Zebrafish in toxicology and drug discovery

PUBLICATIONS

Long-term effects of oxytetracycline exposure in zebrafish: A multi-level perspective

Chemosphere

2014-01-01

Highlights

• Zebrafish exposed to oxytetracycline present different feeding behavior pattern.
• Oxytetracycline induced fish boldness and hyperactivity in light periods.
• Energetic reserves and oxidative stress enzymes decrease after long-term exposure.
• Bacterial communities of fish gut and exposure water were affected by oxytetracycline.

Abstract

Oxytetracycline (OTC) is a broad-spectrum antibiotic widely used in livestock production. Like many other pharmaceuticals, OTC is not completely metabolized by the organism and thus, increasing amounts of the compound are being detected in the aquatic environment. The assessment of the environmental risk of pharmaceuticals is hindered by their very low concentrations and specific modes of action and thus relevant exposure scenarios and sensitive endpoints are needed. Thus, this work aimed to study the long-term effect of OTC exposure in zebrafish (at behavior and biochemical levels) and associated bacterial communities (fish gut and water bacterial communities). Results revealed that at behavioral level, boldness increase (manifested by increased exploratory behavior of a new environment) was observed in fish exposed to low OTC concentrations. Moreover, changes in fish swimming pattern were observed in light periods (increased stress response: hyperactivity and freezing) probably due to photo-sensibility conferred by OTC exposure. Effects at biochemical level suggest that long-term exposure to OTC interfere with cellular energy allocation mainly by reducing lipids levels and increasing energy consumption. Moreover, evidences of oxidative damage were also observed (reduced levels of TG, GST and CAT). The analysis of water and gut microbiome revealed changes in the structure and diversity of bacterial communities potentially leading to changes in communities' biological function. Some of the effects were observed at the lowest concentration tested, 0.1 μg/L which is a concentration already detected in the environment and thus clearly demonstrating the need of a serious ecotoxicological assessment of OTC effects on non-target organisms.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0045653519301638

Advancing the zebrafish embryo test for endocrine disruptor screening using micro‐injection: ethinyl estradiol as a case study

Environmental Toxicology and chemistry

2014-01-01

ABSTRACT

Fish (embryo) toxicity test guidelines are mostly based on aquatic exposures. However, in some cases, other exposure routes can be more practical and relevant. Micro‐injection into the yolk of fish embryos could offer a particular advantage for administering hydrophobic compounds, such as many endocrine disruptors. Single dose micro‐injection was compared to continuous aquatic exposure in terms of compound accumulation and biological responses. 17α‐ethinyl estradiol (EE2) was used as a model compound. First, the optimal solvent and droplet size were optimized, and needle variation was assessed. Next, biological endpoints were evaluated. The accumulated internal dose of EE2 decreased over time in both exposure scenarios. Estrogen receptor (ER) activation was concentration/injected dose dependent, increased daily and was related to esr2b transcription. Vtg1 and cyp19a1b transcription was induced in both scenarios, but the cyp19a1b transcription pattern differed between routes. Injection caused an increase of cyp19a1b transcripts from 48 hours post fertilization (hpf) onwards, while after aquatic exposure the main increase occurred between 96 and 120 hpf. Some malformations only occurred after injection, while others were present for both scenarios. We conclude that responses can differ between exposure routes and therefore micro‐injection is not a direct substitute for, but can be complementary to aquatic exposure. Nevertheless, vtg1 and cyp19a1b transcription and ER activation are suitable biomarkers for endocrine disruptor screening in both scenarios.

Link to the publication :

https://setac.onlinelibrary.wiley.com/doi/abs/10.1002/etc.4343

Protecting the environment from psychoactive drugs: Problems for regulators illustrated by the possible effects of tramadol on fish behaviour

Science of the total environment

2014-01-01

Highlights

• Fish were exposed to the psychoactive drugs tramadol and fluoxetine.
• Alteration of fish behaviour was assessed in a novel tank diving test.
• The results were not easy to interpret with confidence.
• Ecotoxicological experts reached different conclusions based on the same results.
• Determining whether or not psychoactive drugs alter the behaviour of fish is difficult.

Abstract

There is concern that psychoactive drugs present in the aquatic environment could affect the behaviour of fish, and other organisms, adversely. There is considerable experimental support for this concern, although the literature is not consistent. To investigate why, fish were exposed to three concentrations of the synthetic opiate tramadol for 23–24 days, and their anxiolytic behaviour in a novel tank diving test was assessed both before and after exposure. The results were difficult to interpret. The positive control drug, the anti-depressant fluoxetine, produced the expected results: exposed fish explored the novel tank more, and swam more slowly while doing so. An initial statistical analysis of the results provided relatively weak support for the conclusion that both the low and high concentrations of tramadol affected fish behaviour, but no evidence that the intermediate concentration did. To gain further insight, UK and Japanese experts in ecotoxicology were asked for their independent opinions on the data for tramadol. These were highly valuable. For example, about half the experts replied that a low concentration of a chemical can cause effects that higher concentrations do not, although a similar number did not believe this was possible. Based both on the inconclusive effects of tramadol on the behaviour of the fish and the very varied opinions of experts on the correct interpretation of those inconclusive data, it is obvious that more research on the behavioural effects of tramadol, and probably all other psychoactive drugs, on aquatic organisms is required before any meaningful risk assessments can be conducted. The relevance of these findings may apply much more widely than just the environmental risk assessment of psychoactive drugs. They suggest that much more rigorous training of research scientists and regulators is probably required if consensus decisions are to be reached that adequately protect the environment from chemicals.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0048969719305716

Quantitative Cross-Species Extrapolation between Humans and Fish: The Case of the Anti-Depressant Fluoxetine

Plos one

2014-10-22

Fish are an important model for the pharmacological and toxicological characterization of human pharmaceuticals in drug discovery, drug safety assessment and environmental toxicology. However, do fish respond to pharmaceuticals as humans do? To address this question, we provide a novel quantitative cross-species extrapolation approach (qCSE) based on the hypothesis that similar plasma concentrations of pharmaceuticals cause comparable target-mediated effects in both humans and fish at similar level of biological organization (Read-Across Hypothesis). To validate this hypothesis, the behavioural effects of the anti-depressant drug fluoxetine on the fish model fathead minnow (Pimephales promelas) were used as test case. Fish were exposed for 28 days to a range of measured water concentrations of fluoxetine (0.1, 1.0, 8.0, 16, 32, 64 µg/L) to produce plasma concentrations below, equal and above the range of Human Therapeutic Plasma Concentrations (HTPCs). Fluoxetine and its metabolite, norfluoxetine, were quantified in the plasma of individual fish and linked to behavioural anxiety-related endpoints. The minimum drug plasma concentrations that elicited anxiolytic responses in fish were above the upper value of the HTPC range, whereas no effects were observed at plasma concentrations below the HTPCs. In vivo metabolism of fluoxetine in humans and fish was similar, and displayed bi-phasic concentration-dependent kinetics driven by the auto-inhibitory dynamics and saturation of the enzymes that convert fluoxetine into norfluoxetine. The sensitivity of fish to fluoxetine was not so dissimilar from that of patients affected by general anxiety disorders. These results represent the first direct evidence of measured internal dose response effect of a pharmaceutical in fish, hence validating the Read-Across hypothesis applied to fluoxetine. Overall, this study demonstrates that the qCSE approach, anchored to internal drug concentrations, is a powerful tool to guide the assessment of the sensitivity of fish to pharmaceuticals, and strengthens the translational power of the cross-species extrapolation.

 

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0110467

Antinociceptive effects of buprenorphine in zebrafish larvae: An alternative for rodent models to study pain and nociception?

Applied Animal Behaviour Science, Volume 152, March 2014, Pages 92-99

2015-03-10

Abstract:

The underlying processes of nociception and pain are, despite the rodent models available, still not fully understood. One of the drawbacks of rodent model systems is the difficulty to screen compound libraries for their influence on nociception, thus slowing down the discovery of novel analgesics for clinical use.

Rodent behavioural tasks have been previously adapted for larval zebrafish in our group and in the current manuscript we investigated the possibilities of zebrafish larvae as an additional model system to study nociception and pain and their underlying mechanisms.

Zebrafish larvae were exposed to different concentrations of diluted acetic acid, a chemical noxious stimulus, and we measured nociceptive-specific behaviours. Cyclooxygenase-2 (cox-2), a gene known to be involved in nociception, was used as a marker for the activation of nociceptive pathways. Upon exposure to diluted acetic acid, five-day old larval zebrafish showed a concentration dependent increase in locomotor activity. This increase in locomotor activity was accompanied by a stimulus dependent increase in cox-2 mRNA expression, demonstrating that nociceptive pathways were indeed activated. Pre-treatment of the larvae with 0.1 µg/ml buprenorphine before exposure to the noxious stimulus, prevented the behavioural changes induced by the diluted acid. Further, the antinociceptive properties of buprenorphine could be reversed by co-treatment with the µ-receptor antagonist naloxone.

In conclusion, our results demonstrate that larval zebrafish as young as five days, show behavioural responses upon exposure to a noxious stimulus. The magnitude of the responses is dependent on the intensity of the stimulus applied and activation of nociceptive pathways was confirmed by altered cox-2 mRNA expression. The analgesic buprenorphine has similar antinociceptive properties in this model as in higher vertebrates and mammals and is able to prevent the behavioural responses induced by the noxious stimulus. We therefore propose zebrafish larvae as a novel model system in nociception and pain related research.

Antinociceptive effects of buprenorphine in zebrafish larvae: An alternative for rodent models to study pain and nociception?

Peter J. Steenbergen, Nabila Bardine, Applied Animal Behaviour ScienceVolume 152, March 2014, Pages 92–99

Part of this publication was performed with ZebraLab and ZebraBox

Discovery of a Natural Product-Like iNOS Inhibitor by Molecular Docking with Potential Neuroprotective Effects In Vivo

DOI: 10.1371/journal.pone.0092905

2015-04-01

Abstract:

In this study, we applied structure-based virtual screening techniques to identify natural product or natural product-like inhibitors of iNOS. The iNOS inhibitory activity of the hit compounds was characterized using cellular assays and an in vivo zebrafish larvae model. The natural product-like compound 1 inhibited NO production in LPS-stimulated Raw264.7 macrophages, without exerting cytotoxic effects on the cells. Significantly, compound 1 was able to reverse MPTP-induced locomotion deficiency and neurotoxicity in an in vivo zebrafish larval model. Hence, compound 1 could be considered as a scaffold for the further development of iNOS inhibitors for potential anti-inflammatory or anti-neurodegenerative applications.

Discovery of a Natural Product-Like iNOS Inhibitor by Molecular Docking with Potential Neuroprotective Effects In Vivo

Hai-Jing Zhong equal contributor, Li-Juan Liu equal contributor, Cheong-Meng Chong, Lihua Lu, Modi Wang, Daniel Shiu-Hin Chan, Philip Wai Hong Chan, Simon Ming-Yuen Lee,  Dik-Lung Ma, Chung-Hang Leung.

Part of this publication was performed with ZebraLab and ZebraBox

Neurobehavioral effects of two metabolites of BDE-47 on zebrafish larvae

Chemosphere

2016-01-01

Highlights

The neurobehavioral toxicity of 6-OH/MeO-BDE47 were evaluated with zebrafish larvae.

6-OH-BDE47 performed inhibiting effects on routine and average turns.

Different from 6-OH-BDE-47, 6-MeO-BDE47 mainly promoted responsive turns.

6-MeO-BDE-47 performed more adverse effects on social activity.


Abstract

Two metabolites, OH-BDEs and MeO-BDEs, of polybrominated diphenyl ethers (PBDEs) were ubiquitously detected in animal tissues and environmental samples, drawing a widely public concern to their toxicity. The comparison of toxicity between PBDEs and their metabolites has been a focus in recent years, however, comparisons seldom involve neurobehavioral toxicity of PBDEs metabolites in published works. In this study, zebrafish larvae were exposed to 6-OH-BDE-47 and 6-MeO-BDE-47 and their neurobehavioral traits (including locomotion, path angle, and social activity) were recorded using the instrument Zebrabox; meanwhile, light illumination was used as stimuli in the test duration. The results showed larvae were more active in dark periods than light periods, and preferred turning right (+) to left (−). Effects of the two metabolites varied in different behavioral indicators. They induced different effects on path angle but did not reverse the left-right asymmetry. 6-OH-BDE-47 did not induce the effects on larval locomotion and social activity, but mainly decreased average and routine turn numbers; 6-MeO-BDE-47 promoted larvae responsive turns but inhibited social activity. This study offered new experimental means to the neurobehavioral toxicity of various PBDE metabolites. Further studies may focus on the toxic mechanisms of specific neurobehavioral traits.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0045653518302662

Toxic effects of the dinoflagellate Karenia mikimotoi on zebrafish (Danio rerio) larval behavior

Harmful Algae

2016-01-01

Highlights

• Ruptured cell solutions (RCS) of K. mikimotoi lowered locomotor activity in zebrafish larvae.
• RCS significantly inhibits AChE, SOD, and CAT activity in zebrafish larvae.
• RCS downregulates genes involved in larval neural development.
• Larvae compensate for K. mikimotoi neurotoxicity via upregulating bdnf and manf.
• Oxidative stress, AChE inhibition, and impeded neurodevelopment cause toxicity.

Abstract

Karenia mikimotoi is a toxic dinoflagellate that forms harmful blooms in coastal waters, threatening aquaculture worldwide. However, we do not know whether K. mikimotoi has a neurotoxic effect on aquatic animal behavior. Thus, this study investigated potential K. mikimotoi neurotoxicity in zebrafish larvae. Cells of K. mikimotoi were collected at the mid-exponential phase from a batch culture to prepare ruptured cell solutions (RCS). At 6 h post-fertilization (hpf), zebrafish embryos were exposed to different RCS concentrations (0, 102, 103, 104, and 2.5 × 104 cells mL−1). After 120 hpf, treated larvae were collected to analyze locomotor behavior; activities of acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT); and expression of genes related to neurodevelopment. We found that RCS did not affect survival rate, but significantly decreased larval locomotion, as well as their AChE, SOD, and CAT activity. Additionally, the examination of the day-night behavioral experiment revealed RCS decreased locomotion only at night. Zebrafish larvae were also significantly hypoactive in response to light and sound stimulations. Of the neurodevelopment genes, three (th, neurog1, and neurod1) were downregulated, while two (bdnf and manf) were upregulated. Our study suggests that K. mikimotoi neurotoxicity occurs through causing oxidative damage, as well as disorders in the cholinergic system and nervous system development. The results provide new insight that K. mikimotoi in low abundance did not cause significant lethal effect but still exhibited significant neurotoxicity on aquatic animals.

 

Link to the publication :

https://www.sciencedirect.com/science/article/abs/pii/S1568988321000238

Measuring Larval Zebrafish Behavior: Locomotion, Thigmotaxis, and Startle

2016-06-19

Zebrafish Behavior and locomotion

Larval zebrafish are genetically tractable, easy to manipulate, and can be generated in large numbers. Their small size also makes them ideal for high-throughput screens for genes or pharmacological compounds that affect behavior. Thus, larval zebrafish are an ideal model system to investigate locomotion and locomotion-based behaviors such as thigmotaxis and startle. In this chapter, I present methods to measure locomotion, thigmotaxis, and response to a startle stimulus in larval zebrafish.f

Linking genomic responses of gonads with reproductive impairment in marine medaka (Oryzias melastigma) exposed chronically to the chemopreventive and antifouling agent, 3,3′-diindolylmethane (DIM)

Science Direct

2017-01-03

Abstract

3,3′-Diindolylmethane (DIM) has been promoted as an effective chemopreventive and antifouling additive. However, the concurrent risks or side effects of DIM are not fully understood, especially on tissues responsive to estrogen. Therefore, this study employed marine medaka (Oryzias melastigma) as a test model to evaluate relative safety and explore mechanisms of toxic action of DIM on development and function of gonad after chronic (28 days) aqueous exposure to relatively low doses (0 μg/L or 8.5 μg/L). Integration of comprehensive toxicogenomic analysis at the transcriptome and proteome levels with apical endpoints, such as production of eggs and swimming performance of larvae, elucidated the molecular linkage in gonad from bottom up along the reproductive adverse outcome pathway. A series of sequential changes at the transcript and protein levels were linked to lesser fecundity and viability of larvae exposed to DIM. Anomalous production of vitellogenin (VTG) and eggshell proteins in testis confirmed the estrogenic potency of DIM. In the ovary, although storage of VTG was greater, lesser expressions of cathepsin enzymes blocked cleavage and incorporation of VTG into oocytes as yolk, which acted together with lower eggshell proteins to inhibit maturation of primary oocyte and thus contributed to impairment of fecundity. Overall, this study demonstrated that exposure to DIM impaired reproductive fitness. Diverse molecular initiating changes in gonads were linked to apical endpoints that could be used in assessment of risks posed by DIM on gametogenesis. In combination with chemical stability and potent endocrine disruption, the results of this study can inform decisions about the use of DIM either as chemopreventive or antifouling agent.

 

http://www.sciencedirect.com/science/article/pii/S0166445X16303794

Vitamin E reduces endosulfan-induced toxic effects on morphology and behavior in early development of zebrafish

Science Direct

2017-01-10

Abstract

The aim of this study was to investigate if vitamin E (α-TOC) modulates the developmental toxicity of the pesticide endosulfan (ESF), using a modified zebrafish embryotoxicity test (ZET). Zebrafish (Danio rerio) embryos were exposed from 6 to 72 h post fertilization (hpf) to either ESF (0.1–50 mg/L) or α-TOC (0.01–3 mM) alone or in combination. The effects of these exposures on embryonic morphology, larval behavior and antioxidant gene expression were analyzed. Phenotypic analysis at 48 hpf showed that ESF led to a dose-dependent increase in embryonic deformities, including axis malformations, pericardial edema and reduced pigmentation. Co-exposure of ESF with α-TOC (1–3 mM) significantly (p < 0.05) reduced ESF-induced embryonic malformations. Exposure to solely α-TOC did not affect rates of survival or malformations. Behavior studies showed that ESF caused hyperactivity at 5 days post fertilization, indicating a developmental neurotoxic effect. The ESF-induced hyperactivity was ameliorated by α-TOC. Elevated ESF concentrations caused down-regulation of the antioxidant genes cuzn-sod, gpx1a and cat, suggesting that ESF promoted oxidative stress in the embryos. α-TOC did not prevent the ESF-induced dysregulation of these genes. These results demonstrate that α-TOC protects against phenotypic and behavioral effects caused by ESF but did not rescue ESF-induced aberrations in antioxidant gene expression.

 

http://www.sciencedirect.com/science/article/pii/S0278691517300042

Oxidative stress-mediated developmental toxicity induced by isoniazide in zebrafish embryos and larvae

Journal of Applied Toxicology

2017-02-04

Abstract

Isoniazide (INH) is an important first-line drug that is used to treat tuberculosis. However, the effect of INH on fetal growth has not yet been elucidated, and the mechanism of INH-induced developmental toxicity is still unknown. In the present study, we employed zebrafish embryos and larvae to investigate the developmental toxicity of INH. The survival rates of the embryos and larvae as well as the hatching rates of embryos were significantly reduced. Morphological abnormalities, including spinal curvature, yolk retention, swimming bladder absence, tail bending and shorter body lengths were induced by INH. Histopathological analysis showed loose cell-to-cell contacts and large vacuoles in the larval hepatocytes. Thin intestinal walls, frayed gut villi and widespread cell lysis were observed in the intestines of the larvae in the higher concentration (8, 16 mm) exposure groups. In addition, exposure to high doses (≥ 6 mm) of INH significantly reduced the locomotor capacity of the zebrafish larvae. INH significantly increased the levels of reactive oxygen species and malondialdehyde and decreased the superoxide dismutase activity in zebrafish larvae, which suggested that oxidative stress was induced and that the antioxidant capacity was inhibited. Superoxide dismutase 1 and liver fatty acid-binding protein mRNA levels were significantly downregulated, while the GSTP2 and cytochrome P450 3A mRNA levels were significantly upregulated in the INH-exposed zebrafish larvae. The overall results indicated that INH caused a dose- and time-dependent increase in developmental toxicity and that oxidative stress played an important role in the developmental toxicity induced by INH in zebrafish larvae. Copyright © 2017 John Wiley & Sons, Ltd.

 

http://onlinelibrary.wiley.com/doi/10.1002/jat.3432/full

Cystinosis (ctns) zebrafish mutant shows pronephric glomerular and tubular dysfunction

Nature

2017-02-21

Abstract

The human ubiquitous protein cystinosin is responsible for transporting the disulphide amino acid cystine from the lysosomal compartment into the cytosol. In humans, Pathogenic mutations of CTNS lead to defective cystinosin function, intralysosomal cystine accumulation and the development of cystinosis. Kidneys are initially affected with generalized proximal tubular dysfunction (renal Fanconi syndrome), then the disease rapidly affects glomeruli and progresses towards end stage renal failure and multiple organ dysfunction. Animal models of cystinosis are limited, with only a Ctns knockout mouse reported, showing cystine accumulation and late signs of tubular dysfunction but lacking the glomerular phenotype. We established and characterized a mutant zebrafish model with a homozygous nonsense mutation (c.706 C > T; p.Q236X) in exon 8 of ctns. Cystinotic mutant larvae showed cystine accumulation, delayed development, and signs of pronephric glomerular and tubular dysfunction mimicking the early phenotype of human cystinotic patients. Furthermore, cystinotic larvae showed a significantly increased rate of apoptosis that could be ameliorated with cysteamine, the human cystine depleting therapy. Our data demonstrate that, ctns gene is essential for zebrafish pronephric podocyte and proximal tubular function and that the ctns-mutant can be used for studying the disease pathogenic mechanisms and for testing novel therapies for cystinosis.

 

http://www.nature.com/articles/srep42583

Functional analysis of the transmembrane and cytoplasmic domains of Pcdh15a in zebrafish hair cells

The Journal of Neuroscience

2017-02-25

Zebrafish behaviour toxicology research

 

Abstract



Protocadherin 15 (PCDH15) is required for mechanotransduction in sensory hair cells as a component of the tip link. Isoforms of PCDH15 differ in their cytoplasmic domains (CD1, CD2, and CD3), but share the extracellular and transmembrane (TMD) domains, as well as an intracellular domain known as the common region (CR). In heterologous expression systems, both the TMD and CR of PCDH15 have been shown to interact with members of the mechanotransduction complex. The in vivo significance of these protein-protein interaction domains of PCDH15 in hair cells has not been determined. Here we examined the localization and function of the two isoforms of zebrafish Pcdh15a (CD1 and CD3) in pcdh15a null mutants by assessing Pcdh15a transgene-mediated rescue of auditory/vestibular behavior, hair-cell morphology and activity. We found that either isoform alone was able to rescue the Pcdh15a-null phenotype, and that the CD1 or CD3 specific regions were dispensable for hair-bundle integrity and labeling of hair cells with FM4-64, which was used as a proxy for mechanotransduction. When either the CR or TMD domain were deleted, the mutated proteins localized to the stereocilial tips, but were unable to rescue FM4-64 labeling. Disrupting both domains led to a complete failure of Pcdh15a to localize to the hair bundle. Our findings demonstrate that the TMD and cytoplasmic CR domains are required for the in vivo function of Pcdh15a in zebrafish hair cells.

 

http://www.jneurosci.org/content/early/2017/02/20/JNEUROSCI.2216-16.2017

Lipid quantitation and metabolomics data from vitamin E-deficient and -sufficient zebrafish embryos from 0 to 120 hours-post-fertilization

Science Direct

2017-02-25

Abstract

The data herein is in support of our research article by McDougal et al. (2017) [1], in which we used our zebrafish model of embryonic vitamin E (VitE) deficiency to study the consequences of VitE deficiency during development. Adult 5D wild-type zebrafish (Danio rerio), fed defined diets without (E–) or with VitE (E+, 500 mg RRR-α-tocopheryl acetate/kg diet), were spawned to obtain E– and E+ embryos and evaluated using metabolomics and specific lipid analyses (each measure at 24, 48, 72, 120 hours-post-fertilization, hpf), neurobehavioral development (locomotor responses at 96 hpf), and rescue strategies. Rescues were attempted using micro-injection into the yolksac using VitE (as a phospholipid emulsion containing d6-α-tocopherol at 0 hpf) or D-glucose (in saline at 24 hpf).

 

http://www.sciencedirect.com/science/article/pii/S2352340917300653

Toxicological and behavioral responses as a tool to assess the effects of natural and synthetic dyes on zebrafish early life

Science Direct

2017-03-03

ABSTRACT

Organic dyes extracted from natural sources have been widely used to develop safety and eco-friendly dyes as an alternative to synthetic ones, since the latter are usually precursors of mutagenic compounds. Thereby, toxicity tests to non-target organisms are critical step to develop harmless dyes to environment and in this context, zebrafish early life stages are becoming an important alternative model. We aimed to assess the toxic effects of the synthetic dye Basic Red 51 (BR51, used in cosmetic industry), the natural dye erythrostominone (ERY, a potential commercial dye extracted from fungi) and its photodegradation product (DERY), using zebrafish early life assays. Developmental malformations on embryos and behavioral impairment on larvae were explored. Our results showed that embryos exposed to BR51 and ERY exhibited a large yolk sac (LOEC=7.5 mg L-1), possibly due to a deformity or delayed resorption. ERY also induced pericardial and yolk sac edemas at high concentrations (LOEC=15 and 30 mg L-1, respectively). Moreover, larvae swan less distance and time when exposed to ERY (LOEC=7.5 mg L-1) and BR51 (LOEC=1.875 mg L-1). The lowest larvae locomotion have been associated with impairment of the yolk sac, important tissue of the energy source. Interestingly, DERY did not affect neither development nor behavior of zebrafish, showing that ERY photodegradation is sufficient to prevent its toxic effects. In conclusion, both natural and synthetic dyes impaired development and behavior of zebrafish early life, therefore, a simple treatment of the natural dye can prevent the aquatic life impact.

 

http://www.sciencedirect.com/science/article/pii/S0045653517303831

Use of Gammarus fossarum (Amphipoda) embryo for toxicity testing: A case study with cadmium

Wiley Online Library

2017-03-07

Abstract

The effects of environmental contaminants on arthropod embryo stages have been poorly investigated in ecotoxicology. Moreover, many of these tests used hatching success as the sole metric, while many more subtle effects could be detected. In the present study, after having finely described embryogenesis in Gammarus fossarum, sublethal effects of cadmium (Cd) exposure during the embryonic development of G. fossarum were studied. For this purpose, embryos were first directly exposed in multiwell plates throughout the entire embryo cycle (i.e. 23 days) to increasing Cd concentrations (0, 1.5 µg/L and 3.0 µg/L; 120 embryos per concentration). Second, to assess the representativeness of the gammarid embryo assay performed in multiwell plates, embryos were exposed to similar Cd concentrations through the maternal open brood pouch. Third, to pinpoint sensitive periods of development, embryos were directly exposed to 3.0 µg/L of Cd for shorter periods of time: during gastrulation, organogenesis and hatching. After hatching, i) body mass, ii) activity of the enzyme phenoloxidase, a key enzyme of the arthropod immune system, and iii) locomotor activity were measured in the newborn individuals. Phenoloxidase activity was strongly inhibited in newborn individuals of embryos exposed – either in multiwell plates or in the maternal brood pouch – to 3.0 µg/L Cd throughout embryonic development. Furthermore, strong detrimental locomotor effects were observed in newborn individuals of embryos directly exposed to 3.0 µg/L. Exposures for shorter periods of time were not sufficient to induce such effects; no sensitive period could be determined. By bringing new insights into a critical time window of exposure, the gammarid embryo assay could provide a novel and interesting addition to existing bioassays in gammarids.

