated (p < 0.05, 2-way ANOVA) proteins in the brain of zebrafish (Danio rerio). Significant protein responses elicited by: MeHg (A), SeMet (B) and MeHg-SeMet (C) interactions. The number in brackets following the Ingenuity Pathway Analysis (IPA) mapped identifier represent the gel master spot number provided in <a href="#ijms-18-00725-f002" class="html-fig">Figure 2</a> and <a href="#app1-ijms-18-00725" class="html-app">Table S1</a>, respectively. Individual proteins found to be regulated in response to (A,B) are labeled with a plus sign (+). Overlapping responses in (A,C) or (B,C) are marked with an asterisk (*) and a hash (#) sign, respectively. Yellow and blue boxes indicate increased and decreased expression levels relative to the treatment groups; the respective log2 fold changes are listed in <a href="#app1-ijms-18-00725" class="html-app">Table S1</a>. Full article ">Figure 4
Canonical pathway analysis. Significantly regulated (p < 0.05, 2-way ANOVA) proteins in the brain of zebrafish (Danio rerio) after exposure to: MeHg (A), Se (B) and MeHg-SeMet (C). Interactions were subjected to IPA. Statistical significance of the overrepresentation of proteins in different “canonical pathways” is shown as a heatmap. Only selected pathways that were significantly (p < 0.05, Fisher’s exact test) enriched in at least one of the exposure conditions are shown alongside their significance values expressed as scores (−log10 p-value). Scores above the cut-off (1,3) are displayed by a color gradient. Scores below the cut-off value are displayed as white boxes. The full data-set including the proteins in each pathway is presented in <a href="#app1-ijms-18-00725" class="html-app">Table S2B</a>. Full article ">Figure 5
Ingenuity Pathway Analysis (IPA) of “upstream regulators” and “diseases and biological functions” of the lists of significantly regulated proteins (p < 0.05, 2-way ANOVA). RICTOR, the rapamycin-insensitive binding partner of the mammalian target of rapamycin (mTOR) was highlighted as a novel master upstream regulator that was predicted to be activated and inhibited in MeHg and SeMet exposed zebrafish brain, respectively. Diseases and biological functions analysis highlighted “cell death” and “necrosis” to be activated by MeHg and inhibited in SeMet exposed zebrafish brain. Activation (displayed in yellow) and inhibition (displayed in blue) of regulators and functions are based on IPA activation z-scores, which combine directional information encoded in the protein expression results with knowledge from the literature to make predictions about likely adverse outcome pathways. Up-regulated proteins are coloured red, down-regulated proteins are coloured green. The full data-sets of the upstream regulator analysis and the diseases and biological functions are presented in <a href="#app1-ijms-18-00725" class="html-app">Table S2C,D</a>. Full article ">

909 KiB

Open AccessArticle

Comparison of the In Vivo Biotransformation of Two Emerging Estrogenic Contaminants, BP2 and BPS, in Zebrafish Embryos and Adults

by Vincent Le Fol, François Brion, Anne Hillenweck, Elisabeth Perdu, Sandrine Bruel, Selim Aït-Aïssa, Jean-Pierre Cravedi and Daniel Zalko

Int. J. Mol. Sci. 2017, 18(4), 704; https://doi.org/10.3390/ijms18040704 - 25 Mar 2017

Cited by 31 | Viewed by 5620

Abstract

Zebrafish embryo assays are increasingly used in the toxicological assessment of endocrine disruptors. Among other advantages, these models are 3R-compliant and are fit for screening purposes. Biotransformation processes are well-recognized as a critical factor influencing toxic response, but major gaps of knowledge exist [...] Read more.

Zebrafish embryo assays are increasingly used in the toxicological assessment of endocrine disruptors. Among other advantages, these models are 3R-compliant and are fit for screening purposes. Biotransformation processes are well-recognized as a critical factor influencing toxic response, but major gaps of knowledge exist regarding the characterization of functional metabolic capacities expressed in zebrafish. Comparative metabolic studies between embryos and adults are even scarcer. Using ³H-labeled chemicals, we examined the fate of two estrogenic emerging contaminants, benzophenone-2 (BP2) and bisphenol S (BPS), in 4-day embryos and adult zebrafish. BPS and BP2 were exclusively metabolized through phase II pathways, with no major qualitative difference between larvae and adults except the occurrence of a BP2-di-glucuronide in adults. Quantitatively, the biotransformation of both molecules was more extensive in adults. For BPS, glucuronidation was the predominant pathway in adults and larvae. For BP2, glucuronidation was the major pathway in larvae, but sulfation predominated in adults, with ca. 40% conversion of parent BP2 and an extensive release of several conjugates into water. Further larvae/adults quantitative differences were demonstrated for both molecules, with higher residue concentrations measured in larvae. The study contributes novel data regarding the metabolism of BPS and BP2 in a fish model and shows that phase II conjugation pathways are already functional in 4-dpf-old zebrafish. Comparative analysis of BP2 and BPS metabolic profiles in zebrafish larvae and adults further supports the use of zebrafish embryo as a relevant model in which toxicity and estrogenic activity can be assessed, while taking into account the absorption and fate of tested substances. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Open AccessArticle

Zebrafish Embryo as an In Vivo Model for Behavioral and Pharmacological Characterization of Methylxanthine Drugs

by Ram Manohar Basnet, Michela Guarienti and Maurizio Memo

Int. J. Mol. Sci. 2017, 18(3), 596; https://doi.org/10.3390/ijms18030596 - 9 Mar 2017

Cited by 35 | Viewed by 9224

Abstract

Zebrafish embryo is emerging as an important tool for behavior analysis as well as toxicity testing. In this study, we compared the effect of nine different methylxanthine drugs using zebrafish embryo as a model. We performed behavioral analysis, biochemical assay and Fish Embryo [...] Read more.

Zebrafish embryo is emerging as an important tool for behavior analysis as well as toxicity testing. In this study, we compared the effect of nine different methylxanthine drugs using zebrafish embryo as a model. We performed behavioral analysis, biochemical assay and Fish Embryo Toxicity (FET) test in zebrafish embryos after treatment with methylxanthines. Each drug appeared to behave in different ways and showed a distinct pattern of results. Embryos treated with seven out of nine methylxanthines exhibited epileptic-like pattern of movements, the severity of which varied with drugs and doses used. Cyclic AMP measurement showed that, despite of a significant increase in cAMP with some compounds, it was unrelated to the observed movement behavior changes. FET test showed a different pattern of toxicity with different methylxanthines. Each drug could be distinguished from the other based on its effect on mortality, morphological defects and teratogenic effects. In addition, there was a strong positive correlation between the toxic doses (TC₅₀) calculated in zebrafish embryos and lethal doses (LD₅₀) in rodents obtained from TOXNET database. Taken together, all these findings elucidate the potentiality of zebrafish embryos as an in vivo model for behavioral and toxicity testing of methylxanthines and other related compounds. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Spontaneous movement quantification at 24 hpf. The following doses of methylxanthine were used: aminophylline 500 mg/L, caffeine 150 mg/L, diprophylline 5000 mg/L, doxofylline 1000 mg/L, etofylline 600 mg/L, IBMX 50 mg/L, pentoxifylline 200 mg/L, theobromine 200 mg/L, and theophylline 200 mg/L. Negative controls were treated with dilution water without any drugs. Data are the mean ± S.D. of three independent experiments. Asterisks indicate statistically significant increase of spontaneous movements compared to negative controls. Significance was determined using ordinary one-way ANOVA, followed by Dunnett’s multiple comparisons test. * p < 0.05; ** p < 0.005; *** p < 0.0001. White columns: methylxanthine treated embryos; grey column: negative controls. Full article ">Figure 2
Touch-and-response test performed at 72 hpf. X-axis shows the distance swam by the embryos, Y-axis shows the percentage of embryos that swam different distances. The following doses of methylxanthine were used: aminophylline 500 mg/L, caffeine 150 mg/L, diprophylline 5000 mg/L, doxofylline 1000 mg/L, etofylline 600 mg/L, IBMX 50 mg/L, pentoxifylline 200 mg/L, theobromine 200 mg/L, and theophylline 200 mg/L. Negative controls were treated with dilution water without any drugs. Data are the mean ± S.D. of three independent experiments. White columns: methylxanthine treated embryos; grey columns: negative controls. Full article ">Figure 3
Cyclic AMP level in whole zebrafish embryos extract. The following doses of methylxanthine were used: aminophylline 500 mg/L, caffeine 150 mg/L, diprophylline 5000 mg/L, doxofylline 1000 mg/L, etofylline 600 mg/L, IBMX 50 mg/L, pentoxifylline 200 mg/L, theobromine 200 mg/L, and theophylline 200 mg/L. Negative controls were treated with dilution water without any drugs. Data are the mean ± S.D. of three independent experiments. Asterisks indicate statistically significant increase of cAMP compared to negative controls. Significance was determined using ordinary one-way ANOVA, followed by Dunnett’s multiple comparisons test. * p < 0.05; ** p < 0.005; *** p < 0.0001. White columns: methylxanthine treated embryos; grey column: negative controls. Full article ">Figure 4
Effect of nine methylxanthine compounds on zebrafish embryos development. X-axis shows the five increasing concentrations of each methylxanthine drug used in the FET test. Data are the mean ± S.D. of three independent experiments. (GMS) General Morphological defects Score; (GTS) General Teratogenicity Score. White circles: mortality rate; white squares: GMS; white triangles: GTS. Full article ">Figure 5
Correlation between TC50 (Toxic Concentration, 50%) of zebrafish (X-axis) and LD50 (Lethal Dose, 50%) of mice (Y-axis). The analysis included seven of the nine tested methylxanthines: diprophylline was excluded because the value was too large and pentoxifylline because it was an outlier. Numbers linked to each dot correspond to a methylxanthine compound, as reported in <a href="#ijms-18-00596-t002" class="html-table">Table 2</a>. Full article ">Figure 6
Correlation between TC50 (Toxic Concentration, 50%) of zebrafish (X-axis) and LD50 (Lethal Dose, 50%) of rat (Y-axis). The analysis included six of the nine tested methylxanthines: diprophylline was excluded because the value was too large and pentoxifylline because it was an outlier; no data were available about IBMX toxicity in rat. Numbers linked to each dot correspond to a methylxanthine compound, as reported in <a href="#ijms-18-00596-t002" class="html-table">Table 2</a>. Full article ">

620 KiB

Open AccessCommunication

Changes in Brain Monoamines Underlie Behavioural Disruptions after Zebrafish Diet Exposure to Polycyclic Aromatic Hydrocarbons Environmental Mixtures

by Caroline Vignet, Verena M. Trenkel, Annick Vouillarmet, Giampiero Bricca, Marie-Laure Bégout and Xavier Cousin

Int. J. Mol. Sci. 2017, 18(3), 560; https://doi.org/10.3390/ijms18030560 - 4 Mar 2017

Cited by 27 | Viewed by 5344

Abstract

Zebrafish were exposed through diet to two environmentally relevant polycyclic aromatic hydrocarbons (PAHs) mixtures of contrasted compositions, one of pyrolytic (PY) origin and one from light crude oil (LO). Monoamine concentrations were quantified in the brains of the fish after six month of [...] Read more.

Zebrafish were exposed through diet to two environmentally relevant polycyclic aromatic hydrocarbons (PAHs) mixtures of contrasted compositions, one of pyrolytic (PY) origin and one from light crude oil (LO). Monoamine concentrations were quantified in the brains of the fish after six month of exposure. A significant decrease in noradrenaline (NA) was observed in fish exposed to both mixtures, while a decrease in serotonin (5HT) and dopamine (DA) was observed only in LO-exposed fish. A decrease in metabolites of 5HT and DA was observed in fish exposed to both mixtures. Several behavioural disruptions were observed that depended on mixtures, and parallels were made with changes in monoamine concentrations. Indeed, we observed an increase in anxiety in fish exposed to both mixtures, which could be related to the decrease in 5HT and/or NA, while disruptions of daily activity rhythms were observed in LO fish, which could be related to the decrease in DA. Taken together, these results showed that (i) chronic exposures to PAHs mixtures disrupted brain monoamine contents, which could underlie behavioural disruptions, and that (ii) the biological responses depended on mixture compositions. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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3436 KiB

Open AccessArticle

Evaluating Complex Mixtures in the Zebrafish Embryo by Reconstituting Field Water Samples: A Metal Pollution Case Study

by Ellen D. G. Michiels, Lucia Vergauwen, An Hagenaars, Erik Fransen, Stefan Van Dongen, Steven J. Van Cruchten, Lieven Bervoets and Dries Knapen

Int. J. Mol. Sci. 2017, 18(3), 539; https://doi.org/10.3390/ijms18030539 - 2 Mar 2017

Cited by 15 | Viewed by 5444

Abstract

Accurately assessing the toxicity of complex, environmentally relevant mixtures remains an important challenge in ecotoxicology. The goal was to identify biological effects after exposure to environmental water samples and to determine whether the observed effects could be explained by the waterborne metal mixture [...] Read more.

Accurately assessing the toxicity of complex, environmentally relevant mixtures remains an important challenge in ecotoxicology. The goal was to identify biological effects after exposure to environmental water samples and to determine whether the observed effects could be explained by the waterborne metal mixture found in the samples. Zebrafish embryos were exposed to water samples of five different sites originating from two Flemish (Mol and Olen, Belgium) metal contaminated streams: “Scheppelijke Nete” (SN) and “Kneutersloop” (K), and a ditch (D), which is the contamination source of SN. Trace metal concentrations, and Na, K, Mg and Ca concentrations were measured using ICP-MS and were used to reconstitute site-specific water samples. We assessed whether the effects that were observed after exposure to environmental samples could be explained by metal mixture toxicity under standardized laboratory conditions. Exposure to “D” or “reconstituted D” water caused 100% mortality. SN and reconstituted SN water caused similar effects on hatching, swim bladder inflation, growth and swimming activity. A canonical discriminant analysis confirmed a high similarity between both exposure scenarios, indicating that the observed toxicity was indeed primarily caused by metals. The applied workflow could be a valuable approach to evaluate mixture toxicity that limits time and costs while maintaining biological relevance. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Schematic overview of the workflow of this study analogous to the concepts of a Toxicity Identification Evaluation (TIE). The numbered boxes represent the steps that were conducted in this study. Identification of predominant pollutant class(es) corresponds to phase 1 of a TIE and is necessary in cases where no background data are available (e.g., as illustrated by Burgess et al. [<a href="#B18-ijms-18-00539" class="html-bibr">18</a>]). The dashed arrow represents step 6 of the workflow, i.e., comparing the effects between field and reconstituted exposure scenarios. Full article ">Figure 2
Survival curves after exposure to (A) field water samples, (B) reconstituted metal mixtures. Hatching curves after exposure to (C) field water samples and (D) reconstituted metal mixtures. Different small letters in the legend indicate significant differences within each graph. “Control” indicates rearing in standard embryo medium. In (A,C) separate controls (“control D” and “control K”), with pH and conductivity adjusted to those of the field water samples, were included. The pH and conductivity of the Scheppelijke Nete (SN) resembled standard embryo medium; therefore, a separate control was not needed in this case. In (B,D), “control D”, “control SN1” and “control SN2” media were reconstituted based on the actual baseline ionic composition (Na, K, Ca and Mg) measured in the field water samples. Full article ">Figure 3
Sub-lethal effects after exposure to field water samples or to reconstituted metal mixtures. Length of the larvae (A) exposed to field water samples or (B) to reconstituted metal mixtures. Percentages of swim bladder inflation of all hatched larvae at 120 hpf (C) after exposure to field water samples, and (D) after exposure to reconstituted mixtures. Representative photograph: the upper larva has an inflated swim bladder and the bottom one has a non-inflated swim bladder. Average swimming distance of (E) larvae exposed to field water samples, and (F) to reconstituted metal mixtures. For an explanation of the controls, see <a href="#ijms-18-00539-f002" class="html-fig">Figure 2</a>. Different small letters indicate significant differences (p < 0.05) (n is given in parentheses). Full article ">Figure 4
Canonical discriminant analysis of the results of the experiment with field water and the reconstituted mixtures. Small symbols represent the sample scores, and large symbols represent the average scores per experimental group. Filled symbols represent samples of the field experiment and open symbols represent samples of the reconstituted experiment. Control field and control lab represent standard embryo medium. Length, swimming distance and speed are positive effects (i.e., higher values are better), while hatching and swim bladder represent impaired hatching and impaired swim bladder inflation, which are adverse effects. Full article ">Figure 5
Map of the sampling sites. The Scheppelijke Nete is indicated as SN, the ditch as D and the Kneutersloop as K. The arrows indicate the direction of the flow. Full article ">

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Open AccessArticle

Effect of Photon Hormesis on Dose Responses to Alpha Particles in Zebrafish Embryos

by Candy Yuen Ping Ng, Shuk Han Cheng and Kwan Ngok Yu

Int. J. Mol. Sci. 2017, 18(2), 385; https://doi.org/10.3390/ijms18020385 - 11 Feb 2017

Cited by 4 | Viewed by 4932

Abstract

Photon hormesis refers to the phenomenon where the biological effect of ionizing radiation with a high linear energy transfer (LET) value is diminished by photons with a low LET value. The present paper studied the effect of photon hormesis from X-rays on dose [...] Read more.

Photon hormesis refers to the phenomenon where the biological effect of ionizing radiation with a high linear energy transfer (LET) value is diminished by photons with a low LET value. The present paper studied the effect of photon hormesis from X-rays on dose responses to alpha particles using embryos of the zebrafish (Danio rerio) as the in vivo vertebrate model. The toxicity of these ionizing radiations in the zebrafish embryos was assessed using the apoptotic counts at 20, 24, or 30 h post fertilization (hpf) revealed through acridine orange (AO) staining. For alpha-particle doses ≥ 4.4 mGy, the additional X-ray dose of 10 mGy significantly reduced the number of apoptotic cells at 24 hpf, which proved the presence of photon hormesis. Smaller alpha-particle doses might not have inflicted sufficient aggregate damages to trigger photon hormesis. The time gap T between the X-ray (10 mGy) and alpha-particle (4.4 mGy) exposures was also studied. Photon hormesis was present when T ≤ 30 min, but was absent when T = 60 min, at which time repair of damage induced by alpha particles would have completed to prevent their interactions with those induced by X-rays. Finally, the drop in the apoptotic counts at 24 hpf due to photon hormesis was explained by bringing the apoptotic events earlier to 20 hpf, which strongly supported the removal of aberrant cells through apoptosis as an underlying mechanism for photon hormesis. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Figure 1
Net normalized apoptotic counts: (i) NAY+ and NAc+, (ii) NAXY+ and NAXc+ on 24-hpf zebrafish embryos which have been irradiated with (i) alpha-particle dose only, and (ii) alpha-particle dose + 10 mGy X-ray at 5 hpf. Error bars represent the standard errors. The lines joining the data points are drawn to guide the eye only. Comparisons are separately made for the five data points within case (i) or case (ii) through ANOVA. When p ≤ 0.05 from ANOVA, post-hoc t-tests are further performed to assess the difference between tested AY or AXY groups and the corresponding AC or AXC group. Significant differences from post-hoc t-tests are asterisked in <a href="#ijms-18-00385-f001" class="html-fig">Figure 1</a>. * Statistically significant differences between the tested AY or AXY group and the corresponding AC or AXC group. Full article ">Figure 2
Differences between the net normalized apoptotic counts (NAXH(T)+ − NAH(T)+) on zebrafish embryos at 20 hpf (diagonally filled), 24 hpf (filled with diamond), and 30 hpf (filled with dots), which had been irradiated with an alpha-particle dose of 4.4 mGy at 5 hpf and an X-ray dose of 10 mGy, with T = 0, 10, 15, 30, or 60 (min) between the alpha-particle and X-ray irradiations. Error bars represent the standard errors. The error bars represent the standard errors. Full article ">Figure 3
Schematic diagram showing the experimental steps involving embryos in the A1, A2, A4, A8, and AC groups, and AX1, AX2, AX4, AX8, and AXC groups. Full article ">Figure 4
Schematic diagram showing the experimental steps involving embryos in the AX24(T), A24(T), and C24(T) groups, where time gaps T = 0, 10, 15, 30, or 60 (min) between alpha-particle and photon irradiations. Full article ">

1080 KiB

Open AccessArticle

Transcriptional and Behavioral Responses of Zebrafish Larvae to Microcystin-LR Exposure

by Eleni Tzima, Iliana Serifi, Ioanna Tsikari, Ainhoa Alzualde, Ioannis Leonardos and Thomais Papamarcaki

Int. J. Mol. Sci. 2017, 18(2), 365; https://doi.org/10.3390/ijms18020365 - 9 Feb 2017

Cited by 19 | Viewed by 6736

Abstract

Microcystins are cyclic heptapeptides that constitute a diverse group of toxins produced by cyanobacteria. One of the most toxic variants of this family is microcystin-LR (MCLR) which is a potent inhibitor of protein phosphatase 2A (PP2A) and induces cytoskeleton alterations. In this study, [...] Read more.