 

http://onlinelibrary.wiley.com/doi/10.1002/etc.3779/full

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

International Journal of Molecular Sciences

2017-03-07

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 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.

 

http://www.mdpi.com/1422-0067/18/3/539/htm

Effects of Dechlorane Plus exposure on axonal growth, musculature and motor behavior in embryo-larval zebrafish

Science Direct

2017-03-11

Abstract

Developmental neurobehavioral toxicity of Dechlorane Plus (DP) was investigated using the embryo-larval stages of zebrafish (Danio rerio). Normal fertilized embryos were waterborne exposed to DP at 15, 30, 60 μg/L beginning from 6 h post-fertilization (hpf). Larval teratology, motor activity, motoneuron axonal growth and muscle morphology were assessed at different developmental stages. Results showed that DP exposure significantly altered embryonic spontaneous movement, reduced touch-induced movement and free-swimming speed and decreased swimming speed of larvae in response to dark stimulation. These changes occurred at DP doses that resulted no significant teratogenesis in zebrafish. Interestingly, in accord with these behavioral anomalies, DP exposure significantly inhibited axonal growth of primary motoneuron and induced apoptotic cell death and lesions in the muscle fibers of zebrafish. Furthermore, DP exposure at 30 μg/L and 60 μg/L significantly increased reactive oxygen species (ROS) and malondialdehyde (MDA) formation, as well as the mRNA transcript levels of apoptosis-related genes bax and caspase-3. Together, our data indicate that DP induced neurobehavioral deficits may result from combined effects of altered neuronal connectivity and muscle injuries.

 

http://www.sciencedirect.com/science/article/pii/S0269749116318917

Occurrence and ecotoxicological effects of free, conjugated and halogenated steroids of 17α-hydroxypregnanolone and pregnanediol in Swiss wastewater and surface water

ACS Publications - Environmental Science & Technology

2017-05-09

Abstract

Apart from estrogens, the occurrence and ecotoxicity of steroids in the aquatic environment is poorly known. Here we analyzed 33 steroids, including estrogens, androgens, progestins, and glucocorticoids in hospital wastewaters, river water and in municipal wastewater treatment plant (WTP) influents and effluents at different sites in Switzerland. In addition, wastewater from different treatment steps of two WTPs with advanced treatment, such as ozonation or pulverized activated carbon, were analyzed to study the steroid’s behavior during treatment. Considerable levels of different steroids occurred in hospital and raw municipal wastewater but they were low (lower than 1 ng/L) or below detection level in effluents of WTPs and river water. In WTP influents estrogens (estrone, 17β-estradiol, estriol), androgens (androstenedione, androsterone, trans-androsterone, testosterone), progestins and metabolites (medroxyprogesterone acetate, megestrol acetate, mifepristone, pregnanediol, 17α-hydroxypregnanolone, 17α-hydroxyprogesterone, and 21α-hydroxyprogesterone) were detected and removed effectively during biological treatment. Ozonation further removed the steroids. Exposure of zebrafish embryos demonstrated negligible effects of pregnanediol and 17α-hydroxypregnenolone, while mixtures that mimic wastewater and river water composition affected embryo development and led to alteration of steroidogenesis gene transcripts at ng/L concentrations. Although steroid concentrations are low in Swiss rivers, the possibility of additive effects may be of concern.

 

http://pubs.acs.org/doi/abs/10.1021/acs.est.7b01231?journalCode=esthag

Effects of ibuprofen, diclofenac and paracetamol on hatch and motor behavior in developing zebrafish (Danio rerio)

ScienceDirect

2017-05-10

Abstract

Non-steroidal anti-inflammatory drugs (NSAIDs) which are widely used as pain relief medicines are causing increasing environmental concern due to their incomplete removal in wastewater treatment plant and potential toxicity on endocrine, kidney and reproduction in teleost fish. This study focused on the effects of widely used ibuprofen, diclofenac and paracetamol on the hatch and motor ability of early-stage zebrafish, by exposing embryos to the target chemicals at 5, 50 and 500 μg/L starting from 6 h postfertilization (hpf). A significant reduction in hatch rate at 55 hpf was caused by both ibuprofen (-63%) and diclofenac (-58%) at 500 μg/L. Exposure to high concentration of ibuprofen significantly decreased the spontaneous movement by 25%, and reduced the free swimming distance, duration and speed under dark condition by 41%, 29% and 30%, respectively. High concentration of diclofenac also caused 23% decrease in spontaneous movement, and reduced the swimming distance as well as active duration by 17% and 13% under light stimulation. In comparison, the exposure to paracetamol did not cause any notable effect. Among neuron related genes tested, the expression of neurog1 was down-regulated from ibuprofen and diclofenac exposure by 19% and 26%, while the expression of neurod1 was up-regulated only by ibuprofen (31%). These findings indicated that ibuprofen and diclofenac significantly affected embryo locomotivity and were potentially neurotoxic, thus posing threats to zebrafish development.

 

http://www.sciencedirect.com/science/article/pii/S0045653517307579

Zebrafish embryo toxicity of 15 chlorinated, brominated, and iodinated disinfection by-products

ScienceDirect

2017-05-11

Abstract

Disinfection to protect human health occurs at drinking water and wastewater facilities through application of non-selective oxidants including chlorine. Oxidants also transform organic material and form disinfection by-products (DBPs), many of which are halogenated and cyto- and genotoxic. Only a handful of assays have been used to compare DBP toxicity, and researchers are unsure which DBP(s) drive the increased cancer risk associated with drinking chlorinated water. The most extensive data set employs an in vitro model cell, Chinese hamster ovary cells. Traditionally, most DBP research focuses on the threat to human health, but the effects on aquatic species exposed to DBPs in wastewater effluents remain ill defined. We present the developmental toxicity for 15 DBPs and a chlorinated wastewater to a model aquatic vertebrate, zebrafish. Mono-halogenated DBPs followed the in vivo toxicity rank order: acetamides > acetic acids > acetonitriles ~ nitrosamines, which agrees well with previously published mammalian in vitro data. Di- and tri-halogenated acetonitriles were more toxic than their mono-halogenated analogues, and bromine- and iodine-substituted DBPs tended to be more toxic than chlorinated analogues. No zebrafish development effects were observed after exposure to undiluted or non-concentrated, chlorinated wastewater. We find zebrafish development to be a viable in vivo alternative or confirmatory assay to mammalian in vitro cell assays.

 

http://www.sciencedirect.com/science/article/pii/S1001074217301158

Kinetics of glucocorticoid exposure in developing zebrafish: A tracer study

ScienceDirect

2017-05-12

Abstract

In the current study the dynamics of glucocorticoid uptake by zebrafish chorionated embryos from the surrounding medium were studied, using 2.5 μM cortisol or dexamethasone solutions complemented with their tritiated variant. We measured the uptake of radioactive cortisol by embryos during a 1 h submersion. Interestingly, the signal in chorionated embryos was 85% (exposure: 1–2 hpf) or 78% (exposure: 48–49 hpf) of the signal present in an equal volume medium. By comparing embryos measured without chorion, we found that 18–20% of the radioactivity present in chorionated embryos is actually bound to the chorion or located in the perivitelline space. Consequently, embryonic tissue contains radioactivity levels of 60% of a similar volume of medium after 1 h incubation. During early developmental stages (1–48 hpf) exposure of more than 24 h in cortisol was needed to achieve radioactivity levels similar to an equal volume of medium within the embryonic tissue and more than 48 h for dexamethasone. In glucocorticoid-free medium, radioactivity dropped rapidly below 10% for both glucocorticoids, suggesting that the major portion of the embryonic radioactivity was a result of simple diffusion. During later developmental stages (48–96 hpf) initial uptake dynamics were similar, but showed a decrease of tissue radioactivity to 20% of an equal volume of medium after hatching, probably due to development and activation of the hypothalamic pituitary interrenal axis. Uptake is dependent on the developmental stage of the embryo. Furthermore, the presence of the chorion during exposure should be taken into account even when small lipophilic molecules are being tested.

 

http://www.sciencedirect.com/science/article/pii/S0045653517307634

Toxicant induced behavioural aberrations in larval zebrafish are dependent on minor methodological alterations

ScienceDirect

2017-05-18

Abstract

Alterations in zebrafish motility are used to identify neurotoxic compounds, but few have reported how methodology may affect results. To investigate this, we exposed embryos to bisphenol A (BPA) or tetrabromobisphenol A (TBBPA) before assessing larval motility. Embryos were maintained on a day/night cycle (DN) or in constant darkness, were reared in 96 or 24 well plates (BPA only), and behavioural tests were carried out at 96, 100, or 118 (BPA only) hours post fertilisation (hpf). We found that the prior photo-regime, larval age, and/or arena size influence behavioural outcomes in response to toxicant exposure. For example, methodology determined whether 10 μM BPA induced hyperactivity, hypoactivity, or had no behavioural effect. Furthermore, the minimum effect concentration was not consistent between different methodologies. Finally, we observed a mechanism previously used to explain hyperactivity following BPA exposure does not appear to explain the hypoactivity observed following minor alterations in methodology. Therefore, we demonstrate how methodology can have notable implications on dose responses and behavioural outcomes in larval zebrafish motility following identical chemical exposures. As such, our results have significant consequences for human and environmental risk assessment.

 

http://www.sciencedirect.com/science/article/pii/S0378427417301984

Evaluation of the sensitivity spectrum of a video tracking system with zebrafish (Danio rerio) exposed to five different toxicants

Environmental Science and Pollution Research

2017-05-23

Abstract

The aim of this study was to develop a biological early warning system for the detection of aquatic toxicity and test it with five toxicants with distinct chemical nature. This was done in order to verify the spectrum of sensitivities of the proposed system, as well as the potential identification capability of the tested contaminants, using only the analysis of zebrafish’s behavior. Six experimental conditions were tested: negative control and five toxicants (bleach, lindane, tributyltin, mercury, and formaldehyde). The exposure time was 45 min, and the concentrations used corresponded to 9% of LC50’s-96 h for the tested compounds, to ensure ecologically relevant results. A total of 108 fish were used, with each individual experimental condition being tested 18 times. A statistical model of diagnosis was used, combining self-organizing map and correspondence analysis. The values of sensitivity, specificity, accuracy, false positive, false negative, positive predictive value (PPV), and negative predictive value (NPV) were calculated. The objectives of the work were accomplished and the system showed a good overall diagnostic performance with 79% in accuracy, 77% in sensitivity, and 88% in specificity. The lowest result of the predictive values was 78% (lindane and mercury), in the case of the NPV, and 86% (bleach and lindane), in the case of the PPV. The best result of the predictive values was 100% (bleach and tributyltin), for the NPV, and 89% (tributyltin), for the PPV. Regarding the five tested toxicants, the system was able to correctly identify the agent responsible for the contamination in 40% of the positive diagnoses.

 

https://link.springer.com/article/10.1007/s11356-017-9262-4

Transgenerational endocrine disruption and neurotoxicity in zebrafish larvae after parental exposure to binary mixtures of decabromodiphenyl ether (BDE-209) and lead

ScienceDirect

2017-06-22

Abstract

Polybrominated diphenyl ethers (PBDEs) and heavy metals are two key groups of electric and electronic equipment contaminants. Despite their co-occurrence in aquatic environments, their combined effects remain largely unknown, particularly under a chronic exposure regime. In the present study, adult zebrafish (Danio rerio) were exposed to environmentally relevant concentrations of BDE-209 and lead (Pb), or their binary mixtures, for 3 months. After chronic parental exposure, increased transfer of BDE-209 and Pb to the offspring eggs was activated in the coexposure groups, with BDE-197 being the predominant PBDE congener, indicating the dynamic metabolism of BDE-209 in parental zebrafish. In the presence of Pb, culturing the eggs in clean water until 5 days post-fertilization (dpf) further accelerated the debromination of BDE-209 towards BDE-197 in the offspring, caused by the preferential removal of bromine atoms at meta positions. BDE-209 and Pb combinations induced reproductive and thyroid endocrine disruption in adults, which resulted in an imbalanced deposition of hormones in the eggs. However, compared with single chemical exposure, the larval offspring at 5 dpf from the coexposure groups had reversed the adverse influences from maternal origin. In addition, the interaction between BDE-209 and Pb led to transgenerational developmental neurotoxicity in the larval offspring, where inhibited neuronal growth and neurotransmitter signaling, disorganized muscular assembly, and impaired visual function contributed to the observed neurobehavioral deficits. Overall, depending on specific biological events, the complex interaction between BDE-209 and Pb under chronic exposure resulted in significant alterations in their environmental fate and toxicological actions, thus complicating the accurate evaluation of ecological risks and constituting an unquantified threat to environmental safety.

 

http://www.sciencedirect.com/science/article/pii/S0269749117304268

Long-term exposure to elevated carbon dioxide does not alter activity levels of a coral reef fish in response to predator chemical cues

Behavioral Ecology and Sociobiology

2017-07-05

Abstract

Levels of dissolved carbon dioxide (CO2) projected to occur in the world’s oceans in the near future have been reported to increase swimming activity and impair predator recognition in coral reef fishes. These behavioral alterations would be expected to have dramatic effects on survival and community dynamics in marine ecosystems in the future. To investigate the universality and replicability of these observations, we used juvenile spiny chromis damselfish (Acanthochromis polyacanthus) to examine the effects of long-term CO2 exposure on routine activity and the behavioral response to the chemical cues of a predator (Cephalopholis urodeta). Commencing at ~3–20 days post-hatch, juvenile damselfish were exposed to present-day CO2 levels (~420 μatm) or to levels forecasted for the year 2100 (~1000 μatm) for 3 months of their development. Thereafter, we assessed routine activity before and after injections of seawater (sham injection, control) or seawater-containing predator chemical cues. There was no effect of CO2 treatment on routine activity levels before or after the injections. All fish decreased their swimming activity following the predator cue injection but not following the sham injection, regardless of CO2 treatment. Our results corroborate findings from a growing number of studies reporting limited or no behavioral responses of fishes to elevated CO2.

 

https://link.springer.com/article/10.1007/s00265-017-2337-x

Calpain inhibition is protective in Machado-Joseph disease zebrafish due to induction of autophagy

JNeurosci

2017-07-07

Abstract

The neurodegenerative disease Machado-Joseph disease (MJD), also known as spinocerebellar ataxin-3, affects neurons of the brain and spinal cord, disrupting control of the movement of muscles. We have successfully established the first transgenic zebrafish (Danio rerio) model of MJD, which express human ataxin-3 protein containing either 23 glutamines (23Q, wild-type) or 84Q (MJD-causing), within neurons. Phenotypic characterization of the zebrafish (male and female) revealed that the ataxin-3-84Q zebrafish have decreased survival compared to ataxin-3-23Q and develop ataxin-3 neuropathology, ataxin-3 cleavage fragments and motor impairment. Ataxin-3-84Q zebrafish swim shorter distances than ataxin-3-23Q zebrafish as early as 6 days old, even if expression of the human ataxin-3 protein is limited to motor neurons. This swimming phenotype provides a valuable readout for drug treatment studies. Treating the EGFP-Ataxin-3 84Q zebrafish with the calpain inhibitor compound calpeptin decreased levels of ataxin-3 cleavage fragments, but also removed all human ataxin-3 protein (confirmed by ELISA) and prevented the early MJD zebrafish motor phenotype. We identified that this clearance of ataxin-3 protein by calpeptin treatment resulted from an increase in autophagic flux (indicated by decreased p62 levels and increased LC3II). Co-treatment with the autophagy inhibitor chloroquine blocked the decrease in human ataxin-3 levels and the improved movement produced by calpeptin treatment. This study demonstrates that this first transgenic zebrafish model of MJD is a valuable tool for testing potential treatments for MJD. Calpeptin treatment is protective in this model of MJD and removal of human ataxin-3 through by macro-autophagy plays an important role in this beneficial effect.

SIGNIFICANCE STATEMENT

We have established the first transgenic zebrafish model of the neurodegenerative disease MJD, and identified relevant disease phenotypes, including impaired movement from an early age, which can be used in rapid drug testing studies. We have found that treating the MJD zebrafish with the calpain inhibitor calpeptin produces complete removal of human ataxin-3 protein, due to induction of the autophagy quality control pathway. This improves the movement of the MJD zebrafish. Artificially blocking the autophagy pathway prevents the removal of human ataxin-3 and improved movement produced by calpeptin treatment. These findings indicate that induction of autophagy, and removal of ataxin-3 protein, plays an important role in the protective effects of calpain inhibition for the treatment of MJD.

 

http://www.jneurosci.org/content/early/2017/07/07/JNEUROSCI.1142-17.2017

Measuring drug absorption improves interpretation of behavioral responses in a larval zebrafish locomotor assay for predicting seizure liability

ScienceDirect - Journal of Pharmacological and Toxicological Methods

2017-07-13

Abstract


Introduction

Unanticipated effects on the central nervous system are a concern during new drug development. A larval zebrafish locomotor assay can reveal seizure liability of experimental molecules before testing in mammals. Relative absorption of compounds by larvae is lacking in prior reports of such assays; having those data may be valuable for interpreting seizure liability assay performance.


Methods

Twenty-eight reference drugs were tested at multiple dose levels in fish water and analyzed by a blinded investigator. Responses of larval zebrafish were quantified during a 30 min dosing period. Predictive metrics were calculated by comparing fish activity to mammalian seizure liability for each drug. Drug level analysis was performed to calculate concentrations in dose solutions and larvae. Fifteen drug candidates with neuronal targets, some having preclinical convulsion findings in mammals, were tested similarly.


Results

The assay has good predictive value of established mammalian responses for reference drugs. Analysis of drug absorption by larval fish revealed a positive correlation between hyperactive behavior and pro-convulsive drug absorption. False negative results were associated with significantly lower compound absorption compared to true negative, or true positive results. The predictive value for preclinical toxicology findings was inferior to that suggested by reference drugs.


Discussion

Disproportionately low exposures in larvae giving false negative results demonstrate that drug exposure analysis can help interpret results. Due to the rigorous testing commonly performed in preclinical toxicology, predicting convulsions in those studies may be more difficult than predicting effects from marketed drugs.

 

http://www.sciencedirect.com/science/article/pii/S1056871917301028

Cortisol elevation post-hatch affects behavioural performance in zebrafish larvae

ScienceDirect - General and Comparative Endocrinology

2017-07-13

Abstract

Maternal cortisol is essential for cortisol stress axis development and de novo production of this steroid commences only after hatch in zebrafish (Danio rerio). However, very little is known about the effect of elevated cortisol levels, during the critical period of stress axis activation, on larval performance. We tested the hypothesis that elevated cortisol levels post-hatch affect behavioural performance and this is mediated by glucocorticoid receptor (GR) activation in zebrafish larvae. The behavioural response included measuring larval activity in response to alternating light and dark cycles, as well as thigmotaxis. Zebrafish larvae at 3 days post-fertilization were exposed to waterborne cortisol for 24 h to mimic a steroid response to an early-life stressor exposure. Also, larvae were exposed to waterborne RU-486 (a GR antagonist) either in the presence or absence of cortisol to confirm GR activation. Co-treatment with RU-486 completely abolished the upregulation of cortisol-induced 11β-hydroxysteroid dehydrogenase type 2 transcript abundance, confirming GR signalling. Cortisol-exposed larvae displayed increased locomotor activity irrespective of light condition, but showed no changes in thigmotaxis. This cortisol-mediated behavioural response was not affected by co-treatment with RU-486. Cortisol exposure also did not modify the transcript abundances of GR and mineralocorticoid receptor (MR) in zebrafish larvae. Altogether, cortisol stress axis activation post-hatch increases locomotor activity in zebrafish larvae. Our results suggest that GR signalling may not be involved in this behavioural response, leading to the proposal that cortisol action via MR signalling may influence locomotor activity in zebrafish larvae.

 

http://www.sciencedirect.com/science/article/pii/S0016648017304975

Endocrine, teratogenic and neurotoxic effects of cyanobacteria detected by cellular in vitro and zebrafish embryos assays

Chemosphere

2017-07-27

Abstract

Cyanobacteria contain various types of bioactive compounds, which could cause adverse effects on organisms. They are released into surface waters during cyanobacterial blooms, but there is little information on their potential relevance for effects in vivo. In this study presence of bioactive compounds was characterized in cyanobacteria Microcystis aeruginosa (Chroococcales), Planktothrix agardhii (Oscillatoriales) and Aphanizomenon gracile (Nostocales) with selected in vitro assays. The in vivo relevance of detected bioactivities was analysed using transgenic zebrafish embryos tg(cyp19a1b-GFP). Teratogenic potency was assessed by analysis of developmental disorders and effects on functions of the neuromuscular system by video tracking of locomotion. Estrogenicity in vitro corresponded to 0.95–54.6 ng estradiol equivalent (g dry weight (dw))−1. In zebrafish embryos, estrogenic effects could not be detected potentially because they were masked by high toxicity. There was no detectable (anti)androgenic/glucocorticoid activity in any sample. Retinoid-like activity was determined at 1–1.3 μg all-trans-retinoic acid equivalent (g dw)−1. Corresponding to the retinoid-like activity A. gracile extract also caused teratogenic effects in zebrafish embryos. Furthermore, exposure to biomass extracts at 0.3 g dw L−1 caused increase of body length in embryos. There were minor effects on locomotion caused by 0.3 g dw L−1 M. aeruginosa and P. agardhii extracts. The traditionally measured cyanotoxins microcystins did not seem to play significant role in observed effects. This indicates importance of other cyanobacterial compounds at least towards some species or their developmental phases. More attention should be paid to activity of retinoids, estrogens and other bioactive substances in phytoplankton using in vitro and in vivo bioassays.

 

http://www.sciencedirect.com/science/article/pii/S0045653514009370

Species specific anaesthetics for fish anaesthesia and euthanasia

Univ of Bristol

2017-08-02

Amphibians: anesthesia of Fish


There is a need to ensure that the care and welfare for fish maintained in the laboratory are to the highest standards. This extends to the use of anaesthetics for both scientific study and humane killing and euthanasia at end of life. An anaesthetic should not induce negative behaviours and fish should not seek to avoid the anaesthetic. Surprisingly little information is available to facilitate a humane choice of anaesthetic agent for fish despite over 100 years of use and the millions of fish currently held in thousands of laboratories worldwide.

Using a chemotaxic choice chamber we found different species specific behavioural response among four closely related fish species commonly held in the laboratory, exposed to three widely used anaesthetic agents. As previously found for zebrafish (Danio rerio), the use of MS-222 and benzocaine also appears to induce avoidance behaviours in medaka (Oryzias latipes); but etomidate could provide an alternative choice. Carp (Cyprinus carpio), although closely related to zebrafish showed avoidance behaviours to etomidate, but not benzocaine or MS-222; and rainbow trout (Oncorhynchus mykiss) showed no avoidance to the three agents tested. We were unable to ascertain avoidance responses in fathead minnows (Pimephales promelas) and suggest different test paradigms are required for that species.

 

http://research-information.bristol.ac.uk/files/122652284/Gareth_paper_II_SR_2017.pdf

Inhibition of glutamate decarboxylase (GAD) by ethyl ketopentenoate (EKP) induces treatment-resistant epileptic seizures in zebrafish

2017-08-03

Abstract

Epilepsy is a chronic brain disorder characterized by recurrent seizures due to abnormal, excessive and synchronous neuronal activities in the brain. It affects approximately 65 million people worldwide, one third of which are still estimated to suffer from refractory seizures. Glutamic acid decarboxylase (GAD) that converts glutamate into GABA is a key enzyme in the dynamic regulation of neural network excitability. Importantly, clinical evidence shows that lowered GAD activity is associated with several forms of epilepsy which are often treatment resistant. In the present study, we synthetized and explored the possibility of using ethyl ketopentenoate (EKP), a lipid-permeable GAD-inhibitor, to induce refractory seizures in zebrafish larvae. Our results demonstrate that EKP evoked robust convulsive locomotor activities, excessive epileptiform discharges and upregulated c-fos expression in zebrafish. Moreover, transgenic animals in which neuronal cells express apoaequorin, a Ca2+-sensitive bioluminescent photoprotein, displayed large luminescence signals indicating strong EKP-induced neuronal activation. Molecular docking data indicated that this proconvulsant activity resulted from the direct inhibition of both gad67 and gad65. Limited protective efficacy of tested anti-seizure drugs (ASDs) demonstrated a high level of treatment resistance of EKP-induced seizures. We conclude that the EKP zebrafish model can serve as a high-throughput platform for novel ASDs discovery.

 

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543107/

Effects of copper oxide nanoparticles and copper ions to zebrafish (Danio rerio) cells, embryos and fry

Toxicology in Vitro

2017-08-15

Abstract

The use of engineered metal nanoparticles (NPs) is continuously increasing and so is the need for information regarding their toxicity. This study compares the toxicity of CuO NPs with ionic Cu in three zebrafish model systems; zebrafish hepatoma cell line (ZFL), fish embryo toxicity test (FET) and fry locomotion. In the ZFL tests, no significant cytotoxicity (cell death, decreased metabolic or cell membrane integrity) was detected for either treatment, though both significantly affected reactive oxygen species (ROS) production. Embryo mortality was affected by both Cu ions and CuO NPs with similar concentration-response relationships, whereas only Cu ions affected fry mortality (24 h LC50 ≈ 30 μM, ≈ 2 mg Cu L− 1 for Cu ions and no significant mortality observed at up to 200 μM, 12.7 mg Cu L− 1 for CuO NP). Both Cu forms increased fry swimming activity during light cycles and decreased activity during dark cycles: Cu ions had significant impact at lower concentrations than CuO NPs. The implications are that Cu ions generally are more toxic than CuO NPs to embryos and fry but there is a marked difference in toxicity among the different zebrafish model systems. Metal NPs release into the environment may have adverse effects on fish and other aquatic organisms.

 

http://www.sciencedirect.com/science/article/pii/S0887233317302266

The Power of Zebrafish in Personalised Medicine

Advances in Experimental Medicine and Biology

2017-08-25

Abstract

The goal of personalised medicine is to develop tailor-made therapies for patients in whom currently available therapeutics fail. This approach requires correlating individual patient genotype data to specific disease phenotype data and using these stratified data sets to identify bespoke therapeutics. Applications for personalised medicine include common complex diseases which may have multiple targets, as well as rare monogenic disorders, for which the target may be unknown. In both cases, whole genome sequence analysis (WGS) is discovering large numbers of disease associated mutations in new candidate genes and potential modifier genes. Currently, the main limiting factor is the determination of which mutated genes are important for disease progression and therefore represent potential targets for drug discovery. Zebrafish have gained popularity as a model organism for understanding developmental processes, disease mechanisms and more recently for drug discovery and toxicity testing. In this chapter, we will examine the diverse roles that zebrafish can make in the expanding field of personalised medicine, from generating humanised disease models to xenograft screening of different cancer cell lines, through to finding new drugs via in vivo phenotypic screens. We will discuss the tools available for zebrafish research and recent advances in techniques, highlighting the advantages and potential of using zebrafish for high throughput disease modeling and precision drug discovery.