Microcystins are cyclic heptapeptides that constitute a diverse group of toxins produced by cyanobacteria. One of the most toxic variants of this family is microcystin-LR (MCLR) which is a potent inhibitor of protein phosphatase 2A (PP2A) and induces cytoskeleton alterations. In this study, zebrafish larvae exposed to 500 μg/L of MCLR for four days exhibited a 40% reduction of PP2A activity compared to the controls, indicating early effects of the toxin. Gene expression profiling of the MCLR-exposed larvae using microarray analysis revealed that keratin 96 (krt96) was the most downregulated gene, consistent with the well-documented effects of MCLR on cytoskeleton structure. In addition, our analysis revealed upregulation in all genes encoding for the enzymes of the retinal visual cycle, including rpe65a (retinal pigment epithelium-specific protein 65a), which is critical for the larval vision. Quantitative real-time PCR (qPCR) analysis confirmed the microarray data, showing that rpe65a was significantly upregulated at 50 μg/L and 500 μg/L MCLR in a dose-dependent manner. Consistent with the microarray data, MCLR-treated larvae displayed behavioral alterations such as weakening response to the sudden darkness and hypoactivity in the dark. Our work reveals new molecular targets for MCLR and provides further insights into the molecular mechanisms of MCLR toxicity during early development. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Figure 1

Figure 1
Effects of microcystin-LR (MCLR) exposure on protein phosphatase 2A (PP2A) activity. (A) PP2A activity was measured in control and larvae exposed to 500 μg/L of MCLR, as described in the Materials and Methods section. Protein phosphatase 2A activity was expressed as nmol of free PO4−3 released per min using increasing amounts of total protein (μg). The data are indicated as mean ± SE obtained from three independent experiments (p < 0.05); (B) Relative PP2A specific activity of MCLR group vs. control. Control was set to 100%. Full article ">Figure 2
Gene expression profiling of MCLR exposed larvae (A) microarray analysis. Differentially expressed genes in zebrafish larvae treated with 500 μg/L of MCLR involved in the retinoid visual cycle, p < 0.05. (B) The retinoid visual cycle. Briefly, absorption of light by visual pigments causes isomerization of 11-cis-retinal to all-trans-retinal, resulting in phototransduction. All-trans-retinal is reduced to all-trans-retinol and transported into the retinal pigment epithelium (RPE). LRAT esterifies all-trans-retinol to all-trans-retinyl esters, which are further processed to 11-cis-retinol by RPE65. RLBP1 removes 11-cis-retinol from the reaction site to speed the isomerization. RDH5 converts 11-cis-retinol to 11-cis-retinal. STRA6 receptor mediates all-trans-retinol uptake through the plasma membrane. Red check marks indicate the visual cycle-related genes detected in MCLR-treated larvae by microarray analysis. (C) Validation of microarray data by quantitative real-time PCR (qPCR) analysis. Relative mRNA expression of rpe65a, rlbp1a, rlbp1b, rgrb, lrata, stra6, and inpp5kb (inositol polyphosphate-5-phosphatase Kb) at a four-day exposure of larvae to 50 μg/L and 500 μg/L of MCLR. Three biological replicates were run in triplicate. The mRNA expression levels are expressed in relation to the control which is set to 1. * p < 0.05 and ** p < 0.01. RBP: Retinol binding protein. Full article ">Figure 3
Behavioral patterns of MCLR-treated zebrafish larvae. The locomotor activity displayed by controls (0.05% ethanol) and treated larvae with 100 µg/L and 500 µg/L of MCLR is represented by the total distance covered in 2 min time bins. Tracking was performed under variable lighting conditions. Light phases correspond to 0–10 min and 20–30 min and dark phases to 10–20 min and 30–40 min time periods. * p < 0.05, ** p < 0.01, and *** p < 0.001 comparing each treatment to the control group. Full article ">

5560 KiB

Open AccessArticle

Cell Imaging Counting as a Novel Ex Vivo Approach for Investigating Drug-Induced Hepatotoxicity in Zebrafish Larvae

by Xuan-Bac Nguyen, Stanislav Kislyuk, Duc-Hung Pham, Angela Kecskés, Jan Maes, Deirdre Cabooter, Pieter Annaert, Peter De Witte and Annelii Ny

Int. J. Mol. Sci. 2017, 18(2), 356; https://doi.org/10.3390/ijms18020356 - 8 Feb 2017

Cited by 12 | Viewed by 6442

Abstract

Drug-induced liver injury (DILI) is the most common reason for failures during the drug development process and for safety-related withdrawal of drugs from the pharmaceutical market. Therefore, having tools and techniques that can detect hepatotoxic properties in drug candidates at an early discovery [...] Read more.

Drug-induced liver injury (DILI) is the most common reason for failures during the drug development process and for safety-related withdrawal of drugs from the pharmaceutical market. Therefore, having tools and techniques that can detect hepatotoxic properties in drug candidates at an early discovery stage is highly desirable. In this study, cell imaging counting was used to measure in a fast, straightforward, and unbiased way the effect of paracetamol and tetracycline, (compounds known to cause hepatotoxicity in humans) on the amount of DsRed-labeled hepatocytes recovered by protease digestion from Tg(fabp10a:DsRed) transgenic zebrafish. The outcome was in general comparable with the results obtained using two reference methods, i.e., visual analysis of liver morphology by fluorescence microscopy and size analysis of fluorescent 2D liver images. In addition, our study shows that administering compounds into the yolk is relevant in the framework of hepatotoxicity testing. Taken together, cell imaging counting provides a novel and rapid tool for screening hepatotoxicants in early stages of drug development. This method is also suitable for testing of other organ-related toxicities subject to the organs and tissues expressing fluorescent proteins in transgenic zebrafish lines. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Lethality in Tg(fabp10a:DsRed) zebrafish larvae after immersion (a,b); yolk injection (c,d) and pericardial injection (e,f) of paracetamol (a,c,e) and tetracycline (b,d,f). Analysis was performed after three days of treatment (immersion) or three days after treatment (injection), at 6 days post-fertilization (dpf). A total of 10 and 12 larvae were used individually per condition for immersion and injections, respectively. The experiment was performed in triplicate and data were pooled. The results are expressed as percentage of dead larvae relative to the total larvae (mean ± SD). PAR: paracetamol; TET: tetracycline. Full article ">Figure 2
Visual analysis of liver morphology using Tg(fabp10a:DsRed) zebrafish larvae by fluorescence microscopy (original 40× magnification). Larvae with normal liver morphology (a); and abnormal liver morphology (b–d). The liver area is identified as the yellow area. Full article ">Figure 3
Hepatotoxicity assessed by visual analysis of liver morphology (by researchers blinded for the treatment conditions) using fluorescence microscopy after immersion (a,b); yolk injection (c,d) and pericardial injection (e,f) of paracetamol (a,c,e) and tetracycline (b,d,f). Analysis was performed after three days of treatment (immersion) or three days after treatment (injection), at 6 dpf. A total of 10 and 12 larvae were used individually per condition for immersion and injections, respectively. The experiment was performed in triplicate and data were pooled. The results are expressed as the percentage of larvae having normal or abnormal liver morphology. The data were analyzed by Fisher’s exact test. ns: no statistically significant difference; *: p < 0.05; **: p < 0.01: ***: p < 0.001; VHC: vehicle control; PAR: paracetamol; TET: tetracycline. Full article ">Figure 4
Hepatotoxicity assessed by size analysis of fluorescent 2D liver images after immersion (a,b); yolk injection (c,d) and pericardial injection (e,f) of paracetamol (a,c,e) and tetracycline (b,d,f). Analysis was performed after three days of treatment (immersion) or three days after treatment (injection), at 6 dpf. A total of 10 and 12 larvae were used individually per condition for immersion and injections, respectively. The experiment was performed in triplicate and data were pooled. The data were analyzed using one-way ANOVA. ns: no statistically significant difference; *: p < 0.05, ***: p < 0.001; VHC: vehicle control; PAR: paracetamol; TET: tetracycline. Means ± SD are shown. Full article ">Figure 5
DsRed-labeled hepatocytes captured by cell imaging counting (CIC). Yellow dots represent single hepatocytes. Full article ">Figure 6
Hepatotoxicity assessed by quantification of DsRed-labeled hepatocytes using a CIC after immersion (a,b); yolk injection (c,d) and pericardial injection (e,f) of paracetamol (a,c,e) and tetracycline (b,d,f). Analysis was performed after three days of treatment (immersion) or three days after treatment (injection), at 6 dpf. A total of 10 and 12 larvae were used individually per condition for immersion and injections, respectively. The experiment was performed in triplicate and data were pooled. The data were analyzed using one-way ANOVA. ns: no statistically significant difference; *: p < 0.05; ***: p < 0.001; VHC: vehicle control; PAR: paracetamol; TET: tetracycline. Means ± SD of the number of hepatocytes per larva are shown. Full article ">Figure 7
Pharmacokinetics after immersion, yolk and pericardial injection of paracetamol (a) and tetracycline (b). The compounds in larval extracts were quantified by ultra-high performance liquid chromatography with ultraviolet detection (UHPLC-UV) as a function of time (0–24 h post treatment). n = 3, *: consists of two measurements. Means ± SD are shown. Full article ">

1163 KiB

Open AccessArticle

A Mixture of Persistent Organic Pollutants and Perfluorooctanesulfonic Acid Induces Similar Behavioural Responses, but Different Gene Expression Profiles in Zebrafish Larvae

by Abdolrahman Khezri, Thomas W. K. Fraser, Rasoul Nourizadeh-Lillabadi, Jorke H. Kamstra, Vidar Berg, Karin E. Zimmer and Erik Ropstad

Int. J. Mol. Sci. 2017, 18(2), 291; https://doi.org/10.3390/ijms18020291 - 29 Jan 2017

Cited by 42 | Viewed by 6104

Abstract

Persistent organic pollutants (POPs) are widespread in the environment and some may be neurotoxic. As we are exposed to complex mixtures of POPs, we aimed to investigate how a POP mixture based on Scandinavian human blood data affects behaviour and neurodevelopment during early [...] Read more.

Persistent organic pollutants (POPs) are widespread in the environment and some may be neurotoxic. As we are exposed to complex mixtures of POPs, we aimed to investigate how a POP mixture based on Scandinavian human blood data affects behaviour and neurodevelopment during early life in zebrafish. Embryos/larvae were exposed to a series of sub-lethal doses and behaviour was examined at 96 h post fertilization (hpf). In order to determine the sensitivity window to the POP mixture, exposure models of 6 to 48 and 48 to 96 hpf were used. The expression of genes related to neurological development was also assessed. Results indicate that the POP mixture increases the swimming speed of larval zebrafish following exposure between 48 to 96 hpf. This behavioural effect was associated with the perfluorinated compounds, and more specifically with perfluorooctanesulfonic acid (PFOS). The expression of genes related to the stress response, GABAergic, dopaminergic, histaminergic, serotoninergic, cholinergic systems and neuronal maintenance, were altered. However, there was little overlap in those genes that were significantly altered by the POP mixture and PFOS. Our findings show that the POP mixture and PFOS can have a similar effect on behaviour, yet alter the expression of genes relevant to neurological development differently. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Figure 1