 

https://link.springer.com/chapter/10.1007/978-3-319-60733-7_10

No effect of elevated carbon dioxide on reproductive behaviors in the three-spined stickleback

Behavioral Ecology

2017-08-30

Abstract

Ocean acidification, the reduction in ocean pH resulting from anthropogenic emissions of carbon dioxide (CO2), has been predicted to alter the behavior of fishes. During experimental exposure to CO2 concentrations projected for the year 2100 (~1000 µatm), fish have been reported to display disturbances in activity, learning, behavioral lateralization, and even attraction to predator cues. Reproductive behaviors have received far less attention, despite an intensive research effort on ocean acidification and its ecological importance. Here, we investigate whether elevated levels of CO2 affect reproduction in breeding pairs of the three-spined stickleback, Gasterosteus aculeatus, a model species in behavioral, evolutionary ecology, and environmental toxicology. We found that males under both present day levels (400 µatm) and future levels (1000 µatm) of CO2 developed normal sexual ornaments, pursued normal nest building activities, exhibited similar levels of courtship behaviors and displacement fanning, and had the same mating probability. Moreover, fanning behavior during the paternal care period followed what is expected for the species for males from both treatments, and there was no effect of treatment on the numbers of offspring produced. This study is the first to investigate the effect of elevated CO2 on the complete breeding cycle in detail, studying an array of highly fitness-relevant traits. Our study showing surprising resilience of fish reproduction is an important contribution in order to realistically predict the impacts of future ocean acidification.

 

https://academic.oup.com/beheco/article-abstract/doi/10.1093/beheco/arx112/4097644/No-effect-of-elevated-carbon-dioxide-on?redirectedFrom=fulltext

Tris(1,3-dichloro-2-propyl) Disrupts Axonal Growth, Cholinergic System and Motor Behavior in Early Life Zebrafish

Aquatic Toxicology

2017-09-05

Abstract

TDCIPP could have neurotoxic effects and alter motor behaviors in zebrafish (Danio rerio) larvae, however, the underlying mechanisms are still unknown. In this study, zebrafish embryos were subjected to waterborne exposure of TDCIPP at 100, 300, 600, 900 μg/L from 2 to 120-hour post-fertilization (hpf). Behavioral measurements indicate that TDCIPP exposure significantly elevated spontaneous movement, and altered swimming behavior response of larvae to both light and dark stimulation. Interestingly, in accordance with these motor effects, TDCIPP significantly decreased expression of the neuron-specific GFP in transgenic (HuC-GFP) zebrafish larvae as well as decreased expression of the neural marker genes elavl3 and ngn1, inhibited the axonal growth of the secondary motoneurons and altered the expressions of axon-related genes (α1-tubulin, shha and netrin2) in zebrafish larvae. Furthermore, TDCIPP exposure at 900 μg/L significantly increased the activity of acetylcholinesterase (AChE) enzyme, and decreased the total acetylcholine (ACh) concentration. Our data indicate that the alteration in motor neuron and inhibition of cholinergic system could together lead to the TDCIPP induced motor behavior alterations in zebrafish larvae.

 

http://www.sciencedirect.com/science/article/pii/S0166445X17302412

Alterations of larval photo-dependent swimming responses (PDR): New endpoints for rapid and diagnostic screening of aquatic contamination

Ecotoxicology and Environmental Safety

2017-09-19

Abstract

Detection and toxicity assessment of waterborne contaminants are crucial for protecting human health and the environment. Development of easy-to-implement, rapid and cost-effective tools to measure anthropogenic effects on watersheds are critical for responsible management, particularly in times of increasing development and urbanization. Traditionally, environmental toxicology has focused on limited endpoints, such as lethality and fertility, which are directly affecting population levels. However, more sensitive readings are needed to assess sub-lethal effects. Monitoring of contaminant-induced behavior alterations was proposed before, but is difficult to implement in the wild and performing it in aquatic laboratory models seem more suited. For this purpose, we adapted a photo-dependent swimming response (PDR) that was previously described in zebrafish larva. We first asked if PDR was present in other aquatic animals. We measured PDR in larvae from two freshwater prawn species (Macrobrachium rosenbergii, MR, and Macrobrachium carcinus, MC) and from another fish the fathead minnow (FHM, Pimephales promelas). In all, we found a strong and reproducible species-specific PDR, which is arguing that this behavior is important, therefore an environmental relevant endpoint. Next, we measured PDR in fish larvae after acute exposure to copper, a common waterborne contaminant. FHM larvae were hyperactive at all tested concentrations in contrast to ZF larvae, which exhibited a concentration-dependent hyperactivity. In addition to this well-accepted anxiety-like behavior, we examined two more: photo-stimulated startle response (PSSR) and center avoidance (CA). Both were significantly increased. Therefore, PDR measures after acute exposure to this waterborne contaminant provided as sensitive readout for its detection and toxicity assessment. This approach represents an opportunity to diagnostically examine any substance, even when present in complex mixtures like ambient surface waters. Mechanistic studies of toxicity using the extensive molecular tool kit of ZF could be a direct extension of such approaches.

 

http://www.sciencedirect.com/science/article/pii/S0147651317305936

Comparison of cannabinoids with known analgesics using a novel high throughput zebrafish larval model of nociception

2017-09-19

Abstract

It has been established that both adult and larval zebrafish are capable of showing nociceptive responses to noxious stimuli; however, the use of larvae to test novel analgesics has not been fully explored. Zebrafish larvae represent a low-cost, high-throughput alternative to traditional mammalian models for the assessment of product efficacy during the initial stages of drug development. In the current study, a novel model of nociception using zebrafish larvae is described. During the recovery from an acute exposure to low levels of acetic acid, larvae display innate changes in behaviour that may be indicative of nociception. To assess the usefulness of this model for testing potential analgesics, three known synthetic pain medications were assessed (ibuprofen, acetaminophen and tramadol) along with three naturally occurring products (honokiol, tetrahydrocannabinol and cannabidiol). When the effect of each compound on both the acetic acid recovery and control activity was compared there appeared to be both similarities and differences between the compounds. One of the most interesting effects was found for cannabidiol which appeared to oppose the activity change during the recovery period of AA exposed larvae while having a nominal effect on control activity. This would appear to be in line with current research that has demonstrated the nociceptive properties of cannabidiol. Here we have provided a novel model that will complement existing zebrafish models and will expand on the potential use of zebrafish larvae for studying both nociception and new analgesics.

 

http://www.sciencedirect.com/science/article/pii/S0166432817310239

Ecotoxicity of the insensitive munitions compound 3-nitro-1,2,4-triazol-5-one (NTO) and its reduced metabolite 3-amino-1,2,4-triazol-5-one (ATO)

Journal of Hazardous Materials

2017-09-28

Abstract

The insensitive munitions compound 3-nitro-1,2,4-triazol-5-one (NTO) was recently approved by the U.S. Army to replace cyclotrimethylene trinitramine (RDX) in conventional explosives. As its use becomes widespread, concern about the potential toxicity of NTO increases. NTO can undergo microbial reduction to 3-amino-1,2,4-triazol-5-one (ATO), which is recalcitrant in waterlogged soils. In this study, the acute toxicity of NTO and ATO towards various organisms, including microorganisms (i.e., methanogenic archaea, aerobic heterotrophs, and Aliivibrio fischeri (Microtox assay)), the microcrustacean Daphnia magna (ATO only), and zebrafish embryos (Danio rerio), was assessed. NTO was notably more inhibitory to methanogens than ATO (IC50 = 1.2 mM, > 62.8 mM, respectively). NTO and ATO did not cause noteworthy inhibition on aerobic heterotrophs even at the highest concentrations tested (32.0 mM). High concentrations of both NTO and ATO were required to inhibit A. fischeri (IC20 = 19.2, 22.4 mM, respectively). D. magna was sensitive to ATO (LC50 = 0.27 mM). Exposure of zebrafish embryos to NTO or ATO (750 μM) did not cause lethal or developmental effects (22 endpoints tested). However, both compounds led to swimming behavior abnormalities at low concentrations (7.5 μM). The results indicate that the reductive biotransformation of NTO could enhance or lower its toxicity according to the target organism.

 

http://www.sciencedirect.com/science/article/pii/S0304389417307422

Regulation of zebrafish (Danio rerio) locomotor behavior and circadian rhythm network by environmental steroid hormones

Environmental Pollution

2017-10-07

Abstract

Environmental exposure of fish to steroid hormones through wastewater and agricultural runoff may pose a health risk. Thus far, ecotoxicological studies have largely been focused on the disruption of the sex hormone system, but additional effects have been poorly investigated. Here we report on the effects of a series of different natural and synthetic steroid hormones on the locomotor behavior and the transcriptional levels of core clock genes in zebrafish eleuthero-embryos (Danio rerio). Of the 20 steroids analyzed, progestins and corticosteroids, including progesterone and cortisol, significantly decreased the locomotor activities of eleuthero-embryos at concentrations as low as 16 ng/L, while estrogens such as 17β-estradiol led to an increase. Consistently, progestins and corticosteroids displayed similar transcriptional effects on core clock genes, which were remarkably different from those of estrogens. Of these genes, per1a and nr1d2a displayed the most pronounced alterations. They were induced upon exposure to various progestins and corticosteroids and could be recovered using the progesterone receptor/glucocorticoid receptor antagonist mifepristone; this, however, was not the case for estrogens and the estrogen receptor antagonist 4-hydroxy-tamoxifen. Our results suggest that steroid hormones can modulate the circadian molecular network in zebrafish and provide novel insights into their mode of actions and potential environmental risks.

 

http://www.sciencedirect.com/science/article/pii/S0269749117316329

Inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) cause retinal damage in larval zebrafish

Toxicological Sciences OXFORD

2017-10-09

Abstract

Nicotinamide phosphoribosyltransferase (NAMPT) has been investigated as a target for oncology because it catalyzes a rate-limiting step in cellular energy metabolism to produce nicotinamide adenine dinucleotide. Small molecule inhibitors of NAMPT have been promising drug candidates but preclinical development has been hindered due to associated retinal toxicity. Here we demonstrate that larval zebrafish can predict retinal toxicity associated with this mechanism revealing an attractive alternative method for identifying such toxicities. Zebrafish permit higher throughput testing while using far lower quantities of test article compared to mammalian systems. NAMPT inhibitor-associated toxicity manifested in zebrafish as a loss of response to visual cues compared to auditory cues. Zebrafish retinal damage associated with NAMPT inhibitor treatment was confirmed through histopathology. Ranking six NAMPT inhibitors according to their impact on zebrafish vision revealed a positive correlation with their in vitro potencies on human tumor cells. This correlation indicates translatable pharmacodynamics between zebrafish and human NAMPT and is consistent with on-target activity as the cause of retinal toxicity associated with NAMPT inhibition. Together, these data illustrate the utility of zebrafish for identifying compounds that may cause ocular toxicity in mammals, and, likewise, for accelerating development of compounds with improved safety margins.

 

https://academic.oup.com/toxsci/article-abstract/4372457/Inhibitors-of-nicotinamide

Venlafaxine in embryos stimulates neurogenesis and disrupts larval behavior in zebrafish

Environmental Science & Technology

2017-10-11

Abstract

Venlafaxine, a widely prescribed antidepressant, is a selective serotonin and norepinephrine re-uptake inhibitor in humans, and this drug is prevalent in municipal wastewater effluents. While studies have shown that this drug affects juvenile fish behavior, little is known about the developmental impact on non-target aquatic animals. We tested the hypothesis that venlafaxine deposition in the egg, mimicking maternal transfer of this antidepressant, disrupts developmental programming using zebrafish (Danio rerio) as a model. Embryos (1-4 cell stage) were microinjected with either 1 or 10 ng venlafaxine, which led to a rapid reduction (90%) of this drug in the embryo at hatch. There was a concomitant increase in the concentration of the major metabolite o-desmethylvenlafaxine during the same period. Embryonic exposure to venlafaxine accelerated early development, increased hatching rate and produced larger larvae at 5 days post fertilization. Also, there was an increase in neuronal birth in the hypothalamus, dorsal thalamus, posterior tuberculum, and the preoptic region, and this corresponded with a higher spatial expression of nrd4, a key marker of neurogenesis. The venlafaxine-exposed larvae were less active and covered shorter distance in a light and dark behavioural test compared to the controls. Overall, zygotic exposure to venlafaxine disrupts early development, including brain function, and compromises larval behaviour, suggesting impact of this drug on developmental programming in zebrafish.

 

http://pubs.acs.org/doi/abs/10.1021/acs.est.7b04099

Comparative developmental toxicity of a comprehensive suite of polycyclic aromatic hydrocarbons

Archives of Toxicology

2017-11-01

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants that occur in complex mixtures. Several PAHs are known or suspected mutagens and/or carcinogens, but developmental toxicity data is lacking for PAHs, particularly their oxygenated and nitrated derivatives. Such data are necessary to understand and predict the toxicity of environmental mixtures. 123 PAHs were assessed for morphological and neurobehavioral effects for a range of concentrations between 0.1 and 50 µM, using a high throughput early-life stage zebrafish assay, including 33 parent, 22 nitrated, 17 oxygenated, 19 hydroxylated, 14 methylated, 16 heterocyclic, and 2 aminated PAHs. Additionally, each PAH was evaluated for AHR activation, by assessing CYP1A protein expression using whole animal immunohistochemistry (IHC). Responses to PAHs varied in a structurally dependent manner. High-molecular weight PAHs were significantly more developmentally toxic than the low-molecular weight PAHs, and CYP1A expression was detected in five distinct tissues, including vasculature, liver, skin, neuromasts and yolk.

 

https://link.springer.com/article/10.1007/s00204-017-2068-9

A Novel Experimental and Modelling Strategy for Nanoparticle Toxicity Testing Enabling the Use of Small Quantities

Int. J. Environ. Res. Public Health

2017-11-06

Abstract

Metallic nanoparticles (NPs) differ from other metal forms with respect to their large surface to volume ratio and subsequent inherent reactivity. Each new modification to a nanoparticle alters the surface to volume ratio, fate and subsequently the toxicity of the particle. Newly-engineered NPs are commonly available only in low quantities whereas, in general, rather large amounts are needed for fate characterizations and effect studies. This challenge is especially relevant for those NPs that have low inherent toxicity combined with low bioavailability. Therefore, within our study, we developed new testing strategies that enable working with low quantities of NPs. The experimental testing method was tailor-made for NPs, whereas we also developed translational models based on different dose-metrics allowing to determine dose-response predictions for NPs. Both the experimental method and the predictive models were verified on the basis of experimental effect data collected using zebrafish embryos exposed to metallic NPs in a range of different chemical compositions and shapes. It was found that the variance in the effect data in the dose-response predictions was best explained by the minimal diameter of the NPs, whereas the data confirmed that the predictive model is widely applicable to soluble metallic NPs. The experimental and model approach developed in our study support the development of (eco)toxicity assays tailored to nano-specific features.

 

http://www.mdpi.com/1660-4601/14/11/1348/htm

Characterization of a Zebrafish Model of X-linked Centronuclear Myopathy for Therapeutic Drug Development

Molecular Genetics

2017-11-14

Abstract


X-linked centronuclear myopathy (XLCNM) is a congenital skeletal muscle disorder caused by mutations in MTM1. Despite being associated with a high rate of neonatal mortality there are currently no effective therapies for this disorder. MTM1 is a phosphoinositide 3-phosphatase that antagonizes class II and III phosphatidylinositol 3-kinases (PI3K). Loss of Pik3c2b, a class II PI3K, rescues lethality in Mtm1 knockout mice. Importantly, Pik3c2b knockout mice are viable and have no apparent phenotype. Therefore, pharmacological inhibitors of PIK3C2B have the potential to be highly effective in the treatment of XLCNM. I have characterized an mtm1 mutant using the zebrafish, Danio rerio, and found that PIK3C2B inhibition improves the mtm1 mutant phenotype. In parallel, a phenotypic drug screen identified valproic acid as a suppressor of an mtm1 mutant phenotype. The work presented herein demonstrates that PIK3C2B inhibitors and drugs like valproic acid may represent putative therapeutics for translation into MTM1 patients.

 

 

Mechanisms underlying melatonin-mediated prevention of fenvalerate-induced behavioral and oxidative toxicity in zebrafish

Journal of Toxicology and Environmental Health

2017-11-16

ABSTRACT

The neurotoxic effects attributed to the pesticide fenvalerate (FEN) are well-established. The aim of this study was to determine whether melatonin (MLT) was able to protect against FEN-induced behavior, oxidative stress, apoptosis, and neurogenesis using zebrafish (Danio rerio) model. Zebrafish exposed to 100 μg/L FEN for 120 h exhibited decreased swimming activity accompanied by downregulated expression of neurogenesis-related genes (Dlx2, Shha, Ngn1, Elavl3, and Gfap), suggesting that neurogenesis were impaired. In addition, FEN exposure significantly elevated oxidative stress as evidenced by increased malondialdehyde levels, as well as activities of Cu/Zn superoxide dismutase (Cu/Zn SOD), catalase, and glutathione peroxidase. Acridine orange staining demonstrated that embryos treated with FEN for 120 h significantly enhanced apoptosis mainly in the brain. FEN also produced upregulation of the expression of the pro-apoptotic genes (Bax, Fas, caspase 8, caspase 9, and caspase 3) and decreased expression of the anti-apoptotic gene Bcl-2. MLT significantly attenuated the FEN-mediated oxidative stress, modulated apoptotic-regulating genes, and diminished apoptotic responses. Further, MLT blocked the FEN-induced effects on swimming behavior as well as on neurogenesis-related genes. In conclusion, MLT protected against FEN-induced developmental neurotoxicity and apoptosis by inhibiting pesticide-mediated oxidative stress in zebrafish.

 

http://www.tandfonline.com/doi/abs/10.1080/15287394.2017.1384167

Methylated flavonoids as anti-seizure agents: Naringenin 4′,7-dimethyl ether attenuates epileptic seizures in zebrafish and mouse models

Neurochemistry International

2017-11-22

Abstract

Epilepsy is a neurological disease that affects more than 70 million people worldwide and is characterized by the presence of spontaneous unprovoked recurrent seizures. Existing anti-seizure drugs (ASDs) have side effects and fail to control seizures in 30% of patients due to drug resistance. Hence, safer and more efficacious drugs are sorely needed. Flavonoids are polyphenolic structures naturally present in most plants and consumed daily with no adverse effects reported. These structures have shown activity in several seizure and epilepsy animal models through allosteric modulation of GABAA receptors, but also via potent anti-inflammatory action in the brain. As such, dietary flavonoids offer an interesting source for ASD and anti-epileptogenic drug (AED) discovery, but their pharmaceutical potential is often hampered by metabolic instability and low oral bioavailability. It has been argued that their drug-likeness can be improved via methylation of the free hydroxyl groups, thereby dramatically enhancing metabolic stability and membrane transport, facilitating absorption and highly increasing bioavailability. Since no scientific data is available regarding the use of methylated flavonoids in the fight against epilepsy, we studied naringenin (NRG), kaempferol (KFL), and three methylated derivatives, i.e., naringenin 7-O-methyl ether (NRG-M), naringenin 4′,7-dimethyl ether (NRG-DM), and kaempferide (4′-O-methyl kaempferol) (KFD) in the zebrafish pentylenetetrazole (PTZ) seizure model. We demonstrate that the methylated flavanones NRG-DM and NRG-M are highly effective against PTZ-induced seizures in larval zebrafish, whereas NRG and the flavonols KFL and KFD possess only a limited activity. Moreover, we show that NRG-DM is active in two standard acute mouse seizure models, i.e., the timed i.v. PTZ seizure model and the 6-Hz psychomotor seizure model. Based on these results, NRG-DM is proposed as a lead compound that is worth further investigation for the treatment of generalized seizures and drug-resistant focal seizures. Our data therefore highlights the potential of methylated flavonoids in the search for new and improved ASDs.

 

https://www.sciencedirect.com/science/article/pii/S0197018617302243

Automatic quantification of juvenile zebrafish aggression

Journal of Neuroscience Methods

2017-12-21

Abstract


Background

Although aggression is a common symptom of psychiatric disorders the drugs available to treat it are non-specific and can have unwanted side effects. The zebrafish is an ideal model for aggression research. Zebrafish are small, amenable to genetic and pharmacological manipulation, and agonistic behaviour can be measured reliably.


New method

In this study we have established a novel setup to automatically quantify aggression and locomotion in one-month old juvenile zebrafish, a stage at which fish exhibit adult-like behaviour but are small that one camera can film several animals.


Results

We have validated our novel software by comparison to manual quantification of behaviour, characterised the aggression of one-month old fish, and demonstrated that we can detect alterations to aggression caused by mutation or drug application.


Comparison with other methods

The ability to record up to 12 juvenile fish allows us to speed up and standardise data acquisition compared to studies of single fish.


Conclusions

This setup appears to be suitable to screen for drugs that decrease zebrafish aggression as a first step toward developing novel treatments for this behaviour.

 

https://www.sciencedirect.com/science/article/pii/S0165027017304302

Novel Kunitz-like peptides discovered in the zoanthid Palythoa caribaeorum through transcriptome sequencing

Journal of Proteome

2017-12-29

Abstract

Palythoa caribaeorum (class Anthozoa) is a zoanthid that together jellyfishes, hydra, and sea anemones, which are venomous and predatory, belongs to the Phyllum Cnidaria. The distinguished feature in these marine animals is the cnidocytes in the body tissues, responsible for toxin production and injection that are used majorly for prey capture and defense. With exception for other anthozoans, the toxin cocktails of zoanthids have been scarcely studied and are poorly known. Here, based on the analysis of P. caribaeorum transcriptome, numerous predicted venom-featured polypeptides were identified, including allergens, neurotoxins, membrane-active and Kunitz-like peptides (PcKuz). The three predicted PcKuz isotoxins (1 to 3) were selected for functional studies. Through computational processing comprising structural phylogenetic analysis, molecular docking, and dynamics simulation, PcKuz3 was shown to be a potential voltage gated potassium-channel inhibitor. PcKuz3 fitted well as new functional Kunitz-type toxins with strong anti-locomotor activity as in vivo assessed in zebrafish larvae, with weak inhibitory effect toward proteases, as evaluated in vitro. Notably, PcKuz3 can suppress, at low concentration, the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish, which indicated PcKuz3 may have a neuroprotective effect. Taken together, PcKuz3 figures as a novel neurotoxin structure which differs from known homologous peptides expressed in sea anemone. Moreover, the novel PcKuz3 provides an insightful hint for bio-drug development for prospective neurodegenerative disease treatment.

Nonlinear mixed-modelling discriminates the effect of chemicals and their mixtures on zebrafish behavior

Nature

2018-01-12

Abstract

Zebrafish (Danio rerio) early-life stage behavior has the potential for high-throughput screening of neurotoxic environmental contaminants. However, zebrafish embryo and larval behavioral assessments typically utilize linear analyses of mean activity that may not capture the complexity of the behavioral response. Here we tested the hypothesis that nonlinear mixed-modelling of zebrafish embryo and larval behavior provides a better assessment of the impact of chemicals and their mixtures. We demonstrate that zebrafish embryo photomotor responses (PMRs) and larval light/dark locomotor activities can be fit by asymmetric Lorentzian and Ricker-beta functions, respectively, which estimate the magnitude of activity (e.g., maximum and total activities) and temporal aspects (e.g., duration of the responses and its excitatory periods) characterizing early life-stage zebrafish behavior. We exposed zebrafish embryos and larvae to neuroactive chemicals, including isoproterenol, serotonin, and ethanol, as well as their mixtures, to assess the feasibility of using the nonlinear mixed-modelling to assess behavioral modulation. Exposure to chemicals led to distinct effects on specific behavioral characteristics, and interactive effects on temporal characteristics of the behavioral response that were overlooked by the linear analyses of mean activity. Overall, nonlinear mixed-modelling is a more comprehensive approach for screening the impact of chemicals and chemical mixtures on zebrafish behavior.

Introduction

Zebrafish (Danio rerio) have proven to be an excellent model for ecotoxicological applications1, in part due to their tractability in laboratory settings, high fecundity, and well-understood and precisely-timed ontogeny2. The zebrafish is also an excellent model to study chemical effects on developmental programming, as well as early life-stage behavior3,4,5. For instance, as early as 30 hours post fertilization (hpf), zebrafish embryos exhibit a short photomotor response (PMR) involving a spike in movement over a 10 s period4. The PMR has high plasticity to drug exposure and has been used for high-throughput screening of neuroactive chemicals6,7. Also, after hatch (>72 hpf) the larvae exhibit distinct locomotor activity to alternating dark and light photoperiods8. Locomotor activity is supressed when larvae are held in an illuminated environment, while a switch to complete darkness evokes a spike in locomotor activity lasting around 15 min5,8. Recent studies suggest that this larval behavior may be disrupted by environmental contaminants5,8,9.

Although animal behavioral endpoints hold promise in ecotoxicology for risk assessments, they are not widely used by regulatory agencies. A major challenge in using animal behavior for risk assessment involves the wide-ranging responses that require careful assessment and selection of the most appropriate statistical analyses for biological/ecological relevance10. Animal activity is often measured by averaging the values temporally and/or spatially to make them amenable for linear analyses11,12. However, that greatly simplifies behavioral analyses because activity changes are seldom linear. This is exemplified by the nonlinear embryo PMR and larval locomotor activity profiles (i.e., asymmetric peak- and hump-shaped respectively6,8). These activity profiles reveal behavioral characteristics, including quickness, duration and maximum intensity, which are analogous to common parameters of nonlinear functions13.

Nonlinear mixed-modelling includes both fixed-effects (i.e., model parameters – phenotypical characteristics) and random-effects (i.e., within-subject effects), and allows for phenotype comparisons, while controlling for repeated measurements of activity during the behavioral trials14,15. Also, the inclusion of an interaction term in the fixed-effects component of the model allows for testing interactive effects16. Against this backdrop, our objective was to test the feasibility of using nonlinear mixed-modelling to assess the effects of chemicals either alone or as mixtures on zebrafish embryo and larval activity. Specifically, we tested the hypothesis that nonlinear mixed-modelling is a more sensitive representation of chemical effects and their interactions on early life-stage zebrafish behavior compared to linear modelling of mean activity. As a proof of concept, we carried out zebrafish embryo PMR and larval locomotor activity trials with fish exposed to model chemicals that either stimulate or suppress embryo and larvae activity. We also co-exposed embryos and larvae to a mixture of the stimulant and suppressor to demonstrate the capacity of nonlinear mixed-modelling in testing and describing mixture toxicity.

Methods

Zebrafish maintenance and embryo collection

Adult zebrafish (Tupfel long fin strain) were cultured in 10 L polypropylene tanks at 28.5 °C, pH 7.6, and 740 µS conductivity on recirculating systems (Pentair Aquatic Habitats, Apopka, Florida). The recirculating systems were housed in an animal care facility at the University of Calgary with a 14 h:10 h light:dark daily light cycle. Animals were fed with ZieglerTM adult zebrafish diet (Pentair) and live Artemia (San Francisco Bay Brand, Inc, Newark, CA, USA) in the morning and evening respectively. Zebrafish were bred and the early life-stages maintained in E3 medium17 at 28.5 °C as described previously5. The animal maintenance and all experiments were approved by the animal care committee at the University of Calgary (AC17-0079) and were in accordance with the Canadian Council on Animal Care guidelines.