Figure 1
Swimming speed in zebrafish larvae exposed to a mixture of environmental pollutants, sub-mixtures and individual perfluorinated compounds. (A) swimming speed in zebrafish larvae upon exposure to five different concentrations of total persistent organic pollutant (POP) mixture; (B) swimming speed in zebrafish larvae upon exposure to sub-mixtures at the concentration equal to 100× higher than that found in human serum; (C) swimming speed after exposing the zebrafish to individual perfluorinated compounds (100× human serum level) compared to PF mixture; (D) PFOS and POPs sensitivity test (100× human serum level). (Pf) Perfluorinated mixture; (Br) Brominated mixture; (Cl) Chlorinated mixture; (Pf + Br) binary mixture of perfluorinated and brominated compounds; (Pf + Cl) binary mixture of perfluorinated and chlorinated compounds; (Br + Cl) binary mixture of brominated and chlorinated compounds. (+) contained; (−) not contained. Data are means ± SD. An asterisk identifies values that are significantly different from the solvent (0.05% DMSO) control (LME, * = p < 0.017, ** = p < 0.0017, *** = p < 0.0001). Full article ">Figure 2
Euclidean distance and ward clustering on log2 normalized expression values. The heat map shows the differences in expression of 21 genes related to neurodevelopmental processes between the solvent control (0.05% DMSO) and exposed samples in 96 hpf zebrafish. Cluster analysis was performed on log2 expression values of five biological replicates. Full article ">Figure 3
Transcription levels in genes relevant to behaviour following POP mixture and PFOS exposure. The line at zero indicates the gene expression in control groups (DMSO 0.05%). Data are presented as mean ± SD relative to control. An asterisk identifies genes expression levels that were significantly different from the solvent control (one-way ANOVA test, * = p < 0.05, ** = p < 0.005). Full article ">Figure 4
Swimming speed at 96 hpf in zebrafish larvae. Dechorionated and intact zebrafish embryos were exposed to perfluorooctanesulfonic acid (PFOS) (100× human serum level) between 24 to 48 hpf. Data presented as mean ± SD. Full article ">Figure 5
Swimming speed in 96 hpf zebrafish larvae after exposure to POP and PFOS at two different concentrations (10× and 70× higher than human serum level) between 48 to 96 hpf. In order to expose the zebrafish for gene expression analyses, concentrations were adjusted to 10× and 70× human serum level as the highest dose at which there was no significant effect on swimming speed (HNSS) or the lowest dose that consistently and significantly increased the swimming speed (LISS), respectively. Data presented as mean ± SD. An asterisk indicates a significant difference between the exposure group and the solvent control (LME, * = p < 0.017, ** = p < 0.0017, *** = p < 0.0001). Full article ">

915 KiB

Open AccessArticle

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

by Evy Verbueken, Derek Alsop, Moayad A. Saad, Casper Pype, Els M. Van Peer, Christophe R. Casteleyn, Chris J. Van Ginneken, Joanna Wilson and Steven J. Van Cruchten

Int. J. Mol. Sci. 2017, 18(1), 217; https://doi.org/10.3390/ijms18010217 - 22 Jan 2017

Cited by 20 | Viewed by 6329

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Resorufin formation (pmol/min/mg microsomal protein) by microsomes of zebrafish embryos (ZEM) at 72 and 96 h post-fertilization (hpf) and by liver microsomes from adult female zebrafish (ZLM) after incubation with benzyloxy-methyl-resorufin (BOMR). The dots are the reaction velocities for each batch. Each dot represents the mean value of three technical replicates. The mean reaction velocities for human liver microsomes (HLM) and CYP3A4 Baculosomes® (CYP3A4 BAC) were added to the graph as positive controls. The horizontal dotted line represents the lower limit of quantification (LLOQ). Significant differences (p < 0.05) between age groups are indicated by different letters (A and B). Full article ">Figure 2
The effect of various concentrations of ketoconazole and CYP3cide on the biotransformation of BOMR. The dots in the graphs represent the percentage ratios of reaction velocity in case of pre-incubation of the microsomes with the respective inhibitor, divided by the control velocity without inhibitor. Graphs (a–c) show the results for pre-incubation with ketoconazole with (a) demonstrating the mean of the results for Batch 1 and Batch 2 of ZLM; whereas (b,c) show the mean values of the technical replicates with human liver microsomes (HLM) and CYP3A4 Baculosomes® (CYP3A4 BAC), respectively; Graphs (d–f) show the outcome for pre-incubation with 0–4 µM of CYP3cide (data for 0–2 µM of CYP3cide not shown) with (d) representing the mean of the results for Batch 1 and Batch 2 of ZLM; while (e,f) demonstrate the mean values of the technical replicates with HLM and CYP3A4 BAC, respectively. In case of inhibition, the IC50 values and their 95% confidence intervals are added. Full article ">Figure 2 Cont.
The effect of various concentrations of ketoconazole and CYP3cide on the biotransformation of BOMR. The dots in the graphs represent the percentage ratios of reaction velocity in case of pre-incubation of the microsomes with the respective inhibitor, divided by the control velocity without inhibitor. Graphs (a–c) show the results for pre-incubation with ketoconazole with (a) demonstrating the mean of the results for Batch 1 and Batch 2 of ZLM; whereas (b,c) show the mean values of the technical replicates with human liver microsomes (HLM) and CYP3A4 Baculosomes® (CYP3A4 BAC), respectively; Graphs (d–f) show the outcome for pre-incubation with 0–4 µM of CYP3cide (data for 0–2 µM of CYP3cide not shown) with (d) representing the mean of the results for Batch 1 and Batch 2 of ZLM; while (e,f) demonstrate the mean values of the technical replicates with HLM and CYP3A4 BAC, respectively. In case of inhibition, the IC50 values and their 95% confidence intervals are added. Full article ">Figure 3
d-Luciferin formation (pmol/min/mg microsomal protein) by liver microsomes from adult female zebrafish. Each dot represents the mean reaction velocity (mean value of three technical replicates) for the corresponding batch of adult zebrafish liver microsomes. The lower horizontal dotted line demonstrates the lower limit of detection (LLOD) and the upper horizontal dash-dotted line represents the lower limit of quantification (LLOQ). Full article ">

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Open AccessArticle

Retinoic Acid Protects and Rescues the Development of Zebrafish Embryonic Retinal Photoreceptor Cells from Exposure to Paclobutrazol

by Wen-Der Wang, Hwei-Jan Hsu, Yi-Fang Li and Chang-Yi Wu

Int. J. Mol. Sci. 2017, 18(1), 130; https://doi.org/10.3390/ijms18010130 - 11 Jan 2017

Cited by 29 | Viewed by 7828

Abstract

Paclobutrazol (PBZ) is a widely used fungicide that shows toxicity to aquatic embryos, probably through rain-wash. Here, we specifically focus on its toxic effect on eye development in zebrafish, as well as the role of retinoic acid (RA), a metabolite of vitamin A [...] Read more.

Paclobutrazol (PBZ) is a widely used fungicide that shows toxicity to aquatic embryos, probably through rain-wash. Here, we specifically focus on its toxic effect on eye development in zebrafish, as well as the role of retinoic acid (RA), a metabolite of vitamin A that controls proliferation and differentiation of retinal photoreceptor cells, in this toxicity. Embryos were exposed to PBZ with or without RA from 2 to 72 h post-fertilization (hpf), and PBZ-treated embryos (2–72 hpf) were exposed to RA for additional hours until 120 hpf. Eye size and histology were examined. Expression levels of gnat1 (rod photoreceptor marker), gnat2 (cone photoreceptor marker), aldehyde dehydrogenases (encoding key enzymes for RA synthesis), and phospho-histone H3 (an M-phase marker) in the eyes of control and treated embryos were examined. PBZ exposure dramatically reduces photoreceptor proliferation, thus resulting in a thinning of the photoreceptor cell layer and leading to a small eye. Co-treatment of PBZ with RA, or post-treatment of PBZ-treated embryos with RA, partially rescues photoreceptor cells, revealed by expression levels of marker proteins and by retinal cell proliferation. PBZ has strong embryonic toxicity to retinal photoreceptors, probably via suppressing the production of RA, with effects including impaired retinal cell division. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Open AccessArticle

Neurotoxicity of a Biopesticide Analog on Zebrafish Larvae at Nanomolar Concentrations

by Ahmed Nasri, Audrey J. Valverde, Daniel B. Roche, Catherine Desrumaux, Philippe Clair, Hamouda Beyrem, Laurent Chaloin, Alain Ghysen and Véronique Perrier

Int. J. Mol. Sci. 2016, 17(12), 2137; https://doi.org/10.3390/ijms17122137 - 19 Dec 2016

Cited by 12 | Viewed by 6239

Abstract

Despite the ever-increasing role of pesticides in modern agriculture, their deleterious effects are still underexplored. Here we examine the effect of A6, a pesticide derived from the naturally-occurring α-terthienyl, and structurally related to the endocrine disrupting pesticides anilinopyrimidines, on living zebrafish larvae. We [...] Read more.

Despite the ever-increasing role of pesticides in modern agriculture, their deleterious effects are still underexplored. Here we examine the effect of A6, a pesticide derived from the naturally-occurring α-terthienyl, and structurally related to the endocrine disrupting pesticides anilinopyrimidines, on living zebrafish larvae. We show that both A6 and an anilinopyrimidine, cyprodinyl, decrease larval survival and affect central neurons at micromolar concentrations. Focusing on a superficial and easily observable sensory system, the lateral line system, we found that defects in axonal and sensory cell regeneration can be observed at much lower doses, in the nanomolar range. We also show that A6 accumulates preferentially in lateral line neurons and hair cells. We examined whether A6 affects the expression of putative target genes, and found that genes involved in apoptosis/cell proliferation are down-regulated, as well as genes reflecting estrogen receptor activation, consistent with previous reports that anilinopyrimidines act as endocrine disruptors. On the other hand, canonical targets of endocrine signaling are not affected, suggesting that the neurotoxic effect of A6 may be due to the binding of this compound to a recently identified, neuron-specific estrogen receptor. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Open AccessArticle

3D Visualization of Developmental Toxicity of 2,4,6-Trinitrotoluene in Zebrafish Embryogenesis Using Light-Sheet Microscopy

by Juneyong Eum, Jina Kwak, Hee Joung Kim, Seoyoung Ki, Kooyeon Lee, Ahmed A. Raslan, Ok Kyu Park, Md Ashraf Uddin Chowdhury, Song Her, Yun Kee, Seung-Hae Kwon and Byung Joon Hwang

Int. J. Mol. Sci. 2016, 17(11), 1925; https://doi.org/10.3390/ijms17111925 - 17 Nov 2016

Cited by 15 | Viewed by 7867

Abstract

Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in [...] Read more.

Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Open AccessArticle

Programmed Effects in Neurobehavior and Antioxidative Physiology in Zebrafish Embryonically Exposed to Cadmium: Observations and Hypothesized Adverse Outcome Pathway Framework

by Sander Ruiter, Josefine Sippel, Manon C. Bouwmeester, Tobias Lommelaars, Piet Beekhof, Hennie M. Hodemaekers, Frank Bakker, Evert-Jan Van den Brandhof, Jeroen L. A. Pennings and Leo T. M. Van der Ven

Int. J. Mol. Sci. 2016, 17(11), 1830; https://doi.org/10.3390/ijms17111830 - 2 Nov 2016

Cited by 25 | Viewed by 5540

Abstract

Non-communicable diseases (NCDs) are a major cause of premature mortality. Recent studies show that predispositions for NCDs may arise from early-life exposure to low concentrations of environmental contaminants. This developmental origins of health and disease (DOHaD) paradigm suggests that programming of an embryo [...] Read more.

Non-communicable diseases (NCDs) are a major cause of premature mortality. Recent studies show that predispositions for NCDs may arise from early-life exposure to low concentrations of environmental contaminants. This developmental origins of health and disease (DOHaD) paradigm suggests that programming of an embryo can be disrupted, changing the homeostatic set point of biological functions. Epigenetic alterations are a possible underlying mechanism. Here, we investigated the DOHaD paradigm by exposing zebrafish to subtoxic concentrations of the ubiquitous contaminant cadmium during embryogenesis, followed by growth under normal conditions. Prolonged behavioral responses to physical stress and altered antioxidative physiology were observed approximately ten weeks after termination of embryonal exposure, at concentrations that were 50–3200-fold below the direct embryotoxic concentration, and interpreted as altered developmental programming. Literature was explored for possible mechanistic pathways that link embryonic subtoxic cadmium to the observed apical phenotypes, more specifically, the probability of molecular mechanisms induced by cadmium exposure leading to altered DNA methylation and subsequently to the observed apical phenotypes. This was done using the adverse outcome pathway model framework, and assessing key event relationship plausibility by tailored Bradford-Hill analysis. Thus, cadmium interaction with thiols appeared to be the major contributor to late-life effects. Cadmium-thiol interactions may lead to depletion of the methyl donor S-adenosyl-methionine, resulting in methylome alterations, and may, additionally, result in oxidative stress, which may lead to DNA oxidation, and subsequently altered DNA methyltransferase activity. In this way, DNA methylation may be affected at a critical developmental stage, causing the observed apical phenotypes. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Open AccessArticle

Screening the Toxicity of Selected Personal Care Products Using Embryo Bioassays: 4-MBC, Propylparaben and Triclocarban

by Tiago Torres, Isabel Cunha, Rosário Martins and Miguel M. Santos

Int. J. Mol. Sci. 2016, 17(10), 1762; https://doi.org/10.3390/ijms17101762 - 21 Oct 2016

Cited by 53 | Viewed by 7359

Abstract

Recently, several emerging pollutants, including Personal Care Products (PCPs), have been detected in aquatic ecosystems, in the ng/L or µg/L range. Available toxicological data is limited, and, for certain PCPs, evidence indicates a potential risk for the environment. Hence, there is an urgent [...] Read more.

Recently, several emerging pollutants, including Personal Care Products (PCPs), have been detected in aquatic ecosystems, in the ng/L or µg/L range. Available toxicological data is limited, and, for certain PCPs, evidence indicates a potential risk for the environment. Hence, there is an urgent need to gather ecotoxicological data on PCPs as a proxy to improve risk assessment. Here, the toxicity of three different PCPs (4-Methylbenzylidene Camphor (4-MBC), propylparaben and triclocarban) was tested using embryo bioassays with Danio rerio (zebrafish) and Paracentrotus lividus (sea urchin). The No Observed Effect Concentration (NOEC) for triclocarban was 0.256 µg/L for sea urchin and 100 µg/L for zebrafish, whereas NOEC for 4-MBC was 0.32 µg/L for sea urchin and 50 µg/L for zebrafish. Both PCPs impacted embryo development at environmentally relevant concentrations. In comparison with triclocarban and 4-MBC, propylparaben was less toxic for both sea urchin (NOEC = 160 µg/L) and zebrafish (NOEC = 1000 µg/L). Overall, this study further demonstrates the sensitivity of embryo bioassays as a high-throughput approach for testing the toxicity of emerging pollutants. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Figure 1
Larval length (%) and total abnormalities (%) of Paracentrotus lividus exposed to different concentrations of 4-Methylbenzylidene Camphor (4-MBC) (A), propylparaben (PP) (B), and triclocarban (TCC) (C) for 48 h. Controls and solvent controls were grouped. Results of first and second assays are normalized to the respective control assay, for both endpoints. Bars with the letter (a) or the symbol (*) indicate significant differences from controls (p < 0.05). The percentage of larval length inhibition data are expressed as mean ± SE (n = 480 for controls; n = 240 for 5 µg/L 4-MBC, 1000 µg/L PP, 10 µg/L TCC and 0.64 µg/L TCC; n = 120 for the other groups). The percentage of total abnormalities data are expressed as mean ± SE (n = 32 for controls; n = 16 for 5 µg/L 4-MBC, 1000 µg/L PP, 10 µg/L TCC and 0.64 µg/L TCC; n = 8 for the other groups). Full article ">Figure 1 Cont.
Larval length (%) and total abnormalities (%) of Paracentrotus lividus exposed to different concentrations of 4-Methylbenzylidene Camphor (4-MBC) (A), propylparaben (PP) (B), and triclocarban (TCC) (C) for 48 h. Controls and solvent controls were grouped. Results of first and second assays are normalized to the respective control assay, for both endpoints. Bars with the letter (a) or the symbol (*) indicate significant differences from controls (p < 0.05). The percentage of larval length inhibition data are expressed as mean ± SE (n = 480 for controls; n = 240 for 5 µg/L 4-MBC, 1000 µg/L PP, 10 µg/L TCC and 0.64 µg/L TCC; n = 120 for the other groups). The percentage of total abnormalities data are expressed as mean ± SE (n = 32 for controls; n = 16 for 5 µg/L 4-MBC, 1000 µg/L PP, 10 µg/L TCC and 0.64 µg/L TCC; n = 8 for the other groups). Full article ">Figure 2
Embryonic stages and developmental abnormalities of the sea urchin Paracentrotus lividus observed in this study after 48 h of incubation under controlled conditions of temperature and light: (A) normal larva at pluteus stage; (B) gastrula stage; (C) prism larvae; (D) separated tip; (E,F) crossed tip; (F,G) fused arms; (G) general abnormal larvae; (H,I) abnormal arms orientation; (J) deformed arms; (K) arms absence; and (K,L) asymmetric arms. Full article ">

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Open AccessArticle

Exposure to Zinc Sulfate Results in Differential Effects on Olfactory Sensory Neuron Subtypes in Adult Zebrafish

by James T. Hentig and Christine A. Byrd-Jacobs

Int. J. Mol. Sci. 2016, 17(9), 1445; https://doi.org/10.3390/ijms17091445 - 31 Aug 2016

Cited by 25 | Viewed by 8002

Abstract

Zinc sulfate is a known olfactory toxicant, although its specific effects on the olfactory epithelium of zebrafish are unknown. Olfactory organs of adult zebrafish were exposed to zinc sulfate and, after 2, 3, 5, 7, 10 or 14 days, fish were processed for [...] Read more.

Zinc sulfate is a known olfactory toxicant, although its specific effects on the olfactory epithelium of zebrafish are unknown. Olfactory organs of adult zebrafish were exposed to zinc sulfate and, after 2, 3, 5, 7, 10 or 14 days, fish were processed for histological, immunohistochemical, ultrastructural, and behavioral analyses. Severe morphological disruption of the olfactory organ was observed two days following zinc sulfate exposure, including fusion of lamellae, epithelial inflammation, and significant loss of anti-calretinin labeling. Scanning electron microscopy revealed the apical surface of the sensory region was absent of ciliated structures, but microvilli were still present. Behavioral analysis showed significant loss of the ability to perceive bile salts and some fish also had no response to amino acids. Over the next several days, olfactory organ morphology, epithelial structure, and anti-calretinin labeling returned to control-like conditions, although the ability to perceive bile salts remained lost until day 14. Thus, exposure to zinc sulfate results in rapid degeneration of the olfactory organ, followed by restoration of morphology and function within two weeks. Zinc sulfate appears to have a greater effect on ciliated olfactory sensory neurons than on microvillous olfactory sensory neurons, suggesting differential effects on sensory neuron subtypes. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
The effects of various doses of zinc sulfate applied intranasally were examined 3 days after exposure in hematoxylin and eosin-stained horizontal sections. (A) Unlesioned control olfactory organs had sensory (S) and non-sensory (NS) regions lining the lamellae and pigment cells (P) dispersed throughout the lamina propria. There were no obvious effects of 0.2 M (B) or 0.5 M (C) zinc sulfate. At both doses, the sensory (large arrows) and non-sensory (small arrows) epithelia had a uniform appearance. There were effects apparent when the olfactory organs were exposed to 1 M zinc sulfate (D). Although the non-sensory epithelium (small arrow) looked unaffected, the sensory epithelium (large arrows) appeared thin and was occasionally fused (asterisk). The 5 M zinc sulfate concentration had an even more disruptive effect on the tissue (E). Very little structure remained and it was not possible to distinguish sensory and non-sensory epithelia. Pigment (arrowheads) was dispersed throughout the disorganized tissue. Scale bar = 50 µm (A–E). Full article ">Figure 2
The morphology of the olfactory organ and distribution of olfactory sensory neurons were examined with histological and immunohistochemical techniques. Unlesioned control olfactory organs sectioned in the horizontal plane displayed a semi-symmetrical shape with radiating lamellae (A) and typical olfactory epithelium appearance at higher magnification (A′); there was dense anti-calretinin labeling along the sensory epithelium ((B), arrows). Pigment cells were apparent in the lamina propria (arrowheads); (C) olfactory organs 2 days after intranasal infusion with 1 M zinc sulfate exhibited inflammation and fusion of lamellae (asterisks), and pigment was more dispersed (arrowheads). (C′) Higher magnification revealed vacuoles (v) and a generally disorganized epithelium; (D) anti-calretinin labeling (arrows) was diminished and confined to the apical surface of the epithelium. By 5 days after 1 M zinc sulfate exposure, the sensory epithelium was noticeably thinner than control tissue ((E), double arrows), and anti-calretinin labeling showed there were numerous olfactory sensory neurons dispersed throughout the tissue ((F), arrows). Pigment cells were again confined to the lamina propria (arrowheads). The morphology of the olfactory organ 10 days after 1 M zinc sulfate application more closely resembled that of control (G), and anti-calretinin labeling appeared similar to control levels in amount and intensity ((H), arrows). Scale bar = 100 μm (A–H) or 25 µm (A′,C′). Full article ">Figure 3
Anti-calretinin immunoreactivity was compared between treated and internal control olfactory organs using the mean percent difference in optical density. There was a significant decrease in anti-calretinin labeling at 2 and 3 days after zinc sulfate irrigation, compared to unlesioned control fish. By 5 days after chemical exposure, the amount of anti-calretinin labeling was not different from controls, and this continued at 7 and 10 days. * p < 0.05. Full article ">Figure 4
Scanning electron microscopy allowed analysis of the effects of zinc sulfate exposure on surface structures. (A) The sensory (S) and non-sensory (NS) regions of a lamella are distinguished with a defined separation (white arrows) in control fish; (B) the surface of control sensory epithelia was densely packed with cilia from OSNs, which obscured viewing of the shorter microvilli that are also present on the apical surface; (C) at 2 days after zinc sulfate exposure, the sensory epithelial surface appeared to contain only microvilli, with no evidence of cilia (*). Cilia in the non-sensory epithelium remained (black arrows); (D) an alternate morphology with no cilia or microvilli was seen at 2 days in 25% of the specimens examined. In these specimens, only microridges were apparent; (E) on the surface of the sensory epithelium of fish examined 5 days after infusion with the toxicant, intermittent cilia (black arrows) were present across the mat of microvilli; (F) by 10 days of recovery, the sensory epithelium appeared to be densely packed with cilia and microvilli, similar to control tissue. Scale bar in (A) = 100 μm; scale bar in (F) = 7 μm (B–F). Full article ">Figure 5
Behavioral responses were compared before (pre-odor) and after (odor trial) delivery of an amino acid (A) or bile salt (B) mixture. Control fish made significantly more turns after exposure to either mixture. (A) At 2 days following zinc sulfate treatment, fish did not show a statistically significant response to amino acids; however, there appeared to be a subset of fish that showed some response to the odor (2 days) and others who exhibited no change in behavior in response to the odor (2 days’). By 10 and 14 days after chemical exposure, fish made more turns during the amino acid odor trial; (B) Two days after exposure to zinc sulfate, fish did not respond to the bile salts mixture. Even after 10 days, the response to bile salts was not different from the pre-odor behavior. However, when given 14 days following zinc sulfate exposure the ability to perceive bile salts was regained. * p < 0.05. Full article ">

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Open AccessArticle

Exogenous Nitric Oxide Suppresses in Vivo X-ray-Induced Targeted and Non-Targeted Effects in Zebrafish Embryos

by E.Y. Kong, W.K. Yeung, T.K.Y. Chan, S.H. Cheng and K.N. Yu

Int. J. Mol. Sci. 2016, 17(8), 1321; https://doi.org/10.3390/ijms17081321 - 12 Aug 2016

Cited by 15 | Viewed by 6222

Abstract

The present paper studied the X-ray-induced targeted effect in irradiated zebrafish embryos (Danio rerio), as well as a non-targeted effect in bystander naïve embryos partnered with irradiated embryos, and examined the influence of exogenous nitric oxide (NO) on these targeted and [...] Read more.