Zebrafish PMR

We followed the protocol outlined in Kokel et al.6 with slight modifications. We 3D-printed a custom light emitting diode (LED)-array for use with a ZebraBox behavioral acquisition system (Viewpoint Life Sciences, Montreal, QC, Canada) that allowed for multiple configurations of LEDs (Super Bright LEDs, St. Louis, Missouri, USA) that could be activated with a remote switch. PMR trials were 30 s in duration and were carried out in total darkness, with the exception of two 1 s light pulses at 10 and 20 s6, and were recorded at 30 frames per second. Embryos were dechorionated at 24 hpf in 1 g L−1 Pronase (Sigma) and transferred (6 embryos per well) to the center 48 wells of a 96-well plate (Greiner, Sigma) with 225 µL of embryo medium per well. Only the center 48 wells were used to maximize magnification and resolution of video acquisition for activity measurements. Preliminary work identified that exposure to the β-adrenergic receptor agonist, isoproterenol, and ethanol stimulated and supressed the PMR, respectively. The treatments included 100 µM isoproterenol, 2% ethanol or a combination of both and the exposures began 30 min before each PMR trial. Final well volume was 300 uL following additions of chemical stocks. Treatments were randomly assigned by plate column, and each plate contained 2 columns (i.e., 16 wells) of control, isoproterenol, ethanol, and isoproterenol-ethanol treatments. A total of 360 embryos per treatment were used for the PMR experiments. The PMR trials began at 32 hpf with 20 min dark acclimation periods between plates. Embryo activity was quantified as Δ pixel intensity from each frame.


Zebrafish larval locomotor activity

Larvae were transferred to each well of a 96-well plate at 80 hpf along with 225 µL of embryo medium and maintained overnight at 28.5 °C. Preliminary work identified that exposure to isoproterenol and serotonin suppressed and stimulated larval locomotor activity, respectively. The next day each well received either 20 µM isoproterenol, 100 µM serotonin or a combination of both and each plate contained 24 wells/larvae per treatment (a total of 3 plates). Final well volume was 300 uL following additions of chemical stocks. Immediately following the chemical exposure, plates were transferred to the Zebrabox and behavioral trials commenced as described previously18, following the lighting regime of Emran et al.8. The Zebrabox system includes backlighting in the visible spectrum from 0% to 100% intensity. Our lighting regime included 30 min at 0% intensity (i.e., dark adaptation), 30 min at 100% intensity, and a final 30 min at 0% intensity. Larval activity was calculated as total distance travelled every 30 s as described previously5,18.

 

https://www.nature.com/articles/s41598-018-20112-x

A novel metabolism-based phenotypic drug discovery platform in zebrafish uncovers HDACs 1 and 3 as a potential combined anti-seizure drug target

Brain. A journal of Neurology

2018-01-24

Abstract

Despite the development of newer anti-seizure medications over the past 50 years, 30-40% of patients with epilepsy remain refractory to treatment. One explanation for this lack of progress is that the current screening process is largely biased towards transmembrane channels and receptors, and ignores intracellular proteins and enzymes that might serve as efficacious molecular targets. Here, we report the development of a novel drug screening platform that harnesses the power of zebrafish genetics and combines it with in vivo bioenergetics screening assays to uncover therapeutic agents that improve mitochondrial health in diseased animals. By screening commercially available chemical libraries of approved drugs, for which the molecular targets and pathways are well characterized, we were able to reverse-identify the proteins targeted by efficacious compounds and confirm the physiological roles that they play by utilizing other pharmacological ligands. Indeed, using an 870-compound screen in kcna1-morpholino epileptic zebrafish larvae, we uncovered vorinostat (Zolinza™; suberanilohydroxamic acid, SAHA) as a potent anti-seizure agent. We further demonstrated that vorinostat decreased average daily seizures by ∼60% in epileptic Kcna1-null mice using video-EEG recordings. Given that vorinostat is a broad histone deacetylase (HDAC) inhibitor, we then delineated a specific subset of HDACs, namely HDACs 1 and 3, as potential drug targets for future screening. In summary, we have developed a novel phenotypic, metabolism-based experimental therapeutics platform that can be used to identify new molecular targets for future drug discovery in epilepsy.

Behavioural assay

For drug screening using kcna1 morphants, 5 days post-fertilization (dpf) larval zebrafish maintained in 96-well plates were habituated for 20 min, under ambient light. This was followed by treatment with 20 μM of drugs for 20–30 min (n = 6–7 per compound), and assayed for locomotor activity, burst activity, distance moved, inactive duration and tracking in dark for 20 min in ZebraBox (ViewPoint Life Sciences). For drug screening involving PTZ induction, 5 dpf zebrafish were treated with 10 mM PTZ for 10 min, followed by 20 μM (final concentration) of drug for 20 min under ambient light, and assayed for total locomotor activity, burst activity, distance moved and inactive duration, and tracking under 100% light in ZebraBox for 20 min. All behavioural assays were repeated at least twice. Total swim activity and tracking of total distance moved as a measure of hyperactive swimming behaviour were analysed using ZebraLab V3 software (ViewPoint Life Sciences, Lyon, France).

https://academic.oup.com/brain/advance-article/doi/10.1093/brain/awx364/4823617#109328665

NCBINCBI Logo Skip to main content Skip to navigation Resources How To About NCBI Accesskeys Sign in to NCBI PubMed US National Library of Medicine National Institutes of Health Search database Search term Clear input Advanced Help Result Filters Format: Abstract Send to Chemosphere. 2018 Jan 25;197:611-621. doi: 10.1016/j.chemosphere.2018.01.092. [Epub ahead of print] Developmental toxicity induced by PM2.5 through endoplasmic reticulum stress and autophagy pathway in zebrafish embryos.

Chemosphere

2018-02-01

Abstract

The aims of this study were to investigate the mechanism underlying the developmental toxicity of fine particulate matter (PM2.5) and provide a more thorough understanding of the toxicity of PM2.5 in an ecological environment. Zebrafish embryos at 4 h post-fertilization were exposed to PM2.5 at doses of 200, 300, 400, 500, 600 and 800 μg/mL for 120 h. The mortality, hatching rate, morphology score, body length, locomotor capacity, histological changes, antioxidant defense system, leukocyte migration, inflammation-related gene mRNA expression, endoplasmic reticulum stress (ERS) and autophagy were evaluated to study PM2.5-induced developmental toxicity and its underlying mechanisms. PM2.5 exposure significantly increased the mortality and malformations and reduced the hatching rate and body length of the zebrafish. PM2.5 significantly reduced the locomotor capacity of zebrafish larvae, increased the levels of ROS and disturbed the antioxidant defense system in zebrafish larvae. In addition, a histological examination showed that the heart, liver, intestines and muscle of the PM2.5-treated zebrafish exhibited abnormal changes and a significant increase in cellular autophagic accumulation. RT-PCR showed that the expression of genes related to inflammation (tgfβ and cox2), ERS (hspa5, chop, ire1, xbp1s, and atf6) and autophagy (lc3, beclin1 and atg3) pathways was significantly increased in the PM2.5-treated zebrafish, indicating that PM2.5 induced inflammation and promoted ERS and autophagy responses via the activation of the IRE1-XBP1 and ATF6 pathways. Together, our data indicate that PM2.5 induced a dose- and time-dependent increase in developmental toxicity to zebrafish embryos. Additionally, ERS and autophagy may play important roles in PM2.5-induced developmental toxicity.

 

Link to the publication :

 

https://www.sciencedirect.com/science/article/pii/S0045653518301097

 

 

Effects of UV filter 4-methylbenzylidene camphor during early development of Solea senegalensis Kaup, 1858

Science of The Total Environment

2018-02-20

Highlights

4MBC induced mortality, malformations and reduced growth in S. senegalensis larvae.

Swimming behaviour was affected by 4MBC at low concentrations.

4MBC caused alterations in AChE and LDH biochemical markers.

Assessment of behaviour is recommended in ecotoxicological studies with sole larvae.


Abstract

The inclusion of organic UV filters in personal care products (PCPs) has increased in recent years. 4-Methylbenzylidene camphor (4MBC) is one of the most used UV filters, and thus it is commonly found in aquatic ecosystems, with proved negative effects on aquatic organisms. Effects on early life stages of marine vertebrates are largely unknown. Therefore, the main goal of this work was to evaluate 4MBC effects on Senegalese sole (Solea Senegalensis Kaup, 1858) larvae at different levels of biological organization. S. senegalensis were exposed to increasing concentrations of 4MBC from egg stage until 96 h. Mortality, growth, malformations, behaviour and biochemical responses, including enzymatic biomarkers were studied. The exposure to 4MBC until 96 h post-fertilization (hpf) induced mortality and malformations in a dose-response manner. Besides, reduced growth with increasing concentrations was observed. The exposure to 4MBC also caused alterations on behaviour, including overall lower swimming time during light and dark periods. Biomarkers alterations caused by 4MBC included imbalance of neurotransmission related endpoints (increased acetylcholinesterase activity) and decreased activity of enzymes related to anaerobic metabolism (lower cellular lactate dehydrogenase activity) at the lower concentrations tested. Furthermore, our results suggest that 4MBC do not induce oxidative stress in S. senegalensis larvae, since catalase and lipid peroxidation levels were not significantly altered by 4MBC.

S. senegalensis revealed to be a good model species for vertebrate animal testing in the marine environment. Sub-lethal concentrations of 4MBC induced toxic effects at all organizational levels. Swimming behaviour was a sensitive endpoint and showed that exposure to 4MBC causes impairment on response to light stimulus which is possibly linked with the observed imbalances on cholinesterase activity in larvae. Conservation concerns along distribution range of S. senegalensis should consider that increasing levels of UV filters in marine environment might have impact on the ecology of the species.

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0048969718305023

Development of a high-throughput in vivo screening platform for particulate matter exposures

Environmental Pollution

2018-02-21

Highlights

Standardized methods for PM exposure established in developmental zebrafish model.

PM associated bioactivity included mortality, morphological and behavioral responses.

Concentration dependent cyp1a/GFP expression was observed for PM exposures.

Zebrafish is a sensitive in vivo model of PM toxicity.

High-throughput screening of PM will expand knowledge of PM-health effects.


Abstract

Particulate matter (PM) exposure is a public health burden with poorly understood health effect mechanisms and lacking an efficient model to compare the vast diversity of PM exposures. Zebrafish (Danio rerio) are amenable to high-throughput screening (HTS), but few studies have investigated PM toxicity in zebrafish, despite the multitude of advantages. To develop standardized exposure procedures, the urban PM standard reference material (SRM) 1649b was used to systematically determine sample preparation methods, design experimental controls, determine concentration ranges and evaluation procedures. Embryos (n = 32/treatment) were dechorionated and placed into 96-well plates containing SRM1649b (0–200 μg/mL) at 6 h post fertilization (hpf). Developmental toxicity was assessed at 24 and 120 hpf by evaluating morphological changes, embryonic/larval photomotor behavior, and mortality. Differences from blank medium and particle controls were observed for all biological responses measured. Differences due to SRM1649b concentration and preparation method were also observed. Exposure to SRM1649b from DMSO extraction was associated with changes in morphology and mortality and hypoactivity in photomotor responses compared to the DMSO control for the whole particle suspension (76, 68%) and soluble fraction (59, 54%) during the embryonic and larval stages, respectively. Changes in behavioral responses were not observed following exposure to the insoluble fraction of SRM1649b from DMSO extraction. The toxicity bias from PM preparation provided further impetus to select a single HTS exposure method. Based on the biological activity results, the soluble fraction of SRM1649b from DMSO extraction was selected and shown to have concentration dependent cyp1a/GFP expression. This rapid, sensitive and consistently scalable model is a potentially cost-effective vertebrate approach to study the toxicology of PM from diverse locations, and provides a path to identifying the toxic material(s) in these samples, and discover the mechanisms of toxicity.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0269749117344469

 

Graphene Oxide Quantum Dots Reduce Oxidative Stress and Inhibit Neurotoxicity In Vitro and In Vivo through Catalase-Like Activity and Metabolic Regulation

Advanced Science

2018-03-04

Abstract

Both oxidative stress and neurotoxicity are huge challenges to human health, and effective methods and agents for resisting these adverse effects are limited, especially in vivo. It is shown here that, compared to large graphene oxide (GO) nanosheets, GO quantum dots (GOQDs), as nanozymes, efficiently reduce reactive oxygen species (ROS) and H2O2 in 1-methyl-4-phenyl-pyridinium ion (MPP+)-induced PC12 cells. In addition, GOQDs exert neuroprotective effects in a neuronal cell model by decreasing apoptosis and α-synuclein. GOQDs also efficiently diminish ROS, apoptosis, and mitochondrial damage in zebrafish treated with MPP+. Furthermore, GOQDs-pretreated zebrafish shows increased locomotive activity and Nissl bodies in the brain, confirming that GOQDs ameliorate MPP+-induced neurotoxicity, in contrast to GO nanosheets. GOQDs contribute to neurotoxic amelioration by increasing amino acid metabolism, decreasing tricarboxylic acid cycle activity, and reducing steroid biosynthesis, fatty acid biosynthesis, and galactose metabolic pathway activity, which are related to antioxidation and neurotransmission. Meanwhile, H2O2 decomposition and Fenton reactions suggest the catalase-like activity of GOQDs. GOQDs can translocate into zebrafish brains and exert catalase-mimicking activity to resist oxidation in the intracellular environment. Unlike general nanomaterials, biocompatible GOQDs demonstrate their high potential for human health by reducing oxidative stress and inhibiting neurotoxicity.

 

http://onlinelibrary.wiley.com/doi/10.1002/advs.201700595/full

Systematic developmental neurotoxicity assessment of a representative PAH Superfund mixture using zebrafish

Toxicology and Applied Pharmacology

2018-04-06

Abstract

Superfund sites often consist of complex mixtures of polycyclic aromatic hydrocarbons (PAHs). It is widely recognized that PAHs pose risks to human and environmental health, but the risks posed by exposure to PAH mixtures are unclear. We constructed an environmentally relevant PAH mixture with the top 10 most prevalent PAHs (SM10) from a Superfund site derived from environmental passive sampling data. Using the zebrafish model, we measured body burden at 48 hours post fertilization (hpf) and evaluated the developmental and neurotoxicity of SM10 and the 10 individual constituents at 24 hours post fertilization (hpf) and 5 days post fertilization (dpf). Zebrafish embryos were exposed from 6 to 120 hpf to (1) the SM10 mixture, (2) a variety of individual PAHs: pyrene, fluoranthene, retene, benzo[a]anthracene, chrysene, naphthalene, acenaphthene, phenanthrene, fluorene, and 2-methylnaphthalene. We demonstrated that SM10 and only 3 of the individual PAHs were developmentally toxic. Subsequently, we constructed and exposed developing zebrafish to two sub-mixtures: SM3 (comprised of 3 of the developmentally toxicity PAHs) and SM7 (7 non-developmentally toxic PAHs). We found that the SM3 toxicity profile was similar to SM10, and SM7 unexpectedly elicited developmental toxicity unlike that seen with its individual components. The results demonstrated that the overall developmental toxicity in the mixtures could be explained using the general concentration addition model. To determine if exposures activated the AHR pathway, spatial expression of CYP1A was evaluated in the 10 individual PAHs and the 3 mixtures at 5 dpf. Results showed activation of AHR in the liver and vasculature for the mixtures and some individual PAHs. Embryos exposed to SM10 during development and raised in chemical-free water into adulthood exhibited decreased learning and responses to startle stimulus indicating that developmental SM10 exposures affect neurobehavior. Collectively, these results exemplify the utility of zebrafish to investigate the developmental and neurotoxicity of complex mixtures.

Keywords

Passive sampling; Polycyclic aromatic hydrocarbons; Behavior; Aryl hydrocarbon receptor; Cytochrome P4501A; Biomarker

 

https://www.sciencedirect.com/science/article/pii/S0041008X18301212

Analysis of the Uptake, Metabolism, and Behavioral Effects of Cannabinoids on Zebrafish Larvae

Zebrafish

2018-04-10

Abstract

The Cannabis sativa plant contains numerous phytocannabinoids and terpenes with known or potential biological activity. For decades, plant breeders have been breeding the Cannabis plant to control for a desired ratio of the major cannabinoids. A high-throughput in vivo model to understand the relationship between the chemical composition of different strains and their therapeutic potential then becomes of value. Measuring changes in the behavioral patterns of zebrafish larvae is an established model with which to test the biological activity of neuroactive compounds. However, there is currently little information regarding the uptake kinetics and metabolism of compounds by larvae. In this study, we chose to compare the uptake kinetics and metabolism of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) alone or in combination with their effects on larval behavior. We have shown that both compounds have distinct behavioral patterns and concentration response profiles. Additionally, the uptake kinetics observed for each compound appears to correlate with the change in behavior observed in the behavioral assays. When combinations of THC and CBD were tested there were shifts in both the behavioral activity and the uptake kinetics of each compound compared with when they were tested alone. Finally, the THC/CBD-derived metabolites detected in the larvae are similar to those found in mammalian systems. This study thus provides a model for further testing of additional cannabinoids and potentially plant extracts.

 

Keywords

zebrafish ; cannabinoid ; metabolism ; behavior ; THC ; CBD

 

https://www.liebertpub.com/doi/abs/10.1089/zeb.2017.1541

Effects of ozonated sewage effluent on reproduction and behavioral endpoints in zebrafish (Danio rerio)

Aquatic Toxicology

2018-04-25

Abstract

Pharmaceutical residues and other micro-contaminants may enter aquatic environments through effluent from sewage treatment plants (STPs) and could cause adverse effects in wild fish. One strategy to alleviate this situation is to improve wastewater treatment by ozonation. To test the effectiveness of full-scale wastewater effluent ozonation at a Swedish municipal STP, the added removal efficiency was measured for 105 pharmaceuticals. In addition, gene expression, reproductive and behavioral endpoints were analyzed in zebrafish (Danio rerio) exposed on-site over 21 days to ozonated or non-ozonated effluents as well as to tap water. Ozone treatment (7 g O3/m3) removed pharmaceuticals by an average efficiency of 77% in addition to the conventional treatment, leaving 11 screened pharmaceuticals above detection limits. Differences in biological responses of the exposure treatments were recorded in gene expression, reproduction and behavior. Hepatic vitellogenin gene expression was higher in male zebrafish exposed to the ozonated effluent compared to the non-ozonated effluent and tap water treatments. The reproductive success was higher in fish exposed to ozonated effluent compared to non-ozonated effluent and to tap water. The behavioral measurements showed that fish exposed to the ozonated STP effluent were less active in swimming the first minute after placed in a novel vessel. Ozonation is a capable method for removing pharmaceuticals in effluents. However, its implementation should be thoroughly evaluated for any potential biological impact. Future research is needed for uncovering the factors which produced the in vivo responses in fish.

 

Keywords
Fecundity ; Vitellogenin ; Ozone ; Pharmaceuticals ; Wastewater

 

https://www.sciencedirect.com/science/article/pii/S0166445X18303631

Modulation of Threat Response in Larval Zebrafish

Phenotypic Screening

2018-05-08

Abstract

High-throughput, whole-organism phenotypic drug screening is made possible using live zebrafish larvae. Many human drugs have now been shown to affect zebrafish larvae in similar ways, through homologous molecular mechanisms. At this stage in life, zebrafish are small enough to fit in multi-well, microliter plates, yet developed enough to exhibit complex phenotypes, such as hunting behaviors and avoidance of predators. Importantly, zebrafish larvae can be easily dosed via automated pipetting of chemical compounds directly into their liquid medium, without injection. Only microgram amounts of small molecules are required, making animal husbandry and dosing regimens cost effective. This chapter describes how the stereotyped zebrafish larval responses to darkness and strobe light—which cause hyperactivity and freezing behavior, respectively—can be used to efficiently screen small molecules for brain and behavior-modulating activity.


Key words

Fear Freezing behavior CNS Whole-organism screening Phenotypic screens High-throughput drug discovery Small molecules Strobe light response

 

https://link.springer.com/protocol/10.1007/978-1-4939-7847-2_11

Perfluorododecanoic acid exposure induced developmental neurotoxicity in zebrafish embryos

Environmental Pollution

2018-06-08

Abstract

Perfluorododecanoic acid (PFDoA), an artificial perfluorochemical, has been widely distributed in different ambient media and has been reported to have the potential to cause developmental neurotoxicity. However, the specific mechanism is largely unknown. In the current study, zebrafish embryos were treated with 0, 0.24, 1.2, and 6 mg/L PFDoA for 120 h. Exposure to PFDoA causes serious decreases in hatching delay, body length, as well as decreased locomotor speed in zebrafish larvae. Additionally, the acetylcholine (ACh) content as well as acetylcholinesterase (AChE) activity were determined to be significantly downregulated in PFDoA treatment groups. The level of dopamine was upregulated significantly after treating with 1.2 and 6 mg/L of PFDoA. Gene expressions related to the nervous system development were also analyzed, with the exception of the gene mesencephalic astrocyte-derived neurotrophic factor (manf), which is upregulated in the 6 mg/L treatment group. All other genes were significantly downregulated in larvae in the PFDoA group in different degrees. In general, the results demonstrated that PFDoA exposure could result in the disruption of the cholinergic system, dopaminergic signaling, and the central nervous system.

Keywords

PFDoA; Zebrafish embryo; Acetylcholine; Dopamine; Developmental neurotoxicity

 

https://www.sciencedirect.com/science/article/pii/S0269749117352247

Comparative behavioral toxicology with two common larval fish models: Exploring relationships among modes of action and locomotor responses

Science of The Total Environment

2018-06-18

Abstract

Behavioral responses inform toxicology studies by rapidly and sensitively detecting molecular initiation events that propagate to physiological changes in individuals. These behavioral responses can be unique to chemical specific mechanisms and modes of action (MOA) and thus present diagnostic utility. In an initial effort to explore the use of larval fish behavioral response patterns in screening environmental contaminants for toxicity and to identify behavioral responses associated with common chemical specific MOAs, we employed the two most common fish models, the zebrafish and the fathead minnow, to define toxicant induced swimming activity alterations during interchanging photoperiods. Though the fathead minnow (Pimephales promelas) is a common model for aquatic toxicology research and regulatory toxicology practice, this model has received little attention in behavioral studies compared to the zebrafish, a common biomedical model. We specifically compared behavioral responses among 7 different chemicals (1-heptanol, phenol, R-(-)-carvone, citalopram, diazinon, pentylenetetrazole (PTZ), and xylazine) that were selected and classified based on anticipated MOA (nonpolar narcosis, polar narcosis, electrophile, specific mechanism) according to traditional approaches to examine whether these comparative responses differ among chemicals with various structure-based predicted toxicity. Following standardized experimental guidelines, zebrafish embryos and fathead minnow larvae were exposed for 96 h to each compound then were observed using digital behavioral analysis. Behavioral observations included photomotor responses, distance traveled, and stimulatory, refractory and cruising locomotor activity. Though fathead minnow larvae displayed greater behavioral sensitivity to 1-heptanol, phenol and citalopram, zebrafish were more sensitive to diazinon and R-(-)-carvone. Both fish models were equally sensitive to xylazine and PTZ. Further, the pharmaceuticals citalopram and xylazine significantly affected behavior at therapeutic hazard values, and each of the seven chemicals elicited unique behavioral response profiles. Larval fish behaviors appear useful as early tier diagnostics to identify mechanisms and pathways associated with diverse biological activities for chemicals lacking mechanistic data.

Keywords

High throughput screening; Behavior; Alternative toxicity testing; Chemical bioactivity; Comparative toxicology

 

https://www.sciencedirect.com/science/article/pii/S004896971832059X

Swimming activity in zebrafish larvae exposed to veterinary antiparasitic pharmaceuticals

Environmental Toxicology and Pharmacology

2018-08-25

Abstract

Veterinary antiparasitic pharmaceuticals have been detected in surface waters and several of these pharmaceuticals act on the nervous system on the target organisms implying that neurological effects also might be of concern in non-target animals such as fish. We tested if exposure to antiparasitic pharmaceuticals affect swimming activity in 6 days old zebrafish larvae. The results revealed that most pharmaceuticals did not cause any effects in swimming activity. However, larvae exposed to 0.58 mg/L doramectin displayed reduced swimming activity even though they were classified as normal, having no morphological abnormalities.

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S1382668918302874

The Locus Coeruleus Modulates Intravenous General Anesthesia of Zebrafish via a Cooperative Mechanism

Cell Press

2018-09-01

Highlights

• Intravenous anesthetics induce general anesthesia in larval zebrafish
• Impairment of the locus coeruleus (LC)-norepinephrine system affects anesthesia
• Anesthetics suppress LC neuronal activities via pre- and postsynaptic mechanisms
• Larval zebrafish is an ideal model for investigation of general anesthesia

Summary

How general anesthesia causes loss of consciousness has been a mystery for decades. It is generally thought that arousal-related brain nuclei, including the locus coeruleus (LC), are involved. Here, by monitoring locomotion behaviors and neural activities, we developed a larval zebrafish model for studying general anesthesia induced by propofol and etomidate, two commonly used intravenous anesthetics. Local lesion of LC neurons via two-photon laser-based ablation or genetic depletion of norepinephrine (NE; a neuromodulator released by LC neurons) via CRISPR/Cas9-based mutation of dopamine-β-hydroxylase (dbh) accelerates induction into and retards emergence from general anesthesia. Mechanistically, in vivo whole-cell recording revealed that both anesthetics suppress LC neurons’ activity through a cooperative mechanism, inhibiting presynaptic excitatory inputs and inducing GABAA receptor-mediated hyperpolarization of these neurons. Thus, our study indicates that the LC-NE system plays a modulatory role in both induction of and emergence from intravenous general anesthesia.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S2211124718313329

Ecotoxicological effects, water quality standards and risk assessment for the anti-diabetic metformin

Environmental Pollution

2018-09-08

Abstract

Metformin (MET) is among the most consumed pharmaceuticals worldwide. This compound has been frequently detected in fresh surface water. However, ecotoxicological information for MET is still too limited, particularly regarding chronic and behavioral data. This study aimed to help filling these knowledge gaps, by carrying out both acute and chronic studies with four different test organisms from three different trophic levels. We assessed different endpoints, including the swimming behavior of Danio reriolarvae. We also derived both short-term and long-term environmental quality standards (EQS) for the protection of freshwater pelagic biota towards MET adverse effects. A risk quotient (RQ) was calculated for MET in fresh surface water, considering a worst-case scenario. Daphnia similis was by far the most sensitive species evaluated. An EC10 of 4.4 mg L−1 was obtained from the reproduction test with D. similis. A long-term EQS of 88 μg L−1 was derived and a RQ of 0.38 was obtained. An ecological risk is not expected for the chronic exposure of pelagic freshwater species to MET, considering the endpoints and the standard bioassays usually recommended in standard protocols. However, endocrine disruptive effects and potential interactive effects of MET with other co-occurring contaminants cannot be ruled out. To the best of our knowledge, this study presents the first data related with MET effects on population endpoints of D. similis and Hydra attenuata, as well as on the locomotor activity of D. rerio.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0269749118316294

Automated morphological feature assessment for zebrafish embryo developmental toxicity screens

Toxicological Sciences

2018-10-01

Zebrafish development as a model organism

Abstract


Detection of developmental phenotypes in zebrafish embryos typically involves a
visual assessment and scoring of morphological features by an individual
researcher. Subjective scoring could impact results and be of particular concern
when phenotypic effect patterns are also used as a diagnostic tool to classify
compounds. Here we introduce a quantitative morphometric approach based on
image analysis of zebrafish embryos. A software called FishInspector was developed
to detect morphological features from images collected using an automated system
to position zebrafish embryos. The analysis was verified and compared with visual
assessments of three participating laboratories using three known developmental
toxicants (methotrexate, dexamethasone and topiramate) and two negative
compounds (loratadine and glibenclamide). The quantitative approach exhibited
higher sensitivity and made it possible to compare patterns of effects with the
potential to establish a grouping and classification of developmental toxicants. Our
approach improves the robustness of phenotype scoring and reliability of assay
performance and, hence, is anticipated to improve the predictivity of developmental
toxicity screening using the zebrafish embryo.

 

Link to the publication :

http://scholar.google.fr/scholar_url?url=https://academic.oup.com/toxsci/advance-article-pdf/doi/10.1093/toxsci/kfy250/25949135/kfy250.pdf&hl=fr&sa=X&d=693064754340786427&scisig=AAGBfm1hNXS2Dp3SEr7wAAzh0W_DnrNFOg&nossl=1&oi=scholaralrt

 

Sub-lethal UV radiation during early life stages alters the behaviour, heart rate and oxidative stress parameters in zebrafish (Danio rerio)

Ecotoxicology and Environmental Safety

2018-10-01

Abstract

Environmental UV radiation in sufficient doses, as a possible consequence of climate change, is potent enough to affect living organisms with different outcomes, depending on the exposure life stage. The aim of this project was to evaluate the potentially toxic effects of exposure to sub-lethal and environmentally relevant doses of UVA (9.4, 18. 7, 37.7 J/cm2) and UVB radiation (0.013, 0.025, 0.076 J/cm2) on the development and behaviour in early life stages (4.5–5.5 h post fertilization, hpf) of the zebrafish (Danio rerio). The used doses were all below the median lethal dose (LD50) and caused no significant difference in survival, deformities, or hatching between exposed and control groups. Compared to controls, there were transient UVA and UVB exposure effects on heart rate, with dose dependent reductions at 50 hpf, and at 60 hpf for UVA only. The UVB exposure caused an increasing trend in reactive oxygen species (ROS) formation at the two highest doses, even though only significant at 120 hpf for the second highest dose. Both UVA and UVB caused an increasing trend in lipid peroxidation (LPO) at the highest doses tested at 72 hpf. Furthermore, UVA exposure led to significant reductions in larval movement following exposure to the two highest doses of UVA, i.e., reduction in the time spent active and the total distance moved compared to control at 100 hpf, while no effect on the swimming speed was observed. The lowest dose of UVA had no effect on behaviour. In contrast, the highest dose of UVB led to a possible increase in the time spent active and a slower average swimming speed although these effects were not significant (p = 0.07). The obtained results show that UV doses below LD50 levels are able to cause changes in the behaviour and physiological parameters of zebrafish larvae, as well as oxidative stress in the form of ROS formation and LPO. Further testing is necessary to assess how this type of radiation and the effects observed could affect fish population dynamics.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0147651318309436

Motor Neuron Abnormalities Correlate with Impaired Movement in Zebrafish that Express Mutant Superoxide Dismutase 1

Zebrafish

2018-10-09

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons. ALS can be modeled in zebrafish (Danio rerio) through the expression of human ALS-causing genes, such as superoxide dismutase 1 (SOD1). Overexpression of mutated human SOD1 protein causes aberrant branching and shortening of spinal motor axons. Despite this, the functional relevance of this axon morphology remains elusive. Our aim was to determine whether this motor axonopathy is correlated with impaired movement in mutant (MT) SOD1-expressing zebrafish. Transgenic zebrafish embryos that express blue fluorescent protein (mTagBFP) in motor neurons were injected with either wild-type (WT) or MT (A4V) human SOD1 messenger ribonucleic acid (mRNA). At 48 hours post-fertilization, larvae movement (distance traveled during behavioral testing) was examined, followed by quantification of motor axon length. Larvae injected with MT SOD1 mRNA had significantly shorter and more aberrantly branched motor axons (p < 0.002) and traveled a significantly shorter distance during behavioral testing (p < 0.001) when compared with WT SOD1 and noninjected larvae. Furthermore, there was a positive correlation between distance traveled and motor axon length (R2 = 0.357, p < 0.001). These data represent the first correlative investigation of motor axonopathies and impaired movement in SOD1-expressing zebrafish, confirming functional relevance and validating movement as a disease phenotype for the testing of disease treatments for ALS.

 

Link to the publication :

https://www.liebertpub.com/doi/abs/10.1089/zeb.2018.1588

Novel neurotoxic peptides from Protopalythoa variabilis virtually interact with voltage-gated sodium channel and display anti-epilepsy and neuroprotective activities in zebrafish

Archieves of Toxicology

2018-10-17

Abstract

We previously reported a novel toxic peptide identified from the anthozoan Protopalythoa variabilis transcriptome which is homologous to a novel structural type of sodium channel toxin isolated from a parental species (Palythoa caribaeorum). The peptide was named, according to its homologous, as Pp V-shape α-helical peptide (PpVα) in the present study. Through molecular docking and dynamics simulation, linear and hairpin folded PpVα peptides were shown to be potential voltage-gated sodium channel blockers. Nowadays, sodium channel blockers have been the mainstream of the pharmacological management of epileptic seizures. Also, sodium channel blockers could promote neuronal survival by reducing sodium influx and reducing the likelihood of calcium importation resulting in suppressing microglial activation and protecting dopaminergic neurons from degeneration. The folded PpVα peptide could decrease pentylenetetrazol (PTZ)-induced c-fos and npas4a expression level leading to reverse PTZ-induced locomotor hyperactivity in zebrafish model. In vitro, the folded PpVα peptide protected PC12 cells against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity via activating heme oxygenase-1 (HO-1) and attenuating inducible nitric oxide synthase (iNOS) expression. In vivo, PpVα peptide suppressed the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish and, importantly, prevented the 6-OHDA-induced excessive ROS generation and subsequent dopaminergic neurons loss. This study indicates that the single S–S bond folded PpVα peptide arises as a new structural template to develop sodium channel blockers and provides an insight on the peptide discovery from cnidarian transcriptome to potentially manage epilepsy and neurodegenerative disorders.

 

Link to the publication :

https://link.springer.com/article/10.1007/s00204-018-2334-5

A Zebrafish Behavior Assay for Assessing Anti- Epileptic Drug Efficacy

NeuroQuantology

2018-10-17

ABSTRACT


The availability of zebrafish larvae model of epileptic seizure provides opportunities to identify novel anticonvulsants
for treatment of people with epilepsy. However, the major parameters of zebrafish behavior assay for assessing antiepileptic drug efficacy existed disparity. In this study, we chose the high, medium, slow-speed moved distances and the total distances moved for seizure-like activity quantification in individual wells of a 48-well plate in the dark phase.
Results also showed that the high-speed moved distances were given a more reasonable and sensitive dose-response
curve than the total distances in zebrafish larvae model exposed to 10 mM pentylenetetrazole (PTZ). Moreover, this
seizure-like swimming pattern was alleviated by the addition of either phenytoin (PHT) or valproate sodium (VPA),
two commonly prescribed anti-epileptic drugs (AEDs). They exhibited concentration-dependent inhibition of both
locomotor activity and PTZ-induced c-fos transcription, confirming their anticonvulsant characteristics. Whereas
carbamazepine (CBZ) promoted zebrafish larvae movement by 26% at 30 μM and 116.7% at 100 μM, and sharply
suppressed zebrafish larvae motility about 94.8% at 300 μM. C-fos expression significantly decreased at 30 μM and
300 μM CBZ, which was found to oppose various aspects of the PTZ-induced changes in activity. These results indicated
that this zebrafish larvae model could be useful for studying drug efficacy, predicting drug targets and aiding our
understanding of disease etiology.

 

 

 

Identification of GSK-3 as a Potential Therapeutic Entry Point for Epilepsy

ACS Chemical Neuroscience

2018-10-23

Abstract

In view of the clinical need for new antiseizure drugs (ASDs) with novel modes of action, we used a zebrafish seizure model to screen the anticonvulsant activity of medicinal plants used by traditional healers in the Congo for the treatment of epilepsy, and identified a crude plant extract that inhibited pentylenetetrazol (PTZ)-induced seizures in zebrafish larvae. Zebrafish bioassay-guided fractionation of this anticonvulsant Fabaceae species, Indigofera arrecta, identified indirubin, a compound with known inhibitory activity of glycogen synthase kinase (GSK)-3, as the bioactive component. Indirubin, as well as the more potent and selective GSK-3 inhibitor 6-bromoindirubin-3′-oxime (BIO-acetoxime) were tested in zebrafish and rodent seizure assays. Both compounds revealed anticonvulsant activity in PTZ-treated zebrafish larvae, with electroencephalographic recordings revealing reduction of epileptiform discharges. Both indirubin and BIO-acetoxime also showed anticonvulsant activity in the pilocarpine rat model for limbic seizures and in the 6-Hz refractory seizure mouse model. Most interestingly, BIO-acetoxime also exhibited anticonvulsant actions in 6-Hz fully kindled mice. Our findings thus provide the first evidence for anticonvulsant activity of GSK-3 inhibition, thereby implicating GSK-3 as a potential therapeutic entry point for epilepsy. Our results also support the use of zebrafish bioassay-guided fractionation of antiepileptic medicinal plant extracts as an effective strategy for the discovery of new ASDs with novel mechanisms of action.

High-performance counter-current chromatography isolation and initial neuroactivity characterization of furanocoumarin derivatives from Peucedanum alsaticum L (Apiaceae)

Phytomedicine

2018-10-26

Abstract

Background

Medicinal plants are a proven source of drug-like small molecules with activity towards targets relevant for diseases of the central nervous system (CNS). Plant species of the Apiaceae family have to date yielded a number of neuroactive metabolites, such as coumarin derivatives with acetylcholinesterase inhibitory activity or anti-seizure activity.

Purpose

To accelerate the discovery of neuroactive phytochemicals with potential as CNS drug leads, we sought to rapidly isolate furanocoumarins, primary constituents of the dichloromethane (DCM) extract of the fruits of Peucedanum alsaticum L. (Apiaceae), using high-performance counter-current chromatography (HPCCC) and to evaluate their neuroactivity using both in vitro and in vivo microscale bioassays based on cholinesteraseELISAs and zebrafish epilepsy models.

Research methods and procedure

In this study the DCM extract was subjected to HPCCC for the efficient separation (60 min) and isolation of furanocoumarins. Isolated compounds were identified with TOF-ESI-MS and NMR techniques and examined as inhibitors of AChE and BChE using ELISA microtiter assays. Anti-seizure properties of the extract and of the isolated compounds were evaluated using a zebrafish epilepsy model based on the GABAAantagonistpentylenetetrazol (PTZ), which induces increased locomotor activity and seizure-like behavior.

Results

The solvent system, composed of n-heptane, ethyl acetate, methanol and water (3:1:3:1, v/v/v/v), enabled the isolation of 2.63 mg lucidafuranocoumarin A (purity 98%) and 8.82 mg bergamottin (purity 96%) from 1.6 g crude DCM extract. The crude extract, at a concentration of 100 µg/ml, exhibited a weak inhibitory activity against acetylcholinesterase (AChE) (9.63 ± 1.59%) and a moderate inhibitory activity against butyrylcholinestrase (BChE) (49.41 ± 2.19%). Lucidafuranocoumarin A (100 µg/ml) was inactive against AChE but showed moderate inhibition towards BChE (40.66 ± 1.25%). The DCM extract of P. alsaticum fruits (0.62–1.75 µg/ml) and bergamottin (2–10 µm) exhibited weak anti-seizure activity, while lucidafuranocoumarin A (10–16 µm) was found to significantly inhibit PTZ-induced seizures. The percentage of seizure inhibition for the isolated compounds, at their most bioactive concentration, was 26% for bergamottin and 69% for lucidafuranocoumarin A.

Conclusion

Our findings underscore the utility of HPCCC for the rapid isolation of rare coumarin derivatives, and the potential of microscale in vivo bioassays based on zebrafish disease models for the rapid assessment of neuroactivity of these drug-like natural products.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0944711318305518

Drug repositioning in epilepsy reveals novel antiseizure candidates

Annals of clinical and transnational neurology

2018-10-26

Abstract


Objective: Epilepsy treatment falls short in ~30% of cases. A better understanding
of epilepsy pathophysiology can guide rational drug development in
this difficult to treat condition. We tested a low-cost, drug-repositioning strategy
to identify candidate epilepsy drugs that are already FDA-approved and
might be immediately tested in epilepsy patients who require new therapies.
Methods: Biopsies of spiking and nonspiking hippocampal brain tissue from
six patients with unilateral mesial temporal lobe epilepsy were analyzed by
RNA-Seq. These profiles were correlated with transcriptomes from cell lines
treated with FDA-approved drugs, identifying compounds which were tested
for therapeutic efficacy in a zebrafish seizure assay. Results: In spiking versus
nonspiking biopsies, RNA-Seq identified 689 differentially expressed genes, 148
of which were previously cited in articles mentioning seizures or epilepsy. Differentially
expressed genes were highly enriched for protein–protein interactions
and formed three clusters with associated GO-terms including myelination,
protein ubiquitination, and neuronal migration. Among the 184 compounds, a
zebrafish seizure model tested the therapeutic efficacy of doxycycline, metformin,
nifedipine, and pyrantel tartrate, with metformin, nifedipine, and
pyrantel tartrate all showing efficacy. Interpretation: This proof-of-principle
analysis suggests our powerful, rapid, cost-effective approach can likely be
applied to other hard-to-treat diseases.

Behavioural responses in effect to chemical stress in fish

International Journal of Fisheries and Aquatic Studies

2018-12-03

Fish: biochemical responses to toxicants

 

Abstract :

Due to industrialization and urbanization many pollutants are being introduced directly and indirectly into aquatic ecosystem. Behavioural bioassay have been widely used in toxicity assessment. Bioassay based on behavior is faster, more sensitive and ecologically more relevant as assessing growth and reproduction need longer bioassay. Behavioural bioassay is more promising alternatives than lethality evaluating bioassay which are currently used for the risk assessment of toxicant. Behavioural changes provide early warning signals about the health of exposed population which other standard tests do not take in to consideration. These endpoints may be 10–100 times more sensitive than those derived from acute or chronic tests because chemicals can induce rapid behavioural responses in organisms even at very low concentrations. Behaviour is an organism-level effect defined as the action, reaction, or functioning of a system under a set of specific circumstances. We rationalize that a greater understanding of behavioural responses in effect to chemical stress may increase. Therefore in current scenario there is a need of developing newer and effective methods to study the behavioural responses. Behavioural changes in a fish form an efficient index to measure any alterations in the environmental conditions.

 

Link to the publication:  https://www.researchgate.net/profile/Madhu_Sharma6/publication/330090387_Behavioural_responses_in_effect_to_chemical_stress_in_fish_A_review_Madhu_Sharma/links/5c2cd5b892851c22a35547bc/Behavioural-responses-in-effect-to-chemical-stress-in-fish-A-review-Madhu-Sharma.pdf

Developmental neurotoxicity and immunotoxicity induced by graphene oxide in zebrafish embryos

Environmental Toxicology

2018-12-13

Abstract

Graphene oxide (GO) has emerged as the worldwide promising candidate for biomedical application, such as for drug delivery, bio‐sensing and anti‐cancer therapy. This study was focused on the zebrafish and RAW264.7 cell line as in vivo and in vitro models to assess the potential developmental neurotoxicity and immunotoxicity of GO. No obvious acute developmental toxicity was observed upon treatments with 0.01, 0.1, and 1 μg/mL GO for five consecutive days. However, decreased hatching rate, increased malformation rate, heart beat rate and hypoactivity of locomotor behavior were detected when exposed to 10 μg/mL GO. Also, RT‐PCR analysis revealed that expressions of genes related to the nervous system were up‐regulated. The potential risk of GO for developmental neurotoxicity may be ascribed to the high level of oxidative stress induced by high concentration of GO. Most importantly, the mRNA levels of immune response associated genes, such as interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor‐α (TNFα), interferon‐γ (IFN‐γ) were significantly increased under environmental concentration exposure. The activation of pro‐inflammatory immune response was also observed in macrophage cell line. Taken together, our results demonstrated that immunotoxicity is a sensitive indicator for assessment of bio‐compatibility of GO.

 

Link to the publication :

https://onlinelibrary.wiley.com/doi/abs/10.1002/tox.22695

Bioassay-guided isolation of anti-seizure principles from Semen Pharbitidis using a zebrafish pentylenetetrazol seizure model

Journal of Ethnopharmacology

2018-12-17

Abstract

Ethnopharmacological relevance

Semen Pharbitidis, the seeds of Pharbitis nil (Linn.) Choisy (Convolvulaceae) is a well-known traditional Chinese medicinal plant used for treating helminthiasis and epilepsy in China.

Aim of the study

This study aims to identify the anti-seizure components from Semen Pharbitidis.

Methods

A bioassay-guided isolation of anti-seizure compounds from Semen Pharbitidis was performed using a zebrafishpentylenetetrazol seizure model. The structures of active compounds were elucidated by high resolution mass spectrometry. The fragments of active compounds were tested for anti-seizure activity as well.

Results

The bioassay-guided isolation of ethanol extract of Semen Pharbitidis led to a group of resin glucosides, namely pharbitin. One of the fragments of pharbitin, 2-methylbutyric acid, also showed anti-seizure activity.

Conclusions

We provided further experimental scientific evidence to support the traditional use of Semen Pharbitidis for the treatment of epilepsy. Pharbitin was identified to be the main anti-seizure component in Semen Pharbitidis.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0378874118320609

Multigenerational consequences of early-life cannabinoid exposure in zebrafish

Toxicology and Applied Pharmacology

2018-12-26

Highlights

• CBD caused adverse developmental outcomes at much lower concentrations than THC.
• THC and CBD exposure led to expression changes in developmentally imperative genes.
• Sub-lethal concentrations of THC and CBD affected larval and adult zebrafish behavior.
• THC and CBD decreased zebrafish offspring in the F0 but not the F1 generation.

Abstract

While Δ9-tetrahydrocannabinol (THC) has been widely studied in the realm of developmental and reproductive toxicology, few studies have investigated potential toxicities from a second widely used cannabis constituent, cannabidiol (CBD). CBD is popularized for its therapeutic potential for reducing seizure frequencies in epilepsy. This study investigated developmental origins of health and disease (DOHaD) via multigenerational gene expression patterns, behavior phenotypes, and reproductive fitness of a subsequent F1 following an F0 developmental exposure of zebrafish (Danio rerio) to THC (0.024, 0.12, 0.6 mg/L; 0.08, 0.4, 2 μM) or CBD (0.006, 0.03, 0.15 mg/L; 0.02, 0.1, 0.5 μM). Embryonic exposure at these concentrations did not cause notable morphological abnormalities in either F0 or F1 generations. However, during key developmental stages (14, 24, 48, 72, and 96 h post fertilization) THC and CBD caused differential expression of c-fos,brain-derived neurotrophic factor (bdnf), and deleted-in-azoospermia like (dazl), while in F1 larvae only CBD differentially expressed dazl. Larval photomotor behavior was reduced (F0) or increased (F1) by THC exposure, while CBD had no effect on F0 larvae, but decreased activity in the unexposed F1 larvae. These results support our hypothesis of cannabinoid-related developmental neurotoxicity. As adults, F0 fecundity was reduced, but it was not in F1 adults. Conversely, in the adult open field test there were no significant effects in F0 fish, but a significant reduction in the time in periphery was seen in F1 fish from the highest THC exposure group. The results highlight the need to consider long-term ramifications of early-life exposure to cannabinoids.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0041008X18305805

Evaluation of neuroactive effects of ethanol extract of Schisandra chinensis, Schisandrin, and Schisandrin B and determination of underlying mechanisms by zebrafish behavioral profiling

Chinese Journal of Chinese neurosciences

2018-12-27

Abstract

Schisandra chinensis, a traditional Chinese medicine (TCM), has been used to treat sleep disorders. Zebrafish sleep/wake behavioral profiling provides a high-throughput platform to screen chemicals, but has never been used to study extracts and components from TCM. In the present study, the ethanol extract of Schisandra chinensis and its two main lignin components, schisandrin and schisandrin B, were studied in zebrafish. We found that the ethanol extract had bidirectional improvement in rest and activity in zebrafish. Schisandrin and schisandrin B were both sedative and active components. We predicted that schisandrin was related to serotonin pathway and the enthanol extract of Schisandra chinensis was related to seoronin and domapine pathways using a database of zebrafish behaviors. These predictions were confirmed in experiments using Caenorhabditis elegans. In conclusion, zebrafish behavior profiling could be used as a high-throughput platform to screen neuroactive effects and predict molecular pathways of extracts and components from TCM.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S187553641830133X

Evaluation of neuroactive effects of ethanol extract of Schisandra chinensis, Schisandrin, and Schisandrin B and determination of underlying mechanisms by zebrafish behavioral profiling

Chinese Journal of Natural Medicines

2018-12-27

Abstract

Schisandra chinensis, a traditional Chinese medicine (TCM), has been used to treat sleep disorders. Zebrafish sleep/wake behavioral profiling provides a high-throughput platform to screen chemicals, but has never been used to study extracts and components from TCM. In the present study, the ethanol extract of Schisandra chinensis and its two main lignin components, schisandrin and schisandrin B, were studied in zebrafish. We found that the ethanol extract had bidirectional improvement in rest and activity in zebrafish. Schisandrin and schisandrin B were both sedative and active components. We predicted that schisandrin was related to serotonin pathway and the enthanol extract of Schisandra chinensis was related to seoronin and domapine pathways using a database of zebrafish behaviors. These predictions were confirmed in experiments using Caenorhabditis elegans. In conclusion, zebrafish behavior profiling could be used as a high-throughput platform to screen neuroactive effects and predict molecular pathways of extracts and components from TCM.

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S187553641830133X

Ferredoxin 1b deficiency leads to testis disorganization, impaired spermatogenesis and feminization in zebrafish

Endocrinology

2019-01-19

Abstract

The roles of steroids in zebrafish sex differentiation, gonadal development and function of the adult gonad are poorly understood. Herein, we have employed a ferredoxin 1b (fdx1b) mutant zebrafish to explore such processes. Fdx1b is an essential electron-providing cofactor to mitochondrial steroidogenic enzymes, which are crucial for glucocorticoid and androgen production in vertebrates. Fdx1b-/- zebrafish mutants develop into viable adults, in which concentrations of androgens and the glucocorticoid, cortisol, are significantly reduced. Adult fdx1b-/- mutant zebrafish display predominantly female secondary sex characteristics but may possess either ovaries or testes, confirming that androgen signaling is dispensable for testicular differentiation in this species, as previously demonstrated in androgen receptor mutant zebrafish. Adult male fdx1b-/- mutant zebrafish do not exhibit characteristic breeding behaviors, and sperm production is reduced, resulting in infertility in standard breeding scenarios. However, eggs collected from wild-type females can be fertilized by the sperm of fdx1b-/- mutant males by IVF. The testes of fdx1b-/- mutant males are disorganized and lack defined seminiferous tubule structure. Expression of several pro-male and spermatogenic genes is decreased in the testes of fdx1b-/-mutant males, including pro-male transcription factor SRY-box 9a (sox9a) and spermatogenic genes insulin-like growth factor 3 (igf3) and insulin-like 3 (insl3). This study establishes an androgen- and cortisol-deficient fdx1bzebrafish mutant as a model for understanding the impacts of steroid deficiency on sex development and reproductive function. This model will be particularly useful for further investigation of the roles of steroids in spermatogenesis, gonadal development and regulation of reproductive behavior, thus enabling further elucidation of the physiological consequences of endocrine disruption in vertebrates.

 

Link to the publication : https://academic.oup.com/endo/advance-article-abstract/doi/10.1210/en.2019-00068/5535648

Embryonic development, locomotor behavior, biochemical, and epigenetic effects of the pharmaceutical drugs paracetamol and ciprofloxacin in larvae and embryos of Danio rerio when exposed to environmental realistic levels of both drugs

Environmental Toxicology

2019-01-19

Abstract

For several years, the scientific community has been concerned about the presence of pharmaceuticals in the wild, since these compounds may have unpredictable deleterious effects on living organisms. Two examples of widely used pharmaceuticals that are present in the environment are paracetamol and ciprofloxacin. Despite their common presence in the aquatic environment due to their poor removal by sewage treatment plants, knowledge concerning their putative toxic effects is still scarce. This work aimed to characterize the effects of paracetamol (0.005, 0.025, 0.125, 0.625, and 3.125 mg/L) and ciprofloxacin (0.005, 0.013, 0.031, 0.078, 0.195, and 0.488 μg/L) in zebrafish embryos and larvae, exposed to environmentally relevant levels, close to the real concentrations of these pharmaceuticals in surface waters and effluents. The adopted toxic end points were developmental, a behavioral parameter (total swimming time), and a biomarker‐based approach (quantification of the activities of catalase, glutathione‐S‐transferase, cholinesterases, glutathione peroxidase, and lipid peroxidation levels) combined with epigenetic analysis (immunohistochemical detection of 5‐methylcytidine). Exposure to paracetamol had effects on all of the adopted toxic end points; however, ciprofloxacin only caused effects on behavioral tests and alterations in biomarkers. It is possible to ascertain the occurrence of oxidative stress following exposure to both drugs, which was more evident regarding paracetamol, an effect that may be related to the observed epigenetic modifications.

 

Link to the publication : https://onlinelibrary.wiley.com/doi/abs/10.1002/tox.22819

Varying the exposure period and duration of neuroactive pharmaceuticals and their metabolites modulates effects on the visual motor response in zebrafish (Danio rerio) larvae

Neurotoxicology and Teratology

2019-01-31

Highlights

• 7 out of 10 antidepressants altered swimming behavior in zebrafish larvae.
• 2 out of 3 metabolites tested elicited similar effects as parent compounds.
• 2 of 15 compounds altered behavior in repeated, late, and early exposure scenarios.

Abstract

Pharmaceuticals and personal care products are emerging contaminants that are increasingly detected in surface waters around the world. Despite the rise in environmental detections, measured concentrations are still typically low, raising the importance of environmental risk assessments that focus on ecologically relevant sublethal endpoints, such as altered behavior. Neuroactive pharmaceuticals, like mental health medications, pain killers, etc., may be particularly potent in this regard as they are specifically designed to cause behavioral changes without causing physiologic impairment in mammalian systems. We screened 15 different popular neuroactive pharmaceuticals, ranging from antidepressants (including 3 major antidepressant metabolites), anxiety medications, and pain killers, under three different exposure scenarios (repeated, late acute and early transient exposure) to look for behavioral effects in larval zebrafish using the visual motor response (VMR). Drugs were screened at 0, 1, 10, and 100 μg/L in the repeated exposure scenario, and at 0 and 100 μg/L in the late acute and early transient exposure scenarios. Eight of the 15 compounds tested, specifically the antidepressants amitriptyline, fluoxetine, nor-fluoxetine, paroxetine, sertraline, nor-sertraline, venlafaxine, and the antipsychotic drug haloperidol decreased swimming activity by 25% to 40% under repeated exposure conditions. Five of the compounds (amitriptyline, fluoxetine, nor-fluoxetine, paroxetine, and sertraline) also significantly decreased activity by 17% to 31% in the late acute exposure paradigm. Three compounds (fluoxetine, paroxetine and venlafaxine) significantly altered swimming activity with early transient exposure, however creating a hyperactive response and increasing activity from 24% to 28%, while haloperidol significantly decreased activity by 31%. This paper is, to our knowledge, the first to screen so many neuroactive pharmaceuticals, including major metabolites, in parallel under multiple exposure conditions. We show that antidepressants most consistently alter VMR swimming activity. Additionally, we show that major antidepressant metabolites can potentially alter behavior as much as their parent compounds. Furthermore, we show that the magnitude and direction of behavioral effect is dependent on the exposure duration and period, indicating that a more diverse experimental approach might be needed to more accurately assess the risk these compounds pose to the environment.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0892036218301466

Toxicity and neurotoxicity profiling of contaminated sediments from Gulf of Bothnia (Sweden): a multi-endpoint assay with Zebrafish embryos

Environmental Sciences Europe

2019-02-04

Abstract

Background

The toxicological characterization of sediments is an essential task to monitor the quality of aquatic environments. Many hazardous pollutants may accumulate in sediments and pose a risk to the aquatic community. The present study provides an attempt to integrate a diagnostic whole mixture assessment workflow based on a slightly modified Danio rerio embryo acute toxicity test with chemical characterization. Danio rerio embryos were directly exposed to sieved sediment (≤ 63 μm) for 96 h. Sediment samples were collected from three polluted sites (Kramfors, Sundsvall and Örnsköldsvik) in the Gulf of Bothnia (Sweden) which are characterized by a long history of pulp and paper industry impact. Effect data were supported by chemical analyses of 237 organic pollutants and 30 trace elements.