The present paper studied the X-ray-induced targeted effect in irradiated zebrafish embryos (Danio rerio), as well as a non-targeted effect in bystander naïve embryos partnered with irradiated embryos, and examined the influence of exogenous nitric oxide (NO) on these targeted and non-targeted effects. The exogenous NO was generated using an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). The targeted and non-targeted effects, as well as the toxicity of the SNAP, were assessed using the number of apoptotic events in the zebrafish embryos at 24 h post fertilization (hpf) revealed through acridine orange (AO) staining. SNAP with concentrations of 20 and 100 µM were first confirmed to have no significant toxicity on zebrafish embryos. The targeted effect was mitigated in zebrafish embryos if they were pretreated with 100 µM SNAP prior to irradiation with an X-ray dose of 75 mGy but was not alleviated in zebrafish embryos if they were pretreated with 20 µM SNAP. On the other hand, the non-targeted effect was eliminated in the bystander naïve zebrafish embryos if they were pretreated with 20 or 100 µM SNAP prior to partnering with zebrafish embryos having been subjected to irradiation with an X-ray dose of 75 mGy. These findings revealed the importance of NO in the protection against damages induced by ionizing radiations or by radiation-induced bystander signals, and could have important impacts on development of advanced cancer treatment strategies. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Open AccessArticle

Longitudinal Effects of Embryonic Exposure to Cocaine on Morphology, Cardiovascular Physiology, and Behavior in Zebrafish

by Eric J. Mersereau, Cody A. Boyle, Shelby Poitra, Ana Espinoza, Joclyn Seiler, Robert Longie, Lisa Delvo, Megan Szarkowski, Joshua Maliske, Sarah Chalmers, Diane C. Darland and Tristan Darland

Int. J. Mol. Sci. 2016, 17(6), 847; https://doi.org/10.3390/ijms17060847 - 31 May 2016

Cited by 12 | Viewed by 6502

Abstract

A sizeable portion of the societal drain from cocaine abuse results from the complications of in utero drug exposure. Because of challenges in using humans and mammalian model organisms as test subjects, much debate remains about the impact of in utero cocaine exposure. [...] Read more.

A sizeable portion of the societal drain from cocaine abuse results from the complications of in utero drug exposure. Because of challenges in using humans and mammalian model organisms as test subjects, much debate remains about the impact of in utero cocaine exposure. Zebrafish offer a number of advantages as a model in longitudinal toxicology studies and are quite sensitive physiologically and behaviorally to cocaine. In this study, we have used zebrafish to model the effects of embryonic pre-exposure to cocaine on development and on subsequent cardiovascular physiology and cocaine-induced conditioned place preference (CPP) in longitudinal adults. Larval fish showed a progressive decrease in telencephalic size with increased doses of cocaine. These treated larvae also showed a dose dependent response in heart rate that persisted 24 h after drug cessation. Embryonic cocaine exposure had little effect on overall health of longitudinal adults, but subtle changes in cardiovascular physiology were seen including decreased sensitivity to isoproterenol and increased sensitivity to cocaine. These longitudinal adult fish also showed an embryonic dose-dependent change in CPP behavior, suggesting an increased sensitivity. These studies clearly show that pre-exposure during embryonic development affects subsequent cocaine sensitivity in longitudinal adults. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Graphical abstract

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Full article ">Figure 1
Experimental design of cocaine treatment and analysis of five-day larval zebrafish. A schematic showing the time course of embryonic drug exposure, imaging and longitudinal analysis is shown in (A); (B) shows an example of body length and eye diameter measurements made under bright field illumination; (C) shows the same fish under fluorescence illumination, focusing specifically on the brain at higher magnification. The tracing outlines the telencephalon (Tel), the diencephalon (Dien, which actually includes the optic tectum, midbrain and cerebellum), and the hindbrain (Hind, which includes the rhombencephalon), with sample area measurements given for each region. Full article ">Figure 2
Cocaine treatment decreases telencephalic area of zebrafish larvae. There was a readily observed decrease in telencephalic size seen after treatment with the highest doses of cocaine. Panels A and B provide a comparison between untreated fish (A) and siblings treated with 20 mg/L cocaine (B); In (C), results from several experiments were combined, with brain size expressed as a percentage of untreated controls. Brain size was reduced on average 7% by treatment with 20 mg/L cocaine. Error bars signify ± SEM, * p < 0.05 relative to control and ** p < 0.01 relative to untreated control. Full article ">Figure 3
Cocaine treatment during development alters heart rate in zebrafish larvae. The histogram shows data from one of three experiments measuring heart rate in zebrafish larvae exposed to different doses of cocaine for three days and then allowed one day of recovery before testing. Cocaine induced a bell-shaped dose response curve in larval baseline heart rate, with the maximal effect seen after treatment with 10 mg/L (error bars indicate ± SEM, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001 compared to untreated controls, n = 8 for each condition). Full article ">Figure 4
Embryonic cocaine exposure alters cardiovascular sensitivity to isoproterenol (A) and cocaine (B) in longitudinal adults. The heart rate of longitudinal adults was determined by measuring the ECGs. Baseline heart rate in longitudinal adults treated with cocaine as larvae was no different from untreated controls (<a href="#ijms-17-00847-t002" class="html-table">Table 2</a>). However, the response to isoproterenol, a ß1 agonist, was lower in longitudinal fish that were treated with cocaine as larvae than in fish that did not receive cocaine. The histogram in panel A shows the isoproterenol-induced change from baseline heart rate in one of three experiments all showing similar results. The effect of larval pre-exposure to cocaine on the adult isoproterenol response was U-shaped, with a maximal inhibition seen in fish previously treated with 10 mg/L cocaine (Error bars indicate ± SEM, * indicates p < 0.05, ** indicates p < 0.01, compared to untreated controls, n = 7 for each condition). Longitudinal fish from the different embryonic treatment groups were also challenged with 5 mg/L cocaine and their ECGs measured. The histogram in panel B shows the effect of larval pre-exposure to cocaine on subsequent adult cardiovascular response to the drug. The response by the different longitudinal groups is bell-shaped, with a maximal effect seen in fish pre-exposed to 10 mg/L cocaine as larvae (Bars indicate ± SEM, * indicates p < 0.01 when compared to fish pre-exposed to 20 mg/L, n = 18 for 0 and 20 mg/L cocaine, n = 16 for 2.5 and 10 mg/L, and n = 15 for 5 mg/L cocaine). Full article ">Figure 5
Pre-exposure to cocaine in larval fish affects cocaine-induced conditioned place preference (CPP) in longitudinal adults. This histogram represents data from four experiments combined testing CPP in longitudinal adults raised from larvae exposed to different doses of cocaine. Fish from all embryonic treatment groups not treated with cocaine as adults were combined into a single group for comparative purposes (Unt CPP). CPP was tested using 5 mg/L for all embryonic treatment groups and is expressed as a change in the percentage of time spent in the conditioning chamber before and after drug exposure. Cocaine pre-exposure during development results in a bell-shaped response curve for CPP in longitudinal adults. All longitudinal groups showed significantly higher CPP than untreated controls except for fish previously exposed to 20 mg/L cocaine. The maximal response observed in fish previously exposed to 10 mg/L and this group had the lowest p-value and was also significantly higher than the 20 mg/L group (p < 0.05). (Error bars represent ± SEM, * indicates p < 0.05, *** indicates p < 0.001 when compared to the untreated control group, n = 33 for untreated controls, n = 44 for 0 mg/L cocaine, n = 22 for 2.5 mg/L, n = 22 for 5 mg/L, n =30 for 10 mg/L, and n = 37 for 20 mg/L). Full article ">

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Open AccessReview

The Olfactory System of Zebrafish as a Model for the Study of Neurotoxicity and Injury: Implications for Neuroplasticity and Disease

by Erika Calvo-Ochoa and Christine A. Byrd-Jacobs

Int. J. Mol. Sci. 2019, 20(7), 1639; https://doi.org/10.3390/ijms20071639 - 2 Apr 2019

Cited by 57 | Viewed by 16620

Abstract

The olfactory system, composed of the olfactory organs and the olfactory bulb, allows organisms to interact with their environment and through the detection of odor signals. Olfaction mediates behaviors pivotal for survival, such as feeding, mating, social behavior, and danger assessment. The olfactory [...] Read more.