Results

The results show that malformations and neurotoxic compounds are the main drivers of differentiation in chemical and effects analyses, respectively. Specific spinal cord malformations and delayed hatching were observed only in sediments from Kramfors while light hyperactivity was seen only after exposure to sediments from Sundsvall.

Conclusions

Our experiments demonstrate that specific chemical profiles lead to specific effect patterns in Danio rerio embryos. In fact, behavioral endpoints could help detect the exposure to neurotoxins, and the observation of body malformations seems to be a potential tool for the identification of site-specific pollutants as polychlorinated biphenyl (PCBs), brominated flame retardants (BFRs) and several pesticides. Overall, results show the suitability of Danio rerio embryos for the fast screening of sediment samples.

2,4-Dichlorophenoxyacetic acid herbicide effects on zebrafish larvae: development, neurotransmission and behavior as sensitive endpoints

Environmental Science and Pollution Research

2019-02-05

Abstract

Assessment of pesticides toxicity using zebrafish early life stages is relevant for aquatic systems safety. This study aimed to evaluate the short-term effects of 2,4-dichlorophenoxyacetic acid (2,4-D) on zebrafish (Danio rerio) embryos from 3 h post fertilization to 96 hpf. A set of 2,4-D concentrations ranging from 0.32 to 80 mg/L were tested and median lethal concentration (LC50) at 96-h was calculated as 2.86 mg/L. A sub-teratogenic concentrations range from 0.02 to 0.8 mg/L was then used to assess effects at ontogenic, biochemical, and behavioral levels. The main developmental defects were tail deformities and pericardial edema at concentrations equal or above 0.32 mg/L. Cholinesterase activity (at 96 hpf) and larvae swimming behavior (at 120 hpf) were affected even at the lowest tested dose (0.02 mg/L). The behavior analysis was a sensitive endpoint, with a decrease in the swimming distance of exposed larvae during light period. The effect of 2,4-D in ChE was translated by an inhibition of the enzyme activity in all treated groups. These findings demonstrate that 2,4-D can alter the cholinergic system by affecting ChE activity which may be involved in the locomotion reduction of exposed larvae and emphasize the potential of neurotransmission and behavioral endpoints as early warning signs of herbicides contamination in aquatic ecosystems.

 

Link to the publication : https://link.springer.com/article/10.1007/s11356-019-04488-5

Synergistic effects of Pb and repeated heat pulse on developmental neurotoxicity in zebrafish

Ecotoxicology and Environmental Safety

2019-02-06

Highlights

• Repeated heat pulse increases Pb-induced developmental toxicity in zebrafish.
• Synergistic effects of repeated heat pulse on Pb-induced abnormal neurodevelopment.
• Repeated heat pulse enhances Pb-induced suppression on zebrafish locomotor capacity.
• Caspase 3 regulated apoptosis is involved in this synergistic process.

Abstract

Pollutant discharges to the aquatic environment often contain multiple environmental stressors, affecting aquatic organisms. To mimic the discharges from nuclear and industry facilities, the combined effects of two independent types of stressors, heavy metal Pb and repeated heat pulse were addressed in this study. We investigated the developmental toxicity of combined treatment, especially its toxic effects on zebrafish neurodevelopment. The normal embryos at 4 hpf were exposed to 0.2 mM of Pb dissolved in the bathing medium with different temperatures (30, 32, and 34 °C) and then maintained in an incubator at 28 °C. After performing above treatment once every 24 h for 6 days, we found that combined treatment significantly affected neural development, including loss of dopaminergic (DA) neurons and brain vasculature, disruption of locomotor activity and neurodevelopmental genes expression in a temperature-dependent manner as compared to the Pb alone exposure group, indicating that repeated heat pulse enhances these negative impacts induced by Pb. In contrast, no apparent toxicity was observed in repeated heat pulse alone groups, suggesting that Pb treatment reduces thermal tolerance in zebrafish, which emphasized the importance to evaluate synergistic effects of Pb and repeated heat pulse. Moreover, repeated heat pulse aggravated Pb-induced apoptosis in the zebrafish brain. Further study of the underlying mechanism suggested that Caspase 3 regulated apoptosis was involved in this process. Taken together, our findings shed light on the full understanding of toxic effects of discharges from industrial applications on living organisms and its environmental impact.

Normalization of large-scale behavioural data collected from zebrafish

Plos One

2019-02-15

Abstract

Many contemporary neuroscience experiments utilize high-throughput approaches to simultaneously collect behavioural data from many animals. The resulting data are often complex in structure and are subjected to systematic biases, which require new approaches for analysis and normalization. This study addressed the normalization need by establishing an approach based on linear-regression modeling. The model was established using a dataset of visual motor response (VMR) obtained from several strains of wild-type (WT) zebrafish collected at multiple stages of development. The VMR is a locomotor response triggered by drastic light change, and is commonly measured repeatedly from multiple larvae arrayed in 96-well plates. This assay is subjected to several systematic variations. For example, the light emitted by the machine varies slightly from well to well. In addition to the light-intensity variation, biological replication also created batch-batch variation. These systematic variations may result in differences in the VMR and must be normalized. Our normalization approach explicitly modeled the effect of these systematic variations on VMR. It also normalized the activity profiles of different conditions to a common baseline. Our approach is versatile, as it can incorporate different normalization needs as separate factors. The versatility was demonstrated by an integrated normalization of three factors: light-intensity variation, batch-batch variation and baseline. After normalization, new biological insights were revealed from the data. For example, we found larvae of TL strain at 6 days post-fertilization (dpf) responded to light onset much stronger than the 9-dpf larvae, whereas previous analysis without normalization shows that their responses were relatively comparable. By removing systematic variations, our model-based normalization can facilitate downstream statistical comparisons and aid detecting true biological differences in high-throughput studies of neurobehaviour.

Linarin improves the dyskinesia recovery in Alzheimer's disease zebrafish by inhibiting the acetylcholinesterase activity

Life Sciences

2019-02-22

Abstract

Background

Due to complex pathogenesis of Alzheimer's disease (AD), currently there is no effective disease-modifying treatment. Acetylcholinesterase (AChE) has introduced itself as an important target for AD therapy. Linarin as the representative active ingredient of flavonoid glycoside in Flos chrysanthemi indici has been found to have anti-acetylcholinesterase effect.

Aims

The present study intended to explore the potential effect of linarin for treatment of AD.

Main methods

In this study, molecular docking simulation was used to evaluate whether linarin could dock with AChE and decipher the mechanism of linarin as an AChE inhibitor. After molecular docking simulation, AlCl3-induced Alzheimer's disease zebrafish model was established. Effects of linarin on treating AD zebrafish dyskinesia and AChE inhibition were compared with donepezil (DPZ) which was used as a positive control drug.

 

Link to the publication :

 

https://www.sciencedirect.com/science/article/abs/pii/S0024320519301365

Zebrafish neurobehavioral phenomics applied as the behavioral warning methods for fingerprinting endocrine disrupting effect by lead exposure at environmentally relevant level

Chemosphere

2019-05-20

Highlights

 

• Pb of environmentally relevant concentration are of potential reproductive toxicity.
• Pb disturbed expression of genes involved in testicular steroidogenesis and apoptosis.
• Pb exerted biphasic effects on courtship behavior and endocrine regulation in male.

 

Abstract

Environmental lead (Pb) exposure is a great hazard to the public health. Although environmentally relevant Pb poisoning is preventable, insidious Pb contaminants are still a major threat to human health. Herein, we reported that exposure to Pb at environmentally relevant concentration level (1 μg/L, 10 μg/L and 100 μg/L), disturbed the courtship behavior of adult male zebrafish and further altered the transcriptional patterns of key genes involved in testicular steroidogenesis (igf3, amh, piwil1, lhcgr, fshr, cyp11c1star, cyp19a1a, cyp19a1b) and apoptosis (bax, cytoC, caspase 9, caspase 3, puma). Both the behavioral and the transcriptional profiles share a similar biphasic dose response, with stimulatory effects after low-level exposure and inhibitory effects after high-level exposure. This results revealed the endocrine disrupting effects of Pb even at an environmentally relevant level within the concentration range of ambient water quality criteria (AWQC) and the reliability of locomotion fingerprint as the indicator for detecting the risk induced by Pb pollution. Current research, for the first time, employed the ZebraTower system as the biological early warning system (BEWS) to find that Pb exerted biphasic effects on the courtship behavior and endocrine regulation of male adult zebrafish. Methodologically, we firstly propose an efficient solution to monitor and assess the risk of Pb exposure by combining the (BEWS) and data analyzing methods such as zebrafish phenomics, which would make a contribution to the detection and prevention of environmentally relevant Pb poisoning.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0045653519310586

Neurodevelopmental toxicity assessments of alkyl phenanthrene and Dechlorane Plus co-exposure in zebrafish

Ecotoxicology and Environmental Safety

2019-05-30

Highlights

 

• Neurotoxicity of 3-MP and DP co-exposure in zebrafish was firstly studied.
• Co-exposure with 3-MP and DP had synergistic effects on neurobehavioral deficits.
• Behavioral anomalies were due to altered neuronal connectivity and muscle function.

 

Abstract

Alkyl phenanthrene (A-Phen) and Dechlorane Plus (DP) are ubiquitous environmental pollutants that widely co-exist in the environment. It has been established that both A-Phen and DP elicit neurotoxicity, but the potential interactive toxicity of these contaminants is not well-known. To determine whether a mixture of A-Phen and DP would exhibit interactive effects on neurodevelopment, we co-exposed 3-methylphenanthrene (3-MP), a representative of A-Phen, with DP. Our results illustrated that exposure to 5 or 20 μg/L 3-MP alone or in combination with 60 μg/L DP caused neurobehavioral anomalies in zebrafish. In accordance with the behavioral deficits, 3-MP alone or co-exposed with DP significantly decreased axonal growth of secondary motoneurons, altered intracellular Ca2+ homeostasis and induced cell apoptosis in the muscle of zebrafish. Additionally, 3-MP alone or co-exposed with DP significantly increased reactive oxygen species (ROS) and the mRNA levels of apoptosis-related genes. These findings indicate that 3-MP alone or co-exposed with DP induces neurobehavioral deficits through the combined effects on neuronal connectivity and muscle function. Chemical analysis revealed significant increases in 3-MP and DP bioaccumulation in zebrafish co-exposed with 3-MP and DP. Elevated bioaccumulation resulting from mixture exposure may represent a significant contribution of the synergistic effects observed in combined chemical exposure.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0147651319306074

Impact of co-exposure to titanium dioxide nanoparticles and Pb on zebrafish embryos

Chemosphere

2019-06-03

Highlights

 

• n-TiO2-Pb complexes were formed in co-exposure suspension.
• Bioconcentration and bioavailability of Pb was increased by n-TiO2 in zebrafish embryos.
• Neurotoxicity of Pb was enhanced in the presence of n-TiO2.

 

Abstract

Complex interactions have been established between nanoparticles (NPs) and heavy metals in real environments. Herein we used zebrafish embryos to investigate the influence of titanium dioxide NPs (n-TiO2) on the uptake, bioconcentration, and depuration, and toxicity of Pb. The formation of n-TiO2–Pb complexes was confirmed in an exposure suspension. An increase in Pb bioconcentration was observed in zebrafish embryos upon co-exposure to n-TiO2 and Pb; moreover, n-TiO2–Pb complexes could be found in the embryos, indicating the bioavailability of NPs. However, there was no difference in the depuration rates of Pb in the presence of n-TiO2. Metallothionein (MT) content was significantly increased upon exposure to Pb alone, and the content significantly increased even further upon co-exposure. A downregulation in the expression levels of the neurodevelopment-related genes gfapsyn2α, and elavl3 was observed in the embryos, and we also noted a reduction in the swimming speed of and the total distance traveled by the larvae. To summarize, our results indicate that n-TiO2 can act as an effective carrier of Pb to enhance its uptake, bioavailability, and toxicity in zebrafish embryos.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/pii/S0045653519312299

 

Long‑term acclimation to near‑future ocean acidification has negligible effects on energetic attributes in a juvenile coral reef fsh

Oecologia

2019-06-04

Abstract

Increased levels of dissolved carbon dioxide (CO2) drive ocean acidification and have been predicted to increase the energy use of marine fishes via physiological and behavioural mechanisms. This notion is based on a theoretical framework suggesting that detrimental effects on energy use are caused by plasma acid–base disruption in response to hypercapnic acidosis, potentially in combination with a malfunction of the gamma aminobutyric acid type A (GABAA) receptors in the brain. However, the existing empirical evidence testing these effects primarily stems from studies that exposed fish to elevated CO2 for a few days and measured a small number of traits. We investigated a range of energetic traits in juvenile spiny chromis damselfish (Acanthochromis polyacanthus) over 3 months of acclimation to projected end-of-century CO2 levels (~ 1000 µatm). Somatic growth and otolith size and shape were unaffected by the CO2 treatment across 3 months of development in comparison with control fish (~ 420 µatm). Swimming activity during behavioural assays was initially higher in the elevated CO2 group, but this effect dissipated within ~ 25 min following handling. The transient higher activity of fish under elevated CO2 was not associated with a detectable difference in the rate of oxygen uptake nor was it mediated by GABAA neurotransmitter interference because treatment with a GABAA antagonist (gabazine) did not abolish the CO2 treatment effect. These findings contrast with several short-term studies by suggesting that end-of-century levels of CO2 may have negligible direct effects on the energetics of at least some species of fish.

 

Link to the publication :

https://link.springer.com/article/10.1007/s00442-019-04430-z

Combined transcriptomic and proteomic analysis reveals a diversity of venom-related and toxin-like peptides expressed in the mat anemone Zoanthus natalensis (Cnidaria, Hexacorallia)

Archives of Toxicology

2019-06-15

Abstract

Venoms from marine animals have been recognized as a new emerging source of peptide-based therapeutics. Several peptide toxins from sea anemone have been investigated as therapeutic leads or pharmacological tools. Venom complexity should be further highlighted using combined strategies of large-scale sequencing and data analysis which integrated transcriptomics and proteomics to elucidate new proteins or peptides to be compared among species. In this work, transcriptomic and proteomic analyses were combined to identify six groups of expressed peptide toxins in Zoanthus natalensis. These include neurotoxin, hemostatic and hemorrhagic toxin, protease inhibitor, mixed function enzymes, venom auxiliary proteins, allergen peptides, and peptides related to the innate immunity. Molecular docking analysis indicated that one expressed Zoanthus Kunitz-like peptide, ZoaKuz1, could be a voltage-gated potassium channels blocker and, hence, it was selected for functional studies. Functional bioassays revealed that ZoaKuz1 has an intrinsic neuroprotective activity in zebrafish model of Parkinson’s disease. Since pharmacological blockade of KV channels is known to induce neuroprotective effects, ZoaKuz1 holds the potential to be developed in a therapeutic tool to control neural dysfunction by slowing or even halting neurodegeneration mediated by ion-channel hyperactivity.

 

Link to the publication : 

https://link.springer.com/article/10.1007/s00204-019-02456-z

Effects of antidepressants with different modes of action on early life stages of fish and amphibians

Environmental Pollution

2019-08-06

Highlights

 

• Lethal effects were not observed at environmentally relevant concentrations.
• Behaviour was effected only at higher tested concentrations.
• mRNA expression was impacted at environmentally relevant concentrations.

 

Abstract

Drugs are excreted from the human body as both original substances and as metabolites and enter aquatic environment through waste water. The aim of this study was to widen the current knowledge considering the effects of waterborne antidepressants with different modes of action—amitriptyline, venlafaxine, sertraline—on embryos of non-target aquatic biota—fish (represented by Danio rerio) and amphibians (represented by Xenopus tropicalis). The tested concentrations were 0.3; 3; 30; 300 and 3000 μg/L in case of amitriptyline and venlafaxine and 0.1; 1; 10; 100 and 1000 μg/L for sertraline. Test on zebrafish embryos was carried out until 144 h post fertilization, while test on Xenopus embryos was terminated after 48 h. Lethal and sublethal effects as well as swimming alterations were observed at higher tested concentrations that are not present in the environment. In contrast, mRNA expression of genes related to heart, eye, brain and bone development (nkx2.5, otx 2, bmp4 and pax 6) seems to be impacted also at environmentally relevant concentrations. In a wider context, this study reveals several indications on the ability of antidepressants to affect non target animals occupying environments which may be contaminated by such compounds.

 

Link to the publication : https://www.sciencedirect.com/science/article/pii/S0269749119304257

Coexposure to environmental concentrations of cis-bifenthrin and graphene oxide: Adverse effects on the nervous system during metamorphic development of Xenopus laevis

Journal of Hazardous Materials

2019-08-12

Highlights

 

• The adverse effects of coexposure of Xenopus laevis to cis-BF and GO were studied.
• GO increased the bioconcentration of cis-BF and the fraction of 1S-enantiomer.
• Coexposure to cis-BF and GO resulted in activation of neurotransmitters.
• cis-BF and GO coexposure led to metamorphic development delay and neurotoxicity.

 

Abstract

Despite the great concerns associated with the combined biological effects of nanoparticles and insecticides, the current understanding of the corresponding ecological risks remains limited. Xenopus laevis (X. laevis) tadpoles were exposed to various concentrations of typical pyrethroid (cis-bifenthrin; cis-BF), either alone or in combination with graphene oxide (GO), for 21 days. The presence of GO resulted in increased bioconcentration of cis-BF and a higher 1S-enantiomer fraction. Exposure to cis-BF and GO caused further reduction in pre-metamorphic developmental rates and activated dopaminergic, noradrenergic, and serotonergic neurotransmitter systems. Reduced tadpole activity and levels of genomic DNA methylation at cytosine nucleotides (5hmC) were observed in the coexposure groups. These results indicate that GO enhance the bioconcentration of cis-BF and promote the conversion of its 1R-enantiomer to the 1S form, which lead to disruption of neurotransmitter systems as well as interference in metamorphic development.

 

Link to the publication : https://www.sciencedirect.com/science/article/pii/S0304389419309495

Behavioral effects of citalopram, tramadol, and binary mixture in zebrafish (Danio rerio) larvae

Chemosphere

2019-08-13

Highlights

 

• Citalopram and tramadol binary mixture effects studied in zebrafish (Danio rerio).
• Embryo-larvae were exposed from fertilization until six days post-fertilization.
• Heart rate, spontaneous tail coiling, and death/malformation incidence unaffected.
• Anxiolytic effects in citalopram (EC50: 471 μg L−1) and tramadol (EC50: 411 μg L−1).
• The mixture was less anxiolytic (EC50: 713 μg L−1), indicating interaction effects.

 

Abstract

Pharmaceuticals are emerging as environmentally problematic compounds. As they are often not appropriately removed by sewage treatment plants, pharmaceutical compounds end up in surface water environments worldwide at concentrations in the ng to μg L−1 range. There is a need to further explore single compound and mixture effects using e.g. in vivo test model systems. We have investigated, for the first time, behavioral effects in larval zebrafish (Danio rerio) exposed to a binary mixture of an antidepressant drug (citalopram) and a synthetic opioid (tramadol). Citalopram and tramadol have a similar mode of action (serotonin reuptake inhibition) and are known to produce drug-drug interactional effects resulting in serotonin syndrome (SS) in humans. Zebrafish embryo-larvae were exposed to citalopram, tramadol and 1:1 binary mixture from fertilization until 144 h post-fertilization. No effects on heart rate, spontaneous tail coiling, or death/malformations were observed in any treatment at tested concentrations. Behavior (hypoactivity in dark periods) was on the other hand affected, with lowest observed effect concentrations (LOECs) of 373 μg L−1 for citalopram, 320 μg L−1 for tramadol, and 473 μg L−1 for the 1:1 mixture. Behavioral EC50 was calculated to be 471 μg L−1 for citalopram, 411 μg L−1 for tramadol, and 713 μg L−1 for the 1:1 mixture. The results of this study conclude that tramadol and citalopram produce hypoactivity in 144 hpf zebrafish larvae. Further, a 1:1 binary mixture of the two caused the same response, albeit at a higher concentration, possibly due to SS.

 

Link to the publication : https://www.sciencedirect.com/science/article/pii/S0045653519318119

Testing the Translational Power of the Zebrafish: An Interspecies Analysis of Responses to Cardiovascular Drugs

Frontiers in Pharmacology

2019-08-16

The zebrafish is rapidly emerging as a promising alternative in vivo model for the detection of drug-induced cardiovascular effects. Despite its increasing popularity, the ability of this model to inform the drug development process is often limited by the uncertainties around the quantitative relevance of zebrafish responses compared with nonclinical mammalian species and ultimately humans. In this test of concept study, we provide a comparative quantitative analysis of the in vivo cardiovascular responses of zebrafish, rat, dog, and human to three model compounds (propranolol, losartan, and captopril), which act as modulators of two key systems (beta-adrenergic and renin–angiotensin systems) involved in the regulation of cardiovascular functions. We used in vivo imaging techniques to generate novel experimental data of drug-mediated cardiovascular effects in zebrafish larvae. These data were combined with a database of interspecies mammalian responses (i.e., heart rate, blood flow, vessel diameter, and stroke volume) extracted from the literature to perform a meta-analysis of effect size and direction across multiple species. In spite of the high heterogeneity of study design parameters, our analysis highlighted that zebrafish and human responses were largely comparable in >80% of drug/endpoint combinations. However, it also revealed a high intraspecies variability, which, in some cases, prevented a conclusive interpretation of the drug-induced effect. Despite the shortcomings of our study, the meta-analysis approach, combined with a suitable data visualization strategy, enabled us to observe patterns of response that would likely remain undetected with more traditional methods of qualitative comparative analysis. We propose that expanding this approach to larger datasets encompassing multiple drugs and modes of action would enable a rigorous and systematic assessment of the applicability domain of the zebrafish from both a mechanistic and phenotypic standpoint. This will increase the confidence in its application for the early detection of adverse drug reactions in any major organ system.

 

Introduction

A considerable number of drug candidates have the potential to alter cardiovascular functions at therapeutically relevant concentrations. Predicting those effects as early as possible during drug development is critically important to ensure the progression of safer compounds through the pipeline and to minimize the risk of cardiovascular safety liabilities emerging at later stages of development (Laverty et al., 2011Cook et al., 2014Lester and Olbertz, 2016). The fast-paced advancements ongoing in the development of human-based in silico and in vitro predictive approaches hold great promise for improving the early detection of drug-induced cardiovascular alterations, including cardiotoxicity (Clements et al., 2015Colatsky et al., 2016Gintant et al., 2016Land et al., 2017Passini et al., 2017). However, to date, the use of in vivo preclinical models is still a key aspect of cardiovascular efficacy and safety assessment (Fliegner et al., 2015Vargas et al., 2015Berridge et al., 2016), mainly because of the ability of in vivo testing to capture integrated multiscale processes that cannot be observed outside an intact organism. These processes include pharmacokinetic-dependent and metabolism-mediated effects, chronic or delayed toxicity, vascular and hemodynamic alterations, as well as interaction between cardiovascular, nervous, and renal systems (Holzgrefe et al., 2014).

In this context, the identification of the most suitable preclinical animal model represents a central challenge for the design of a successful testing strategy, as this choice can profoundly affect the translational value of each experiment and, in turn, data interpretation and subsequent decision-making (Denayer et al., 2014Holzgrefe et al., 2014). From a cardiovascular perspective, dog and nonhuman primates (e.g., cynomolgus monkey) are the most commonly used nonrodent models, as their physiology is considered the most relevant to humans (Leishman et al., 2012Holzgrefe et al., 2014). Other test species include minipig (Bode et al., 2010), marmoset (Tabo et al., 2008), and guinea pigs (Marks et al., 2012). Beside these models, small rodent species (i.e., rat and mouse) remain the most popular choice to investigate cardiovascular physiology and disease, genetics, and pharmacology (Camacho et al., 2016). As with any animal model, each species mentioned above has both advantages and limitations (e.g., see Holzgrefe et al. (2014) and Milani-Nejad and Janssen (2014) for extensive reviews of these aspects); however, common limitations include high ethical and financial costs, and low throughput potential.

In recent years, extensive research efforts have been allocated worldwide to identify potential alternative testing approaches that may lead to the reduction, replacement, or refinement (3Rs) of the model species mentioned above. Within this research theme, the zebrafish has emerged as a new, potentially valuable, model for the in vivo assessment of a variety of human-relevant drug-induced effects, including cardiovascular alterations (Parker et al., 2014MacRae and Peterson, 2015). Zebrafish are characterized by a number of valuable features, including relatively inexpensive maintenance costs, amenability to genetic manipulation, high conservation of human drug targets (i.e., >82%; Howe et al., 2013Verbruggen et al., 2017), and of a broad range of human-relevant phenotypes that can be modified by pharmacological treatment (MacRae and Peterson, 2015).

Considering the high impact of unpredicted cardiotoxicity on drug development (Laverty et al., 2011), the availability of a simpler vertebrate model, such as zebrafish, may enable cardiovascular profiling of new drugs before commencing mammalian toxicity tests, thus serving as a bridge between early in vitro safety predictions and later in vivo preclinical testing. Several studies have started to explore this potential from a translational perspective, such as Parker et al. (2014)and Cornet et al. (2017). Despite encouraging results, to date, the implementation of zebrafish in existing testing strategies faces resistance not least because of uncertainty around the quantitative aspects of zebrafish cardiovascular responses compared with both mammalian preclinical species and humans. We propose that coordinated efforts to perform quantitative comparative assessment of those responses may help to clarify the translational value of zebrafish and help define its domain of applicability from both mechanistic and phenotypic standpoints.

The aim of the present study was to quantify the degree of similarity in the in vivo cardiovascular responses of zebrafish, rat, dog, and human to three model compounds (propranolol, losartan, and captopril), which act as modulators of two key systems (beta-adrenergic and renin–angiotensin systems) involved in the regulation of cardiovascular functions. To do so, we used in vivo imaging techniques to generate novel zebrafish experimental data. The latter were successively combined with a database of interspecies responses extracted from the literature to perform a meta-analysis of effect size and direction across species (Figure 1).

 

Link to the publication : https://www.frontiersin.org/articles/10.3389/fphar.2019.00893/full

Involvement of peroxisome proliferator-activated receptor γ in anticonvulsant activity of α-asaronol against pentylenetetrazole-induced seizures in zebrafish

Neuropharmacology

2019-09-02

Highlights

 

• α-asaronol, a metabolic product of α-asarone, attenuated seizures in zebrafish.
• RNA-seq revealed the involvement of PPAR γ in anticonvulsant effects of α-asaronol.
• Blocking of PPAR γ reversed the anti-seizures actions of α-asaronol.
• Docking simulation implied the physical interaction between α-asaronol and PPAR γ.

 

Abstract

In mammals, peroxisome proliferators activated receptors (PPARs), the nuclear hormone receptors, have been reported to be involved in seizure control. Selective agonists and antagonists of PPARs raise seizure thresholds and suppress seizures, respectively. In this study, we evaluated the anticonvulsant effects of α-asaronol, a metabolic product of α-asarone, on pentylenetetrazole (PTZ)-induced seizures in zebrafish and investigated the underlying mechanisms. As a result, α-asaronol ameliorated seizures with increase of seizure latency, as well as decrease of seizure-like behavior, c-fos expression, and abnormal neuronal discharge in a concentration dependent manner. By comparing gene expression profiles of zebrafish undergoing seizures and α-asaronol pretreated zebrafish, we found that α-asaronol attenuate seizures through increase of PPAR γ expression, while PPAR γ antagonist GW9662 inhibit the anti-seizures actions of α-asaronol. Moreover, molecular docking simulation implied the physical interaction between α-asaronol and PPAR γ. The overall results indicated that the anticonvulsant effects of α-asaronol are regulated through PPAR γ-mediated pathway, which shed light on development of α-asaronol as a potential antiepileptic drug. In addition, it is for first time to report that PPAR γ is associated with seizures in zebrafish, supporting previous evidence that zebrafish is a suitable alternative for studying seizures.

 

Link to the publication : https://www.sciencedirect.com/science/article/pii/S0028390819303193

Biochemical and behavioral responses of zebrafish embryos to magnetic graphene/nickel nanocomposites

Ecotoxicology and Environmental Safety

2019-10-10

Highlights

• No acute or developmental toxicity of graphene/Ni nanocomposites (G/Ni) was observed.

• G/Ni sublethal toxicity to zebrafish embryos depends on their shape and size.
• Locomotor behavior was the most sensitive parameter evaluated.
• Predicted environmental concentrations (PEC) of these compounds should be calculated.
• Relevance of findings to be determined in light of the PEC values.

Abstract

Graphene nanocomposites are emerging carbon-based materials with interesting electrical, mechanical, optical and magnetic properties, relevant for applications in different fields. Despite this increased use, the impact of graphene nanocomposites residues in the environment has not been properly studied. Thus, the goal of this work was to assess the toxicity of two nickel/graphene nanocomposites (G/Ni1 and G/Ni2) differing in size and shape to Danio rerio embryos. Their toxicity was evaluated using apical (mortality, development and hatching), biochemical [cholinesterase (ChE), glutathione-S-transferase (GST), and catalase (CAT) activities] and behavioral (locomotor activity) endpoints. At the tested concentrations, neither of the nanocomposites presented lethal or developmental effects. Nevertheless, both nanocomposites induced behavioral effects, reducing swimming distances. This effect was, however detected at lower concentrations in the G/Ni1 nanocomposite. At biochemical level, only G/Ni1 nanocomposite showed to interfere with the measured parameters, increasing the activities of ChE, CAT and GST. Differences in the effects induced by the two nanocomposites seem to be related not only with their size, but also with the shape and the ability to continuously release nickel ions to aqueous medium. This work highlights the importance of studying graphene nanocomposites effects to aquatic organisms even when acute toxicity is not expected. The relevance of the effects found in this work need to be further analyzed in light of the consequences to the long-term fitness of the organisms and in light of the environmental concentrations expected for this type of compounds.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0147651319310917

The P450 side chain cleavage enzyme Cyp11a2 facilitates steroidogenesis in zebrafish.

Journal of Endocrinology

2019-11-01

Abstract

The cytochrome P450 side-chain cleavage enzyme, encoded by the CYP11A1 gene, catalyzes the first and rate-limiting step of steroid hormone biosynthesis. Previous morpholino knockdown studies in zebrafish suggested cyp11a2 is a functional equivalent of human CYP11A1 and is essential for interrenal steroidogenesis in zebrafish larvae. The role of Cyp11a2 in adult zebrafish, particularly in gonadal steroidogenesis, remains elusive. To explore the role of Cyp11a2 in adults, we developed zebrafish mutant lines by creating deletions in cyp11a2 using the CRISPR/Cas9 genomic engineering approach. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrenal axis. Furthermore, Cyp11a2-deficient zebrafish demonstrated profound glucocorticoid and androgen deficiencies. Cyp11a2 homozygotes only developed into males with feminized secondary sex characteristics. Adult cyp11a2-/- mutant fish showed a lack of natural breeding behaviors. Histological characterization revealed disorganized testicular structure and significantly decreased numbers of mature spermatozoa. These findings are further supported by the downregulation of the expression of several pro-male genes in the testes of cyp11a2 homozygous zebrafish, including sox9a, dmrt1 and amh. Moreover, the spermatogonia markers nanos2 and piwil1 were upregulated, while the spermatocytes marker sycp3 and spermatids marker odf3b were downregulated in the testes of cyp11a2 homozygous mutants. Our expression analysis is consistent with our histological studies, suggesting that spermatogonia are the predominant cell types in the testes of cyp11a2 homozygous mutants. Our work thus demonstrates the crucial role of Cyp11a2 in interrenal and gonadal steroidogenesis in zebrafish larvae and adults.

Flumazenil-insensitive benzodiazepine binding sites in GABAA receptors contribute to benzodiazepine-induced immobility in zebrafish larvae

Life Sciences

2019-11-04

Abstract

Aims

Benzodiazepines (BZDs) produce various pharmacological actions by binding to and allosterically regulating GABAA receptors. Several in vitro studies have demonstrated diazepam, the prototypic BZD, produces a high-dose action that cannot be countered with the classical BZD-binding site antagonist flumazenil. Here, we investigate the existence and behavioral relevance of non-classical BZD binding sites in zebrafish larvae.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0024320519309609

Zebrafish (Danio rerio) embryo-larvae locomotor activity data analysis: Evaluating anxiolytic effects of the antidepressant compound citalopram

Journal Data in Brief

2019-11-15

Abstract

Newly fertilized zebrafish (Danio rerio) embryos were exposed to increasing concentrations of the selective serotonin reuptake inhibitor (SSRI) citalopram from fertilization until six days post-fertilization (dpf). Locomotor activity data were acquired at six dpf using an automated ZebraBox® infrared tracking system. Individual (n = 32) locomotor activity was recorded during 75 minutes in total during alternating illumination conditions (0% light, i.e. dark periods, and 100% light, i.e. light periods). The first 15 minutes of the test consisted of a dark period, i.e an acclimatization phase. Afterward, six alternating light and dark periods were conducted. Individual zebrafish embryo-larvae locomotion was tracked and aggregated in ten-second bins. The dataset, containing nine locomotor-related quantified endpoints (factors), was parsed, analyzed, and visualized using R software. The dataset and its associated custom R script may be used to further explore locomotor activity outcomes (e.g. anxiolytic or anxiogenic properties) following exposure to citalopram or other neuroactive chemicals.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/pii/S2352340919311679

Pyridox(am)ine 5′-phosphate oxidase (PNPO) deficiency in zebrafish results in fatal seizures and metabolic aberrations

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease

2019-11-21

Highlights

 

• PNPO deficient zebrafish (Danio rerio) and HEK293 cell line were generated using CRISPR/Cas9 gene editing technology.
• PNPO deficiency resulted in strong shortening of the survival and in development of fatal spontaneous seizures.
• PLP treatment increased survival and normalized PLP, pyridoxal, 4-pyridoxic acid, GABA, glutamate and glycine levels.
• PLP treatment did not prevent accumulation of pyridoxamine, pyridoxamine 5’-phosphate and several essential amino acids.
• Accumulation of pyridoxamine 5’-phosphate may play a role in the residual clinical phenotype of PNPO deficiency.

 

Abstract

Pyridox(am)ine 5′-phosphate oxidase (PNPO) catalyzes oxidation of pyridoxine 5′-phosphate (PNP) and pyridoxamine 5′-phosphate (PMP) to pyridoxal 5′-phosphate (PLP), the active form of vitamin B6. PNPO deficiency results in neonatal/infantile seizures and neurodevelopmental delay. To gain insight into this disorder we generated Pnpo deficient (pnpo−/−) zebrafish (CRISPR/Cas9 gene editing). Locomotion analysis showed that pnpo−/− zebrafish develop seizures resulting in only 38% of pnpo−/− zebrafish surviving beyond 20 days post fertilization (dpf). The age of seizure onset varied and survival after the onset was brief. Biochemical profiling at 20 dpf revealed a reduction of PLP and pyridoxal (PL) and accumulation of PMP and pyridoxamine (PM). Amino acids involved in neurotransmission including glutamate, γ-aminobutyric acid (GABA) and glycine were decreased. Concentrations of several, mostly essential, amino acids were increased in pnpo−/− zebrafish suggesting impaired activity of PLP-dependent transaminases involved in their degradation. PLP treatment increased survival at 20 dpf and led to complete normalization of PLP, PL, glutamate, GABA and glycine. However, amino acid profiles only partially normalized and accumulation of PMP and PM persisted. Taken together, our data indicate that not only decreased PLP but also accumulation of PMP may play a role in the clinical phenotype of PNPO deficiency.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/pii/S0925443919303308

Developmental exposure to lead at environmentally relevant concentrations impaired neurobehavior and NMDAR-dependent BDNF signaling in zebrafish larvae

Environmental Pollution

2019-11-21

Highlights

 

• The potential ecological risks of Pb could be underestimated in the real environment.
• Environmental levels of Pb induced neurobehavioral changes in zebrafish larvae.
• The neurotoxicity of Pb could be related with NMDAR-dependent BDNF signaling.

 

Abstract

Lead (Pb) is one of the predominant heavy metals in e-waste recycling arears and recognized as a notorious environmental neurotoxic substance. However, whether Pb at environmentally relevant concentrations could cause neurobehavioral alteration and even what kind of signaling pathway Pb exposure would disrupt in zebrafish were not fully uncovered. In the present study, 6 h postfertilization (hpf) zebrafish embryos were exposed to Pb at the concentrations of 0, 5, 10, and 20 μg/L until 144 hpf. Then the neurobehavioral indicators including locomotor, turnings and social behaviors, and the expressions of selected genes concerning brain-derived neurotrophic factor (BDNF) signaling were investigated. The results showed that significant changes were obtained under 20 μg/L Pb exposure. The hypoactivity of zebrafish larvae in locomotor and turning behaviors was induced during the dark period, while hyperactivity was observed in a two-fish social assay during the light period. The significantly downregulation of genes encoding BDNF, its receptor TrkB, and N-methyl-D-aspartate glutamate receptor (NMDAR) suggested the involvement of NMDAR-dependent BDNF signaling pathway. Overall, our study demonstrated that developmental exposure to Pb at environmentally relevant concentrations caused obvious neurobehavioral impairment of zebrafish larvae by disrupting the NMDAR-dependent BDNF signaling, which could exert profound ecological consequences in the real environment.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0269749119339235

Towards less hazardous industrial compounds: coupling quantum mechanical computations, biomarker responses and behavioral profiles identify bioactivity of SN2 electrophiles in alternative vertebrate models

Chemical research in toxicology

2019-12-02

Abstract

Abstract Image

Sustainable molecular design of less hazardous chemicals promises to reduce risks to public health and the environment. Computational chemistry modeling coupled with alternative toxicology models (e.g., larval fish) present unique high-throughput opportunities to understand structural characteristics eliciting adverse outcomes. Numerous environmental contaminants with reactive properties can elicit oxidative stress, an important toxicological response associated with diverse adverse outcomes (i.e., cancer, diabetes, neurodegenerative disorders, etc.). We examined a common chemical mechanism (bimolecular nucleophilic substitution (SN2)) associated with oxidative stress using property-based computational modeling coupled with acute (mortality) and sublethal (glutathione, photomotor behavior) responses in the zebrafish (Danio rerio) and the fathead minnow (Pimephales promelas) models to identify whether relationships exist among biological responses and molecular attributes of industrial chemicals. Following standardized methods, embryonic zebrafish and larval fathead minnows were exposed separately to eight different SN2 compounds for 96 h. Acute and sublethal responses were compared to computationally derived in silico chemical descriptors. Specifically, frontier molecular orbital energies were significantly related to acute LC50 values and photomotor response (PMR) no observed effect concentrations (NOECs) in both fathead minnow and zebrafish. This reactivity index, LC50 values, and PMR NOECs were also significantly related to whole body glutathione (GSH) levels, suggesting that acute and chronic toxicity results from protein adduct formation for SN2 electrophiles. Shared refractory locomotor response patterns among study compounds and two alternative vertebrate models appear informative of electrophilic properties associated with oxidative stress for SN2 chemicals. Electrophilic parameters derived from frontier molecular orbitals were predictive of experimental in vivo acute and sublethal toxicity. These observations provide important implications for identifying and designing less hazardous industrial chemicals with reduced potential to elicit oxidative stress through bimolecular nucleophilic substitution.

 

Link to the publication : 

https://pubs.acs.org/doi/abs/10.1021/acs.chemrestox.9b00290

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

Journal of Visualized Experiments

2019-12-05

Abstract

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research have uncovered a large spectrum of genes implicated in various forms of epilepsy, highlighting the heterogeneous nature of this disorder. Appropriate animal models are essential for investigating the pathological mechanisms triggered by genetic mutations implicated in epilepsy and for developing specialized, targeted therapies. In recent years, zebrafish has emerged as a valuable vertebrate organism for modeling epilepsies, with the use of both genetic manipulation and exposure to known epileptogenic drugs, such as pentylenetetrazole (PTZ), to identify novel anti-epileptic therapeutics. Deleterious mutations in the mTOR regulator DEPDC5 have been associated with various forms of focal epilepsies and knock-down of the zebrafish orthologue causes hyperactivity associated with spontaneous seizure-like episodes, as well as enhanced electrographic activity and characteristic turn wheel swimming. Here, we described the method involved in generating the DEPDC5 loss-of-function model and illustrate the protocol for assessing motor activity at 28 and 48 h post fertilization (hpf), as well as a method for recording field activity in the zebrafish optic tectum. An illustration of the effect of the epileptogenic drug PTZ on neuronal activity over time is also provided.

 

Link to the publication :

https://www.jove.com/video/58837/behavioral-and-physiological-analysis-in-a-zebrafish-model-of-epilepsy?status=a60843k

Bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate affects lipid metabolism in zebrafish larvae via DNA methylation modification

Environmental Science and Technology

2019-12-05

Abstract

Abstract Image

Bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate (TBPH) is a ubiquitous environmental contaminant, but its toxicity is not fully understood. Accordingly, we investigated the effects of TBPH and its metabolite, mono-(2-ethyhexyl)tetrabromophthalate (TBMEHP), on lipid metabolism using a zebrafish model. The molecular docking study revealed that TBPH and TBMEHP bind to zebrafish peroxisome proliferator-activated receptor γ (PPARγ), with binding energies similar to rosiglitazone, a PPARγ agonist. Zebrafish embryos 0.75 hpf were exposed to TBPH (0.2–2000 nM) or TBMEHP (0.2–2000 nM) until 72 hpf, and their effects on PPARγ-mediated lipid metabolism were evaluated. Significant regional DNA demethylation of the PPARγ promoter was observed in the larvae at 72 hpf. Demethylation of the PPARγ promoter accompanied by upregulation of tet1 and tet2 transcription caused upregulation of PPARγ transcription and certain downstream genes involved in lipid lipolysis, transport, and metabolism. The triglyceride and total cholesterol concentrations in the larvae were significantly reduced following exposure to TBPH or TBMEHP. Furthermore, significant increases in the whole ATP content and locomotor activity in the 120 hpf larvae were observed. The overall results suggest that both TBPH and TBMEHP affect methylation of the PPARγ promoter, subsequently influencing larvae lipid metabolism via the PPARγ signaling pathway and disrupting energy homeostasis.

 

Link to the publication : 

https://pubs.acs.org/doi/abs/10.1021/acs.est.9b05796

Municipal wastewater effluent exposure disrupts early development, larval behavior, and stress response in zebrafish

Environmental Pollution

2019-12-13

Effects of waste watereffluent in zebrafish different life-stages

 

Highlights

 

• Municipal wastewater effluent (MWWE) contains pharmaceutically-active compounds.
• MWWE delays somitogenesis and hatching rates in embryonic zebrafish.
• Short-term exposures to MWWE alters zebrafish embryo and larval behaviors.
• MWWE dulls the cortisol and lactate stress response in juvenile zebrafish.
• Zebrafish at various lifestages are a sensitive model for assessing MWWE toxicity.

 

Abstract

While wastewater treatment standards have been progressively increasing, emerging contaminants such as pharmaceuticals can nonetheless pass through treatment and end up in our watersheds. Pharmaceuticals in the parts-per-billion range can impact fish behavior, survival, and recruitment in the wild. However, the ecological risk posed by whole municipal wastewater effluents (MWWE), a complex mixture, is not clear. This knowledge gap is particularly evident for early lifestages (ELS) of fish, and because effluent discharge events are typically short, the effects of short-term MWWE exposures to ELS fish are particularly important from an environmental perspective. Here we tested the effects of rapid 30-min exposures, and short-term 24- and 72-h exposures to MWWE on development, behaviors, and stress response in zebrafish (Danio rerio) embryos, larvae, and juveniles. We obtained 24-h composite samples of tertiary-treated MWWE that contained a mixture of chemicals with affinities for serotonergic, adrenergic, dopaminergic, and ion-channel receptors. Embryos exposed to 5%, 10%, and 50% MWWE experienced developmental delays in somitogenesis and hatching rate, although there was no effect on survival. Embryonic photomotor responses were affected following 30-min and 24-h exposures to 10% and 50% MWWE, and larval visual motor responses were reduced from 24-h exposure to 10% MWWE. Exposure to 10% MWWE dulled the juvenile cortisol and lactate response following an acute air-exposure. Compromised behavioral and stress performances demonstrate the capacity of MWWE to impact phenotypes critical to the survival of fish in the environment. Taken together, we found that zebrafish were sensitive to toxic effects of MWWE at multiple life-stages.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0269749119350638

Assessment of the ecotoxicity of the pharmaceuticals bisoprolol, sotalol, and ranitidine using standard and behavioral endpoints

Environmental Science and Pollution Research

2019-12-18

Abstract

The pharmaceuticals bisoprolol (BIS), sotalol (SOT), and ranitidine (RAN) are among the most consumed pharmaceuticals worldwide and are frequently detected in different aquatic ecosystems. However, very few ecotoxicity data are available in the literature for them. To help fill these data gaps, toxicity tests with the algae Raphidocelis subcapitata, the macrophyte Lemna minor, the cnidarian Hydra attenuata, the crustacean Daphnia similis, and the fish Danio rerio were performed for assessing the ecotoxicity of these pharmaceuticals. Standard, as well as non-standard endpoint, was evaluated, including the locomotor behavior of D. rerio larvae. Results obtained for SOT and RAN showed that acute adverse effects are not expected to occur on aquatic organisms at the concentrations at which these pharmaceuticals are usually found in fresh surface waters. On the other hand, BIS was classified as hazardous to the environment in the acute III category. Locomotor behavior of D. rerio larvae was not affected by BIS and RAN. A disturbance on the total swimming distance at the dark cycle was observed only for larvae exposed to the highest test concentration of 500 mg L−1 of SOT. D. similis reproduction was affected by BIS with an EC10 of 3.6 (0.1–34.0) mg L−1. A risk quotient (RQ) of 0.04 was calculated for BIS in fresh surface water, considering a worst-case scenario. To the best of our knowledge, this study presents the first chronic toxicity data with BIS on non-target organisms.

 

Link to the publication :

https://link.springer.com/article/10.1007/s11356-019-07322-0

DMSO effects larval zebrafish (Danio rerio) behavior, with additive and interaction effects when combined with positive controls

Science of the Total Environment

2019-12-19

Highlights

 

• Solvents are frequently used during zebrafish toxicity testing but their effects are unknown.
• DMSO affected behavior at a concentration of ≥0.55%
• Different zebrafish strains showed different basal activity, but the same behavioral response to DMSO.
• DMSO had an additive and interaction effects on behavior when co-exposed with positive controls.

 

Abstract

Embryonic and larval zebrafish (Danio rerio) behavior is commonly used to identify neurotoxic compounds. Here, we investigated whether sub-lethal exposures to the common solvents dimethyl sulfoxide (DMSO, 0.01–1%) and methanol (MeOH, 0.01–1%), or the anti-fungal agent methylene blue (MB, 0.0001–0.0005%), can influence larval behavior in a simple light/dark paradigm conducted in 96-well plates. In addition, we tested whether the media volume within the behavioral arena or the zebrafish strain, AB wild type, AB Tübingen (AB/TU), or Tüpfel long-fin (TL), could also influence larval behavior. Following the single exposures, we co-exposed larvae to DMSO and either MB or two other compounds with known behavioral effects in larval zebrafish, flutamide and perfluorooctanesulfonic acid (PFOS). We found ≥0.55% DMSO and 0.0005% MB significantly affected larval behavior, but there was no effect of MeOH. Similarly, TL showed less movement compared to AB and AB/TU strains, whereas lower media volumes also significantly reduced larval movement. However, all strains responded similarly to DMSO and MB. In the co-exposure studies, we found either additive or interaction effects between DMSO and either MB, flutamide, or PFOS, depending on the behavioral endpoint measured. In addition, media volume had no effect on the DMSO concentration response curve, but again we observed additive effects on behavior. In conclusion, methodology can lead to alterations in baseline locomotor activity and compounds can have additive or interaction effects on behavioral endpoints. However, we found no evidence that strain effects should be a concern when deciding on solvents for a simple light/dark behavioral test in larval zebrafish.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S004896971934481X

Isoliquiritigenin triggers developmental toxicity and oxidative stressemediated apoptosis in zebrafish embryos/larvae via Nrf2-HO1/ JNK-ERK/mitochondrion pathway

Chemosphere

2019-12-24

Highlights

 

• ISL triggered developmental toxicity in zebrafish, caused death and malformations.
• ISL exposure instigated developmental toxicity in heart, liver, and nervous system.
• ISL triggered apoptosis in zebrafish via Nrf2-HO1/JNK-ERK/mitochondrion pathway.

 

Abstract

Isoliquiritigenin (ISL) is an emerging natural flavonoid found in the roots of licorice, exhibits antioxidant, anti-cancer, anti-inflammatory, anti-allergic, cardioprotective, hepatoprotective and neuroprotective properties. However, the effect of ISL in embryonic development is yet to be elucidated, and the mechanisms underlying its target-organ toxicity and harmful side effects are still unclear. In the present study, we employed zebrafish embryos to study the developmental toxicity effect of ISL and its underlying mechanisms. Zebrafish embryos upon treatment with either vehicle control (0.1% DMSO) or ISL solutions for 4–96 h post fertilization (hpf) showed that ISL exposure instigated severe developmental toxicity in heart, liver, and nervous system. Mortality and morphological abnormalities were also observed. High concentrations of ISL exposure resulted in abnormal phenotypes and embryonic malformations including pericardial edema, swim bladder defects, yolk retention, curved body shape and shortening of body length. Moreover, ISL exposure led to significant loss of dopaminergic neurons accompanied by reduced locomotor behaviour. Apoptotic cells were predominantly located in the heart area of 96 hpf embryo. Additionally, ISL significantly increased the levels of reactive oxygen species, lipid peroxidation content and decreased antioxidant enzyme activities. The expressions pattern of apoptosis-related genes BadCyto cCaspase-9Caspase-3 and Bax/Bcl-2 indicated that the oxidative stress–induced apoptosis triggered by ISL suggest involvement of Nrf2-HO1/JNK-ERK/mitochondrion pathways. In conclusion, here we provide first evidence that demonstrate ISL-induced dose-dependent developmental toxicity in zebrafish embryos. Furthermore, gene expression patterns in the embryos correlate the above and reveal potential genetic mechanisms of developmental toxicity.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0045653519329686

Anti-Parkinson's disease activity of phenolic acids from Eucommia ulmoides Oliver leaf extracts and its autophagy activation mechanism

Food and Function

2019-12-30

Abstract

Although Parkinson’s disease (PD) is the second most common neurodegenerative disorder, the preventative or therapeutic agents for the treatment of PD are limited. Eucommia ulmoides Oliver (EuO) is widely used as traditional herb to treat various diseases. EuO bark extracts have been reported to possess anti-PD activity. Here, we investigated whether extracts of EuO leaves (EEuOL) also have therapeutic effect on PD since similar components and clinical application have been found between barks and leaves of this tree. We identified the chemical composition of EEuOL by HPLC-Q-TOF-MS and tested the anti-PD effect of EEuOL using zebrafish PD model. As a result, 28 compounds including 3 phenolic acids, 7 flavonoids, and 9 iridoids were identified. EEuOL significantly reversed the loss of dopaminergic neurons and neural vasculature as well as reduced the numbers of apoptotic cells in the zebrafish brain in a concentration-dependent manner. Moreover, EEuOL relieved locomotor impairments in MPTP-modeled PD zebrafish. We also investigated the underlying mechanism and found that EEuOL may activate autophagy, contributing to α-synuclein degradation, therefore alleviate PD-like symptoms. Molecular docking simulation implied the interaction between autophagy regulators (Pink1, Beclin1, Ulk2, and Atg5) and phenolic acids of EEuOL, affirming the involvement of autophagy in EEuOL-exerted anti-PD action. The overall results indicated the anti-PD effect of EEuOL, opening a possibility to use the extract in PD treatment.

 

Link to the publication :

https://pubs.rsc.org/en/content/articlelanding/2020/fo/c9fo02288k#!divAbstract

Toxicity of different zinc oxide nanomaterials and dose-dependent onset and development of Parkinson’s disease-like symptoms induced by zinc oxide nanorods

Environment International

2020-01-01

ABSTRACT

With the increasing applications in various fields, the release and accumulation of zinc oxide (ZnO) nano-materials ultimately lead to unexpected consequences to environment and human health. Therefore, toxicity comparison among ZnO nanomaterials with different shape/size and their adverse effects need better charac-terization. Here, we utilized zebrafish larvae and human neuroblastoma cells SH-SY5Y to compare the toxic effects of ZnO nanoparticles (ZnO NPs), short ZnO nanorods (s-ZnO NRs), and long ZnO NRs (l-ZnO NRs). We found their developmental- and neuro-toxicity levels were similar, where the smaller sizes showed slightly higher toxicity than the larger sizes. The developmental neurotoxicity of l-ZnO NRs (0.1, 1, 10, 50, and 100 μg/mL) was further investigated since they had the lowest toxicity. Our results indicated that l-ZnO NRs induced develop-mental neurotoxicity with hallmarks linked to Parkinsons disease (PD)-like symptoms at relatively high doses, including the disruption of locomotor activity as well as neurodevelopmental and PD responsive genes expres-sion, and the induction of dopaminergic neuronal loss and apoptosis in zebrafish brain. l-ZnO NRs activated reactive oxygen species production, whose excessive accumulation triggered mitochondrial damage and mito-chondrial apoptosis, eventually leading to PD-like symptoms. Collectively, the developmental- and neuro-toxicity of ZnO nanomaterials was identified, in which l-ZnO NRs harbors a remarkably potential risk for the onset and development of PD at relatively high doses, stressing the discretion of safe range in view of nano-ZnO exposure to ecosystem and human beings.