The olfactory system, composed of the olfactory organs and the olfactory bulb, allows organisms to interact with their environment and through the detection of odor signals. Olfaction mediates behaviors pivotal for survival, such as feeding, mating, social behavior, and danger assessment. The olfactory organs are directly exposed to the milieu, and thus are particularly vulnerable to damage by environmental pollutants and toxicants, such as heavy metals, pesticides, and surfactants, among others. Given the widespread occurrence of olfactory toxicants, there is a pressing need to understand the effects of these harmful compounds on olfactory function. Zebrafish (Danio rerio) is a valuable model for studying human physiology, disease, and toxicity. Additionally, the anatomical components of the zebrafish olfactory system are similar to those of other vertebrates, and they present a remarkable degree of regeneration and neuroplasticity, making it an ideal model for the study of regeneration, reorganization and repair mechanisms following olfactory toxicant exposure. In this review, we focus on (1) the anatomical, morphological, and functional organization of the olfactory system of zebrafish; (2) the adverse effects of olfactory toxicants and injury to the olfactory organ; and (3) remodeling and repair neuroplasticity mechanisms following injury and degeneration by olfactory toxicant exposure. Full article

(This article belongs to the Special Issue Zebrafish 2.0: A Model for Toxicological Research)

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Graphical abstract

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Full article ">Figure 1
Anatomical and morphological organization of the zebrafish olfactory system. (A) Localization of the olfactory system in zebrafish. Dorsal side is shown; rostral side is located upwards; (B) Olfactory organ morphology. Olfactory sensory epithelium arranged in lamellae is shown in black; (C) Olfactory epithelium (OE), composed of the following olfactory sensory neurons (OSNs): microvillous (mv); ciliated (cl); crypt (cr); kappe (kp); and pear (pr) OSNs. OSNs extend their axons to the olfactory bulb via the olfactory nerve (ON) to form discrete glomeruli; (D) Olfactory bulb organization in three laminae: olfactory nerve layer (ONL); glomerular layer (GL); and intracellular layer (ICL). Full article ">Figure 2
Odor-mediated behavioral tasks in zebrafish. (A) Odor-elicited swimming behaviors experimental setup. Individual fish are placed in either a rectangular or circular (not shown) experimental tank with two odorant delivery tubes collinearly positioned. An odorant is administered in one tube while water is simultaneously delivered in the other tube. Fish swimming patterns are recorded with a video camera (not shown); (B) Swimming trajectory of zebrafish after (top) odorant or (bottom) water exposure. Both swimming trajectory and time spent in each quadrant can be assessed with this test. This example depicts one of several swimming parameters that can be studied using this experimental setup. Full article ">Figure 3
Toxicant exposure and physical lesioning effects on the olfactory epithelium and its subsequent regeneration. (A) Degeneration and atrophy of the olfactory organ (OO), olfactory epithelium (OE), and olfactory sensory neurons (OSNs) following exposure to some toxicants and injury paradigms, some of which lead to olfactory dysfunction; (B) Effects of olfactory epithelium damage due to exposure to some toxicants and direct injury on the olfactory bulb, some of which lead to olfactory dysfunction; (C) Olfactory epithelium regeneration and repair following damage, leading to olfactory functional recovery; (D) Olfactory bulb regeneration and repair following damage to the OE, leading to olfactory functional recovery. Full article ">

26 pages, 1102 KiB

Open AccessReview

Zebrafish Models of Neurodevelopmental Disorders: Limitations and Benefits of Current Tools and Techniques

by Raquel Vaz, Wolfgang Hofmeister and Anna Lindstrand

Int. J. Mol. Sci. 2019, 20(6), 1296; https://doi.org/10.3390/ijms20061296 - 14 Mar 2019

Cited by 70 | Viewed by 17552

Abstract

For the past few years there has been an exponential increase in the use of animal models to confirm the pathogenicity of candidate disease-causing genetic variants found in patients. One such animal model is the zebrafish. Despite being a non-mammalian animal, the zebrafish [...] Read more.

For the past few years there has been an exponential increase in the use of animal models to confirm the pathogenicity of candidate disease-causing genetic variants found in patients. One such animal model is the zebrafish. Despite being a non-mammalian animal, the zebrafish model has proven its potential in recapitulating the phenotypes of many different human genetic disorders. This review will focus on recent advances in the modeling of neurodevelopmental disorders in zebrafish, covering aspects from early brain development to techniques used for modulating gene expression, as well as how to best characterize the resulting phenotypes. We also review other existing models of neurodevelopmental disorders, and the current efforts in developing and testing compounds with potential therapeutic value. Full article

(This article belongs to the Special Issue Zebrafish 2.0: A Model for Toxicological Research)

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Figure 1

Figure 1
Development of the zebrafish brain. (A) Schematic representation of the embryonic brain (30 hpf), showing the forebrain (in yellow), midbrain (in blue), MHB (in green), and hindbrain (in orange). Coronal and sagittal section schemes show brain structures primordia. Forebrain is subdivided in the telencephalon (in darker gray) and the diencephalon (containing the hypothalamus, lighter grey). (B) Simplified representation of the adult brain and main domains. Drawings not to scale. Adapted from [<a href="#B42-ijms-20-01296" class="html-bibr">42</a>]; C: cerebellum; D: diencephalon; M: midbrain; MHB: midbrain-hindbrain boundary; H: hindbrain; Ha: habenula; Hyp: hypothalamus; OB: olfactory bulb; ON: optic nerve; ORR: optic recess region; OV: otic vesicle; Pal: pallium; PB: pineal body; R: retina; r1–r7: rhombomeres 1 to 7; Sub: sub-pallium; T: telencephalon; and Teg: tegmentum. Full article ">Figure 2
Tests used for assessing behavior in zebrafish larvae. (A) Touch evoked response assay is performed by stimulating swimming via the physical stimulation of larvae as young as 2 days post fertilization (dpf). Response is recorded using a high-speed camera. Other studies are commonly performed on larvae older than 5 dpf and can be performed on several larvae simultaneously: (B) unstimulated swimming behavior, which consists on recording swimming activity for a determined period of time; (C) vibrational startle response consists of recording the response to environmental stimulation, usually a vibration stimulus by tapping the multi-well dish. This can be repeated multiple times, and learning can therefore be tested; and (D) visual motor response test is performed by recording the swimming activity in intermittent bright and dark environments. Full article ">Figure 3
Common behavior tests for adult zebrafish. (A) Learning test, or conditioned place preference (CPP) test, consists of testing the learning capacity by repeatedly exposing the specimen to a reward in a specific location (conditioning area, in green). (B) The novel tank test is used to assess anxiety. The animal is placed in a new arena containing refuge areas (represented by a black box) or risk areas (shallow area), and the swimming pattern is analyzed. Bold fish typically explore the open and shallow areas, while socially impaired fish prefer refuge areas. (C) Social interaction tests. These can be sub-divided into the schoaling test, assessing a fish behavior when in the presence of a group of conspecific fish; the social preference test, by separating the subject fish from conspecific or non-conspecific fish using a physical barrier and recording response; the social interaction test, by placing usually two fish in a tank and assessing behavior; or the mirror test. These tests are useful to test for socialization or aggressive behavior. Full article ">

1219 KiB

Open AccessReview

Zebrafish as a Model Organism for the Development of Drugs for Skin Cancer

by Fatemeh Bootorabi, Hamed Manouchehri, Reza Changizi, Harlan Barker, Elisabetta Palazzo, Annalisa Saltari, Mataleena Parikka, Carlo Pincelli and Ashok Aspatwar

Int. J. Mol. Sci. 2017, 18(7), 1550; https://doi.org/10.3390/ijms18071550 - 18 Jul 2017

Cited by 46 | Viewed by 10924

Abstract

Skin cancer, which includes melanoma and squamous cell carcinoma, represents the most common type of cutaneous malignancy worldwide, and its incidence is expected to rise in the near future. This condition derives from acquired genetic dysregulation of signaling pathways involved in the proliferation [...] Read more.

Skin cancer, which includes melanoma and squamous cell carcinoma, represents the most common type of cutaneous malignancy worldwide, and its incidence is expected to rise in the near future. This condition derives from acquired genetic dysregulation of signaling pathways involved in the proliferation and apoptosis of skin cells. The development of animal models has allowed a better understanding of these pathomechanisms, with the possibility of carrying out toxicological screening and drug development. In particular, the zebrafish (Danio rerio) has been established as one of the most important model organisms for cancer research. This model is particularly suitable for live cell imaging and high-throughput drug screening in a large-scale fashion. Thanks to the recent advances in genome editing, such as the clustered regularly-interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) methodologies, the mechanisms associated with cancer development and progression, as well as drug resistance can be investigated and comprehended. With these unique tools, the zebrafish represents a powerful platform for skin cancer research in the development of target therapies. Here, we will review the advantages of using the zebrafish model for drug discovery and toxicological and phenotypical screening. We will focus in detail on the most recent progress in the field of zebrafish model generation for the study of melanoma and squamous cell carcinoma (SCC), including cancer cell injection and transgenic animal development. Moreover, we will report the latest compounds and small molecules under investigation in melanoma zebrafish models. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Zebrafish as a relevant model for human disease and cancer therapy. The zebrafish genome shows up to 80% similarity with human disease-associated genes. Moreover, thanks to a well-conserved physiology, the pharmacological behavior and metabolism of several drugs have been screened in zebrafish with effects similar to humans. Full article ">Figure 2
Zebrafish model for high-throughput drug screening. Zebrafish is a valuable tool for high-throughput screening assays. In terms of toxicity, zebrafish have been used to evaluate a specific organ or behavior with respect to toxicity. In drug screening, zebrafish is also established as precious platform to multiple systemic phenotype studies simultaneously with metabolic profiles and toxicity reporter lines. Full article ">Figure 3
The tumor suppressor hexamethylene bisacetamide inducible 1 (HEXIM1) gene inhibits melanoma in the zebrafish model. HEXIM1 plays an important role as a melanoma tumor suppressor in response to nucleotide stress. HEXIM1 forms a complex with positive transcription elongation factor (P-TEFb) in order to inhibit the kinase to initiate transcription elongation at tumorigenic genes. Alteration of gene expression, in parallel with anti-tumorigenic RNAs binding to HEXIM1, favors the “anti-cancer” gene expression. Pol II: DNA polymerase II; CDK9: cyclin-dependent kinase; CCNT1: Cyclin-T. Full article ">Figure 4
Drug development and inhibitor screening using selected MEKi and PI3K/mTOR inhibitors. Zebrafish plays an important role for the screening of compounds targeting MEK/ERK and PI3K/mTOR pathways, both alone and in combination. (A) Example of screening of the FDA library molecules using zebrafish embryos; (B) steps showing hit selection after the screening procedure with different drugs and drug dose response using the melanin assay. At the end of this process, 11 hits were detected to be further evaluated in cell culture. MEKi: mitogen-activated protein kinases inhibitor; PI3K: phosphoinositide 3-kinase; mTOR: mechanistic target of rapamycin. Full article ">

801 KiB

Open AccessReview

Utilizing Zebrafish Visual Behaviors in Drug Screening for Retinal Degeneration

by Logan Ganzen, Prahatha Venkatraman, Chi Pui Pang, Yuk Fai Leung and Mingzhi Zhang

Int. J. Mol. Sci. 2017, 18(6), 1185; https://doi.org/10.3390/ijms18061185 - 2 Jun 2017

Cited by 45 | Viewed by 10335

Abstract

Zebrafish are a popular vertebrate model in drug discovery. They produce a large number of small and rapidly-developing embryos. These embryos display rich visual-behaviors that can be used to screen drugs for treating retinal degeneration (RD). RD comprises blinding diseases such as Retinitis [...] Read more.

Zebrafish are a popular vertebrate model in drug discovery. They produce a large number of small and rapidly-developing embryos. These embryos display rich visual-behaviors that can be used to screen drugs for treating retinal degeneration (RD). RD comprises blinding diseases such as Retinitis Pigmentosa, which affects 1 in 4000 people. This disease has no definitive cure, emphasizing an urgency to identify new drugs. In this review, we will discuss advantages, challenges, and research developments in using zebrafish behaviors to screen drugs in vivo. We will specifically discuss a visual-motor response that can potentially expedite discovery of new RD drugs. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Graphical abstract

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Full article ">Figure 1
A typical visual motor response (VMR) experiment. Sufficient zebrafish embryos for an experiment are collected by breeding adult fish. The embryos can be maintained in petri dishes with media, as they develop until they are needed for a VMR assay. (A) At the appropriate stage, embryos can be placed into 96-well plate format to facilitate throughput, storage and, data collection during a VMR assay. It should be noted that there are multiple arrangements possible for placing zebrafish larvae in a 96-well plate, such as row-wise, column-wise, or checkerboard patterns. Larvae in the 96-well plate arrangement can then be placed in a light-proof recording chamber and exposed to light onset or light offset stimulus. The locomotor output of the larvae is recorded and processed. Recorded data can be visualized through programs such as R 3.4.0 [<a href="#B123-ijms-18-01185" class="html-bibr">123</a>] (B) This graph illustrates the VMR of a group of 7-dpf wild-type larvae (black trace) responding to light onset stimulus [<a href="#B106-ijms-18-01185" class="html-bibr">106</a>]. Their response is compared to the VMR from a group of visually-impaired pde6c mutant larvae (red trace). Healthy larvae exhibit a strong startle response to the light onset, while the visually-impaired larvae do not. This lack of response by retinal degeneration (RD) zebrafish models forms the basis for drug screens. Full article ">Figure 2
Zebrafish larvae display different startle escape behaviors. (A) A larva escapes from a touch-stimulus by exhibiting a C-bend. In this response, larvae curve their bodies in a C-shape and swim quickly away from the location of the stimulus [<a href="#B128-ijms-18-01185" class="html-bibr">128</a>]; (B) Larvae orient into an O-bend in response to a dark flash. The larva curves its body approximately 180 degrees to swim in the opposite direction [<a href="#B125-ijms-18-01185" class="html-bibr">125</a>]. Reproduced with permissions from Burgess et al. and Lorent et al. Full article ">

1232 KiB

Open AccessReview

Zebrafish as a Vertebrate Model System to Evaluate Effects of Environmental Toxicants on Cardiac Development and Function

by Swapnalee Sarmah and James A. Marrs

Int. J. Mol. Sci. 2016, 17(12), 2123; https://doi.org/10.3390/ijms17122123 - 16 Dec 2016

Cited by 118 | Viewed by 9720

Abstract

Environmental pollution is a serious problem of the modern world that possesses a major threat to public health. Exposure to environmental pollutants during embryonic development is particularly risky. Although many pollutants have been verified as potential toxicants, there are new chemicals in the [...] Read more.

Environmental pollution is a serious problem of the modern world that possesses a major threat to public health. Exposure to environmental pollutants during embryonic development is particularly risky. Although many pollutants have been verified as potential toxicants, there are new chemicals in the environment that need assessment. Heart development is an extremely sensitive process, which can be affected by environmentally toxic molecule exposure during embryonic development. Congenital heart defects are the most common life-threatening global health problems, and the etiology is mostly unknown. The zebrafish has emerged as an invaluable model to examine substance toxicity on vertebrate development, particularly on cardiac development. The zebrafish offers numerous advantages for toxicology research not found in other model systems. Many laboratories have used the zebrafish to study the effects of widespread chemicals in the environment on heart development, including pesticides, nanoparticles, and various organic pollutants. Here, we review the uses of the zebrafish in examining effects of exposure to external molecules during embryonic development in causing cardiac defects, including chemicals ubiquitous in the environment and illicit drugs. Known or potential mechanisms of toxicity and how zebrafish research can be used to provide mechanistic understanding of cardiac defects are discussed. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Advantages of use of zebrafish in cardiotoxicity research, which provide enormous information within a short time. (A) Dissecting microscope image of 3 hpf zebrafish embryos showing how easily accessible zebrafish embryos are to treat with chemicals at different developmental stages for desired periods; (B,C) Dissecting microscope images showing normal pericardium in the control embryos (yellow arrow) (B) and pericardial edema phenotype in 4 days post-fertilization (dpf) ethanol-treated zebrafish embryos (yellow star) to help predict defective cardiogenesis (C); (D,E) Bright field images of Tg(myl7:GFP) embryos showing normal shaped two-chambered heart in control (D) and an almost linear heart in ethanol-exposed embryos (E), confirming heart malformation after ethanol exposure; (F,G) Confocal images of Tg(myl7:nlsKiKGR) embryos showing nuclei of cardiomyocytes in closely apposed bean-shaped atrium and ventricle in control embryos (F), fewer cardiomyocytes are seen in misshapen chambers of ethanol-treated embryos (G); and (H,I) Confocal images of Tg(fli1:EGFP) embryos show endocardial cells in normal endocardium in control embryos (H), fewer endocardial cells are seen in misshapen endocardium of ethanol-treated embryo (I). Full article ">Figure 2
Zebrafish studies discovered altered valve regulatory pathways due to embryonic ethanol exposure leading to persistent atrioventricular valve defects. (A) Schematic representation of atrio-ventricular canal (AVC) showing myocardium and endocardium layers. Bmp, Notch and Wnt signaling play critical roles during AVC differentiation. Ethanol exposure reduced Notch and Wnt activity at the AVC (represented by small dark brown arrows) during atrioventricular valve formation; (B) Schematic representation of the ventricle showing myocardium and endocardium layers. Ethanol exposure (3–24 hpf) increased Notch activity in the ventricle (represented by big dark brown arrows) during atrioventricular valve formation. Green arrow: normal condition; dark brown arrow: ethanol-exposed condition; (C) Schematic representation of atrium, ventricle and AVC (black arrow) of the control zebrafish heart at 50 hpf (during atrioventricular valve formation). Differentiated valve-forming cells (red) are localized at the AVC. Gray line represents myocardium layer; greenish-yellow line represents endocardial layer; (D) Schematic representation of the atrium, the ventricle and the AVC of the ethanol-treated (3–24 hpf) zebrafish heart at 50 hpf (during atrioventricular valve formation). Note that the shape of the heart is different from control. Differentiated valve-forming cells (pinkish-red), which do not exhibit all characteristics of normal valve cells are not restricted at the AVC. Those cells extend into the ventricle. The distance between myocardium and endocardium (the space containing cardiac jelly; black line) is more in ethanol-treated embryos. Gray line represents myocardium layer; greenish-yellow line represents endocardial layer; and (E,F) Wheat germ agglutinin-stained atrioventricular valves of two-month-old zebrafish shows four well-organized valve cusps in control fish (E), and small, deformed valve cusps in fish treated with ethanol during embryonic development (3–24 hpf). Full article ">

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Open AccessReview

Zebrafish as an In Vivo Model to Assess Epigenetic Effects of Ionizing Radiation

by Eva Yi Kong, Shuk Han Cheng and Kwan Ngok Yu

Int. J. Mol. Sci. 2016, 17(12), 2108; https://doi.org/10.3390/ijms17122108 - 15 Dec 2016

Cited by 14 | Viewed by 5698

Abstract

Exposure to ionizing radiations (IRs) is ubiquitous in our environment and can be categorized into “targeted” effects and “non-targeted” effects. In addition to inducing deoxyribonucleic acid (DNA) damage, IR exposure leads to epigenetic alterations that do not alter DNA sequence. Using an appropriate [...] Read more.

Exposure to ionizing radiations (IRs) is ubiquitous in our environment and can be categorized into “targeted” effects and “non-targeted” effects. In addition to inducing deoxyribonucleic acid (DNA) damage, IR exposure leads to epigenetic alterations that do not alter DNA sequence. Using an appropriate model to study the biological effects of radiation is crucial to better understand IR responses as well as to develop new strategies to alleviate exposure to IR. Zebrafish, Danio rerio, is a scientific model organism that has yielded scientific advances in several fields and recent studies show the usefulness of this vertebrate model in radiation biology. This review briefly describes both “targeted” and “non-targeted” effects, describes the findings in radiation biology using zebrafish as a model and highlights the potential of zebrafish to assess the epigenetic effects of IR, including DNA methylation, histone modifications and miRNA expression. Other in vivo models are included to compare observations made with zebrafish, or to illustrate the feasibility of in vivo models when the use of zebrafish was unavailable. Finally, tools to study epigenetic modifications in zebrafish, including changes in genome-wide DNA methylation, histone modifications and miRNA expression, are also described in this review. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

1102 KiB

Open AccessReview

Zebrafish: A Model for the Study of Toxicants Affecting Muscle Development and Function

by Magda Dubińska-Magiera, Małgorzata Daczewska, Anna Lewicka, Marta Migocka-Patrzałek, Joanna Niedbalska-Tarnowska and Krzysztof Jagla

Int. J. Mol. Sci. 2016, 17(11), 1941; https://doi.org/10.3390/ijms17111941 - 19 Nov 2016

Cited by 60 | Viewed by 13328

Abstract

The rapid progress in medicine, agriculture, and allied sciences has enabled the development of a large amount of potentially useful bioactive compounds, such as drugs and pesticides. However, there is another side of this phenomenon, which includes side effects and environmental pollution. To [...] Read more.

The rapid progress in medicine, agriculture, and allied sciences has enabled the development of a large amount of potentially useful bioactive compounds, such as drugs and pesticides. However, there is another side of this phenomenon, which includes side effects and environmental pollution. To avoid or minimize the uncontrollable consequences of using the newly developed compounds, researchers seek a quick and effective means of their evaluation. In achieving this goal, the zebrafish (Danio rerio) has proven to be a highly useful tool, mostly because of its fast growth and development, as well as the ability to absorb the molecules diluted in water through its skin and gills. In this review, we focus on the reports concerning the application of zebrafish as a model for assessing the impact of toxicants on skeletal muscles, which share many structural and functional similarities among vertebrates, including zebrafish and humans. Full article

(This article belongs to the Special Issue Zebrafish: A Model for Toxicological Research)

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Zebrafish as a tool for toxicological studies. In classical toxicology research, an excessive or non-excessive (e.g., environmentally relevant) dose of the tested compound is administered to an animal e.g., via injection, dietary or waterborne uptake. Evaluation of tested chemical toxicity can be based on various approaches such as lethality assessment, phenotypic screening or gene profiling. Phenotypic screening involves the monitoring of different parameters, e.g., endocrine disrupting compounds (EDCs) can be identified via gonadal morphology and histological comparative analysis. Gene profiling is used in so-called toxicogenomics due to the organism’s susceptibility to different chemicals manifested in the induction of genes, e.g., involved in detoxification or protection against cellular stresses. This method of toxicant identification consists of the assessment of changes in gene-expression profiles by the use of oligonucleotide microarray. Of note, the sensitivity of this assay system is high enough to detect a distinct compound at a concentration that does not cause morphological effects. WT, wild type. Full article ">Figure 2
Transgenic (TG) zebrafish as a biosensor for toxicant identification. The modern toxicological approach takes advantage of biotechnological techniques, which allow the development of various zebrafish transgenic lines equipped with the reporter genes such as green fluorescent protein (GFP). Induction of reporter gene expression is driven by specific response elements providing the possibility for demonstration of the tissue-specific mode of toxicant action. These methods have improved the sensitivity and effectiveness of detection in comparison to traditional toxicological techniques. These types of genetically modified zebrafish lines are excellent biosensors and are used for precise qualitative and quantitative analysis of a wide range of potential toxicants. Full article ">

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