Link to the publication :

https://sci-hub.se/https://www.sciencedirect.com/science/article/pii/S0160412020321346

Effects of Toluene on the Development of the Inner Ear and Lateral Line Sensory System of Zebrafish

Biomedical and environment Sciences

2020-03-14

  Objective  The aim of this study was to explore the ototoxicity of toluene in the early development of zebrafish embryos/larvae.  Methods  Zebrafish were utilized to explore the ototoxicity of toluene. Locomotion analysis, immunofluorescence, and qPCR were used to understand the phenotypes and molecular mechanisms of toluene ototoxicity.  Results  The results demonstrated that at 2 mmol/L, toluene induced zebrafish larvae death at 120 hours post fertilization (hpf) at a rate of 25.79% and inhibited the rate of hatching at 72 hpf. Furthermore, toluene exposure inhibited the distance travelled and average swimming velocity of zebrafish larvae while increasing the frequency of movements. As shown by fluorescence staining of hair cells, toluene inhibited the formation of lateral line neuromasts and middle line 1 (Ml1) neuromasts in 3 days post fertilization larvae in a concentration-dependent manner. Toluene altered the expression level of genes involved in ear development/function in zebrafish, among which the mRNA levels of cd164l2, tekt3, and pcsk5a were upregulated, while the level of otofb was downregulated, according to the qPCR results.  Conclusion  This study indicated that toluene may affect the development of both the inner ear and lateral line systems in zebrafish, while the lateral line system may be more sensitive to toluene than the inner ear.
 
Link to the publciation :
http://www.besjournal.com/en/article/doi/10.3967/bes2021.016

Differential responses of larval zebrafish to the fungicide propamocarb: Endpoints at development, locomotor behavior and oxidative stress

2020-05-14

Authors : Xin Liu, Rui Zhang, Yuanxiang Jin

Institute of standardization, China Jiliang University, Hangzhou 310018, China
College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310032, China

Abstract

The fungicide propamocarb (PM) is widely used to protect cucumbers, tomatoes and other plants from pathogens. According to previous studies, PM could be detected in the aquatic system in some area. However, the toxic effects of PM on zebrafish received very limited attention. In this study, we examined the toxic effects of various concentration of PM on the endpoints of development, locomotor behavior and oxidative stress in larval zebrafish. It was observed that PM exposure delayed embryonic development, inhibited hatchability at 60 and 72 h postfertilization and increased heart rate. After PM exposure, the larval zebrafish showed abnormal free swimming behavior and the swimming behavior in response to light-dark transition, indicating that PM had the potential to induce neurotoxicity. Moreover, PM exposure also affected the enzymatic activity of acetylcholinesterase and dopamine and the transcriptional level of genes related to neurotoxicity. In addition, PM exposure not only affects catalase (CAT), glutathione peroxidase (GPX), and glutathione S-transferase (GST) activity but also affects the transcription level of various genes. We believed that PM induced oxidative stress was also a possible reason to cause neurotoxicity in larval zebrafish. In summary, our results suggested that PM could disturb the endpoints at development, locomotor behavior and oxidative stress in larval zebrafish.

 

https://www.sciencedirect.com/science/article/abs/pii/S004896972032653X

Acute and chronic effects of environmental realistic concentrations of clofibric acid in Danio rerio: Behaviour, oxidative stress, biotransformation and lipid peroxidation endpoints

Environmental Toxicology and Pharmacology

2020-05-15

Highlights

 

• Clofibric acid exposure in zebrafish affected behaviour through hypoactivity.
• Clofibric acid properties caused an increase in all biochemical biomarkers.
• The metabolism of this antihyperlipidemic seems to have modulated behavioral and biochemical results.
• No evidences were found in terms of histology endpoints being altered by clofibric acid.

Abstract

Due to their widespread use, pharmaceuticals can be metabolized, excreted and ultimately discarded in the environment, thereby affecting aquatic organisms. Lipid-regulating drugs are among the most prescribed medications around the world, controlling human cholesterol levels, in more than 20 million patients. Despite this growing use of lipid-regulating drugs, particularly those whose active metabolite is clofibric acid, the potential toxicological effects of these pharmaceuticals in the environment is not fully characterized. This work intended to characterize the toxicity of an acute (120 hours post-fertilization) and chronic (60 days post-fertilization) exposures to clofibric acid in concentrations of 10.35, 20.7, 41.4, 82.8, and 165.6 μg L-1 in zebrafish (Danio rerio). The concentrations which were implemented in both exposures were based on predicted environmental concentrations for Portuguese surface waters. The acute effects were analysed focusing on behavioural endpoints (small and large distance travelled, swimming time and total distance travelled), biomarkers of oxidative stress (activity of the enzymes superoxide dismutase, Cu/Zn- and Mn SOD; catalase, CAT; glutathione peroxidase, Se- and total GPx), biotransformation (activity of glutathione S-transferases, GSTs) and lipid peroxidation (thiobarbituric acid reactive substances, TBARS). Chronically exposed individuals were also histologically analysed for sex determination and gonadal developmental stages. In terms of acute exposure, significant alterations were reported, in terms of behavioural alterations (hypoactivity), followed by an overall increase in all tested biomarkers. Chronically exposed organisms did not show alterations in terms of sex ratio and maturation stages, suggesting that clofibric acid did not act as an endocrine disruptor. Moreover, the metabolism of clofibric acid resulted in increased levels of both forms of SOD activity, especially for animals exposed to higher levels of this drug. An increase of CAT activity was observed in fish exposed to low levels, and a decrease in those exposed to higher amounts of clofibric acid. Both GPx forms had their activities increased. The enzyme of biotransformation GSTs were increased at low levels of clofibric acid but inhibited at higher amounts of this substance. Lipid peroxidation levels were also changed, with an induction of this parameter with increasing amounts of clofibric acid. Changes also occurred in behavioural endpoints and patterns for control organisms and for those exposed to clofibric acid were significantly distinct, for all types (light and darkness) of exposure, and for the two analysed endpoints (small and large distance). Results from this assay allow inferring that clofibric acid can have an ecologically relevant impact in living organisms exposed to this substance, with putative effects on the metabolism of individuals, affecting their behaviour and ultimately their survival.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/pii/S1382668920301447

Visuomotor deficiency in panx1a knockout zebrafish is linked to dopaminergic signaling

2020-06-12

Abstract

Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play roles in the nervous system. The analysis of roles in both standard and pathological conditions benefits from a model organism with rapid development and early onset of behaviors. Such a model was developed by ablating the zebrafish panx1a gene using TALEN technology. Here, RNA-seq analysis of 6 days post fertilization larvae were confirmed by Real-Time PCR and paired with testing visual-motor behavior and in vivo electrophysiology. Results demonstrated that loss of panx1a specifically affected the expression of gene classes representing the development of the visual system and visual processing. Abnormal swimming behavior in the dark and the expression regulation of pre-and postsynaptic biomarkers suggested changes in dopaminergic signaling. Indeed, altered visuomotor behavior in the absence of functional Panx1a was evoked through D1/D2-like receptor agonist treatment and rescued with the D2-like receptor antagonist Haloperidol. Local field potentials recorded from superficial areas of the optic tectum receiving input from the retina confirmed abnormal responses to visual stimuli, which resembled treatments with a dopamine receptor agonist or pharmacological blocking of Panx1a. We conclude that Panx1a functions are relevant at a time point when neuronal networks supporting visual-motor functions undergo modifications preparing for complex behaviors of freely swimming fish.

Introduction

Pannexin 1 (Panx1) is an integral membrane glycoprotein forming ATP release channels in different tissues and cell types1,2,3,4,5, including neurons6,7,8. In the CNS, evidence for physiological functions of Panx1 points at roles in the processing of sensory signals and learning and memory9,10,11. For example, in Panx1 knockout mice, altered retinal contrast sensitivity12 and hearing loss have been found13,14. Also, performance in spatial learning and memory abilities such as object recognition and fear conditioning tasks are decreased9,15,16. Intellectual disabilities, severe hearing loss, primary ovarian failure, kyphoscoliosis, and difficulties navigating in darkness were found in the first human patient identified with a homozygous Panx1 mutation17.

To form a better view of Panx1 functions in the processing of sensory information, we used the zebrafish as model organisms. Two Panx1 genes, panx1a and panx1b, originated from partial genome duplications during early teleost evolution18,19. Although the two genes have been separated for more than 200 million years, principal channel functions are comparable to rodent or human Panx120. In the retina, the panx1a protein is expressed in horizontal cells19,20 and plays essential roles in feedback from horizontal cells to cones in adult zebrafish21,22,23. Here the panx1a gene was edited using transcription activator-like effector nucleases (TALEN). A loss of function mutation allowed to investigate Panx1a in 6 dpf old zebrafish larvae at a developmental stage when neuronal networks for visually guided locomotor behaviors were functional. Transcriptome analysis detected noteworthy expression differences of genes associated with eye development and vision-related processes. When followed up, altered dopaminergic signaling affecting both pre- and postsynaptic proteins were found. The molecular evidence was supported by measuring abnormal responsiveness of panx1a−/− larvae to darkness, or after the abrupt loss of illumination. Pharmacological activation of D1/D2-like dopamine receptors simulated this phenotype. Blocking D2-like receptors with Haloperidol rescued the phenotype. In vivo electrophysiological recording of local field potentials (LOF) from the larval optic tectum in a region receiving input from the retina demonstrated that the dynamic transition from low to higher-frequency brain waves in light and darkness was compromised in panx1a−/− larvae. This phenotype was reproduced by pharmacological blocking of Panx1a, or by treatment with the D1/D2 receptor agonist apomorphine. This research delivers a novel association between Panx1a in the integration of sensory-motor behavior through modulation of dopaminergic signaling.

 

Link to the publication :

https://www.nature.com/articles/s41598-020-66378-y

A multiorganism pipeline for antiseizure drug discovery: Identification of chlorothymol as a novel γ‐aminobutyric acidergic anticonvulsant

Epilepsia

2020-06-14

Abstract

Objective

Current medicines are ineffective in approximately one‐third of people with epilepsy. Therefore, new antiseizure drugs are urgently needed to address this problem of pharmacoresistance. However, traditional rodent seizure and epilepsy models are poorly suited to high‐throughput compound screening. Furthermore, testing in a single species increases the chance that therapeutic compounds act on molecular targets that may not be conserved in humans. To address these issues, we developed a pipeline approach using four different organisms.

Methods

We sequentially employed compound library screening in the zebrafish, Danio rerio, chemical genetics in the worm, Caenorhabditis elegans, electrophysiological analysis in mouse and human brain slices, and preclinical validation in mouse seizure models to identify novel antiseizure drugs and their molecular mechanism of action.

Results

Initially, a library of 1690 compounds was screened in an acute pentylenetetrazol seizure model using D rerio. From this screen, the compound chlorothymol was identified as an effective anticonvulsant not only in fish, but also in worms. A subsequent genetic screen in C elegans revealed the molecular target of chlorothymol to be LGC‐37, a worm γ‐aminobutyric acid type A (GABAA) receptor subunit. This GABAergic effect was confirmed using in vitro brain slice preparations from both mice and humans, as chlorothymol was shown to enhance tonic and phasic inhibition and this action was reversed by the GABAA receptor antagonist, bicuculline. Finally, chlorothymol exhibited in vivo anticonvulsant efficacy in several mouse seizure assays, including the 6‐Hz 44‐mA model of pharmacoresistant seizures.

Significance

These findings establish a multiorganism approach that can identify compounds with evolutionarily conserved molecular targets and translational potential, and so may be useful in drug discovery for epilepsy and possibly other conditions.

 

Key Points

 

  • Compound library screening in zebrafish identifies chlorothymol as an anticonvulsant
  • Genetic screening in nematodes reveals that chlorothymol acts on GABAA receptors
  • Electrophysiological recording from mouse and human neurons confirms chlorothymol's GABAergic mechanism of action
  • Chlorothymol is protective in several mouse seizure assays, including the 6‐Hz 44‐mA model of pharmacoresistant seizures

Link to the publication :

https://onlinelibrary.wiley.com/doi/full/10.1111/epi.16644

Role of GH/IGF axis in arsenite-induced developmental toxicity in zebrafish embryos

Ecotoxicology and Environmental Safety

2020-06-15

Highlights

 

• Arsenite induced abnormal development of zebrafish.
• Arsenite increased the level of GH in zebrafish.
• Arsenite down-regulated the expression of GH/IGF axis related genes.
• Arsenite caused locomotor behavior disorder by affecting abnormal neurodevelopment.

Abstract

Growth hormone (GH)/insulin-like growth factor (IGF) axis plays a critical role in fetal development. However, the effect of arsenite exposure on the GH/IGF axis and its toxic mechanism are still unclear. Zebrafish embryos were exposed to a range of NaAsO2 concentrations (0.0–10.0 mM) between 4 and 120 h post-fertilization (hpf). Development indexes of survival, malformation, hatching rate, heart rate, body length and locomotor behavior were measured. Hormone levels, GH/IGF axis-related genes, and nerve-related genes were also tested. The results showed that survival rate, hatching rate, heart rate, body length and locomotor behavior all decreased, while deformity increased. At 120 hpf, the survival rate of zebrafish in 1.5 mM NaAsO2 group was about 70%, the deformity rate exceeded 20%, and the body length shortened to 3.35 mm, the movement distance of zebrafish decreased approximately 63.6% under light condition and about 52.4% under dark condition. The level of GH increased and those of IGF did not change significantly, while the expression of GH/IGF axis related genes (ghra, ghrb, igf2r, igfbp3, igfbp2a, igfbp5b) and nerve related genes (dlx2, shha, ngn1, elavl3, gfap) decreased. In 1.5 mM NaAsO2 group, the decrease of igfbp3 and igfbp5b was almost obvious, about 78.2% and 72.2%. The expression of nerve genes in 1.5 mM NaAsO2 group all have declined by more than 50%. These findings suggested that arsenite exerted disruptive effects on the endocrine system by interfering with the GH/IGF axis, leading to zebrafish embryonic developmental toxicity.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S014765132030659X

The multi-dimensional embryonic zebrafish platform predicts flame retardant bioactivity

Reproductive Toxicology

2020-06-15

Highlights

 

• Identified the bioactivity of 61 flame retardant chemicals (FRCs).
• The zebrafish assay detects bioactivity of 12 of the 10 known developmental neurotoxic FRCs.
• Classification model using physicochemical properties and 3 zebrafish assays yields a balanced accuracy of 91.7%.

 

Abstract

Flame retardant chemicals (FRCs) commonly added to many consumer products present a human exposure burden associated with adverse health effects. Under pressure from consumers, FRC manufacturers have adopted some purportedly safer replacements for first-generation brominated diphenyl ethers (BDEs). In contrast, second and third-generation organophosphates and other alternative chemistries have limited bioactivity data available to estimate their hazard potential. In order to evaluate the toxicity of existing and potential replacement FRCs, we need efficient screening methods. We built a 61-FRC library in which we systemically assessed developmental toxicity and potential neurotoxicity effects in the embryonic zebrafish model. Data were compared to publicly available data generated in a battery of cell-based in vitro assays from ToxCast, Tox21, and other alternative models. Of the 61 FRCs, 19 of 45 that were tested in the ToxCast assays were bioactive in our zebrafish model. The zebrafish assays detected bioactivity for 10 of the 12 previously classified developmental neurotoxic FRCs. Developmental zebrafish were sufficiently sensitive at detecting FRC structure-bioactivity impacts that we were able to build a classification model using 13 physicochemical properties and 3 embryonic zebrafish assays that achieved a balanced accuracy of 91.7%. This work illustrates the power of a multi-dimensional in vivo platform to expand our ability to predict the hazard potential of new compounds based on structural relatedness, ultimately leading to reliable toxicity predictions based on chemical structure.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0890623820301969

Environmental levels of venlafaxine impact larval behavioural performance in fathead minnows

Chemosphere

2020-06-18

Highlights

 

• Environmental levels of venlafaxine reduce larval growth and activity.
• Larval minnows exhibit altered behavioural responses in light and darkness.
• Thigmotaxis is a reliable measure of larval anxiogenic/anxiolytic behaviour in fathead minnows.
• Venlafaxine did not affect thigmotaxis in larval fathead minnows.

 

Abstract

Venlafaxine, a selective serotonin and norepinephrine reuptake inhibitor, is one of the most abundant antidepressants in municipal wastewater effluents (MWWE). The early life stages are particularly sensitive to contaminant exposure, but few studies have examined whether persistent exposure to venlafaxine impart adverse developmental outcomes. The fathead minnow (Pimephales promelas) is a widely used model for ecotoxicological studies, and this fish is native to Alberta, Canada. We tested the hypothesis that environmental levels of venlafaxine compromises early developmental behavioural performances in fathead minnows. Embryos were exposed to waterborne venlafaxine at either 0, 0.06, 0.33, 0.66, 1.37 or 3 μg L−1 concentration for 7 days. Environmental levels of venlafaxine did not impact the survival, hatch rate or heart rate of fathead minnow embryos and larvae but reduced the growth of larvae even at concentrations as low as 0.06 μg L−1. We validated thigmotaxis as a screen for anxiolytic and anxiogenic behaviour in fathead minnow larvae by exposing them to concentrations of ethanol and caffeine, respectively. Behavioural analyses revealed that early developmental exposure to venlafaxine does not alter thigmotaxis but reduced the activity of fathead minnows. The larval behavioural assays reported here for fathead minnow have the potential to be used as screening tools for the risk assessment of neurotoxic contaminants in MWWE. Overall, we demonstrate for the first time that exposure to environmental levels of venlafaxine during the critical early developmental window does not elicit an anxiogenic response but may adversely affect the larval growth performance of fathead minnows.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0045653520316313

Inhibitory effects of polystyrene microplastics on caudal fin regeneration in zebrafish larvae

Environmental Pollution

2020-06-21

Highlights

 

• Microplastic exposure inhibited the caudal fin regeneration in zebrafish larvae.
• Regeneration-related signalings were altered after microplastic exposure.
• Changes of ROS signaling and immune response contributed to the inhibitory effects.
• Transcriptomic analyses suggested the metabolic pathways were also involved.
• Microplastics might impair the capacity for repair and regeneration in injured fish.

 

Abstract

Microplastic pollution is pervasive in aquatic environments, but the potential effects of microplastics on aquatic organisms are still under debate. Given that tissue damage is unavoidable in fish and the available data mostly concentrate on healthy fish, there is a large chance that the ecotoxicological risk of microplastic pollution is underrated. Therefore, in this study, the effects of microplastics on the regenerative capacity of injured fish were investigated using a zebrafish caudal fin regeneration model. After fin amputation at 72 h post fertilization, the larvae were exposed to polystyrene microplastics (0.1–10 mg/L) with diameters of 50 or 500 nm. Microplastic exposure significantly inhibited fin regeneration, both morphologically and functionally. Furthermore, the signaling networks that regulate fin regeneration, as well as reactive oxygen species signaling and the immune response, both of which are essential for tissue repair and regeneration, were altered. Transcriptomic analyses of the regenerating fin confirmed that genes related to fin regeneration were transcriptionally modulated in response to microplastic exposure and that metabolic pathways were also extensively involved. In conclusion, this study demonstrated for the first time that microplastic exposure could disrupt the regenerative capacity of fish and might eventually impair their fitness in the wild.

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0269749120301524

Impacts of bisphenol A analogues on zebrafish post‐embryonic brain

Journal of Neuroendocrinology

2020-06-21

Abstract

Bisphenol A (BPA) is a widely studied and well‐recognised endocrine‐disrupting chemical, and one of the current issues is its safe replacement by various analogues. Using larva zebrafish as a model, the present study reveals that moderate and chronic exposure to BPA analogues such as bisphenol S, bisphenol F and bisphenol AF may also affect vertebrate neurodevelopment and locomotor activity. Several parameters of embryo‐larval development were investigated, such as mortality, hatching, number of mitotically active cell, as defined by 5‐bromo‐2'‐deoxyuridine incorporation and proliferative cell nuclear antigen labelling, aromatase B protein expression in radial glial cell and locomotor activity. Our results show that exposure to several bisphenol analogues induced an acceleration of embryo hatching rate. At the level of the developing brain, a strong up‐regulation of the oestrogen‐sensitive Aromatase B was also detected in the hypothalamic region. This up‐regulation was not associated with effects on the numbers of mitotically active progenitors nor differentiated neurones in the preoptic area and in the nuclear recessus posterior of the hypothalamus zebrafish larvae. Furthermore, using a high‐throughput video tracking system to monitor locomotor activity in zebrafish larvae, we show that some bisphenol analogues, such as bisphenol AF, significantly reduced locomotor activity following 6 days of exposure. Taken together, our study provides evidence that BPA analogues can also affect the neurobehavioural development of zebrafish.

 

Link to the publication : 

https://onlinelibrary.wiley.com/doi/abs/10.1111/jne.12879

Toxicity of different polycyclic aromatic hydrocarbons (PAHs) to the freshwater planarian Girardia tigrina

Environmental Pollution

2020-07-03

Highlights

 

• Several endpoints can be evaluated in planarians in response to chemical stress.
• Phenanthrene, but not pyrene or B[a]P, caused mortality in planarians.
• Planarian sub-lethal endpoints were more sensitive to pyrene and B[a]P exposures.
• Behavioral endpoints showed the greatest sensitivity for all tested PAHs.
• Planarians can be useful experimental animals for ecotoxicological studies.

 

Abstract

Freshwater planarians have been gaining relevance as experimental animals for numerous research areas given their interesting features, such as high regeneration potential, shared features with the vertebrates’ nervous system or the range of endpoints that can be easily evaluated in response to contaminants. Ecotoxicological research using these animals has been steadily increasing in the past decades, as planarians’ potentialities for this research area are being recognized. In this work, we used polycyclic aromatic hydrocarbons (PAHs) as model contaminants and evaluated effects of exposure to phenanthrene, pyrene and benzo[a]pyrene (B[a]P) in planarians. The freshwater planarian Girardia tigrina was chosen and mortality, cephalic regeneration (during and post-exposure), behavioral endpoints and presence of PAHs in tissues, were evaluated. Mortality was only observed in planarians exposed to phenanthrene, with an estimated LC50 of 830 μg L−1. Results indicate that planarian behavioral endpoints were very sensitive in response to sub-lethal concentrations of PAHs, showing a greater sensitivity towards B[a]P and pyrene. Briefly, post-exposure locomotion and post-exposure feeding were significantly impaired by sub-lethal concentrations of all compounds, whereas regeneration of photoreceptors was only significantly delayed in planarians exposed to pyrene. Moreover, levels of PAH-type compounds in planarian tissues followed a concentration-dependent increase, showing uptake of compounds from experimental solutions. The present results highlight the importance of studying alternative and complementary endpoints, such as behavior, not only because these may be able to detect effects at lower levels of contamination, but also due to their ecological relevance. The simplicity of evaluating a wide range of responses to contaminants further demonstrates the utility of freshwater planarians for ecotoxicological research.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0269749120302244

Impacts of high dose 3.5 GHz cellphone radiofrequency on zebrafish embryonic development

Plos One

2020-07-09

Abstract

The rapid deployment of 5G spectrum by the telecommunication industry is intended to promote better connectivity and data integration among various industries. However, since exposures to radio frequency radiations (RFR) >2.4 GHz are still uncommon, concerns about their potential health impacts are ongoing. In this study, we used the embryonic zebrafish model to assess the impacts of a 3.5 GHz RFR on biology- a frequency typically used by 5G-enabled cell phones and lies within the 4G and 5G bandwidth. We established a plate-based exposure setup for RFRs, exposed developing zebrafish to 3.5 GHz RFR, specific absorption rate (SAR) ≈ 8.27 W/Kg from 6 h post fertilization (hpf) to 48 hpf, and measured a battery of morphological and behavioral endpoints at 120 hpf. Our results revealed no significant impacts on mortality, morphology or photomotor response and a modest inhibition of startle response suggesting some levels of sensorimotor disruptions. This suggests that the cell phone radiations at low GHz-level frequencies are likely benign, with subtle sensorimotor effects. Through this assessment, we have established a robust setup for zebrafish RFR exposures readily amenable to testing various powers and frequencies. Future developmental exposure studies in zebrafish will evaluate a wider portion of the radio frequency spectrum to discover the bioactive regions, the potential molecular targets of RFR and the potential long-term effects on adult behavior.

 

Link to the publication : 

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235869

Ponatinib-induced ischemic stroke in larval zebrafish for drug screening

European Journal of Pharmacology

2020-07-12

Highlights

 

• We developed and characterized a larval zebrafish ischemic stroke model using Ponatinib as an inducer under an optimized exposure concentration and treatment period. The larval zebrafish ischemic stroke was further validated with 6 known human ischemic stroke therapeutics. The pathophysiology of this zebrafish ischemic stroke is closely similar to that of human ischemic stroke and could be used for rapidly identifying the preventive and therapeutic agents.

 

Absract

Conventional mammalian ischemic stroke models for drug screening are technically challenging, laborious and time-consuming. In this study, using Ponatinib as an inducer, we developed and characterized a zebrafish ischemic stroke model. This zebrafish ischemic stroke had the cerebral vascular endothelial injury, thrombosis, reduced blood flow, inflammation and apoptosis as well as the reduced motility. The zebrafish ischemic stroke model was validated with 6 known human therapeutic drugs of ischemic stroke (Aspirin, Clopidogrel, Naoxintong capsules, Edaravone, Xingnaojing injection, Shuxuening injection). The mRNA levels of the neovascularization-related gene (vegfaa) and vascular endothelial growth factor receptor gene (VEGFR), neurodevelopment related genes (mbp and α1-tubulin), brain-derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF) were significantly downregulated; whereas apoptosis-related genes (caspase-3caspase-7caspase-9 and bax/bcl-2), and inflammatory factor genes (IL-1βIL-6IL-10TNF-α and NF-κB) were remarkably upregulated in the model. These results suggest that the pathophysiology of Ponatinib-induced zebrafish ischemic stroke is similar to that of human ischemic stroke patients and this whole animal model could be used to study the complex cellular and molecular pathogenesis of ischemic stroke and to rapidly identify therapeutic agents.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0014299920303848

Xanthotoxin reverses Parkinson’s disease-like symptoms in zebrafish larvae and mice models: a comparative study

Pharmacological Reports

2020-07-22

Abstract

Background

The aim of this study is to preliminary evaluate the antiparkinsonian activity of furanocoumarin—xanthotoxin, in two behavioral animal models, zebrafish larvae treated with 6-hydroxydopamine and mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in order to compare both models.

Methods

Xanthotoxin was isolated from Pastinaca sativa L. (Apiaceae) fruits. Then, the compound was administered by immersion to zebrafish 5 days after fertilization (dpf) larvae or intraperitoneally to male Swiss mice, as a potential therapeutic agent against locomotor impairments.

Results

Acute xanthotoxin administration at the concentration of 7.5 µM reversed locomotor activity impairments in 5-dpf zebrafish larvae. In mice model, acute xanthotoxin administration alleviated movement impairments at the concentration of 25 mg/kg.

Conclusions

The similar activity of the same substance in two different animal models indicates their compatibility and proves the potential of in vivo bioassays based on zebrafish models. Results of our study indicate that xanthotoxin may be considered as a potential lead compound in the discovery of antiparkinsonian drugs.

 

Link to the publication : 

https://link.springer.com/article/10.1007/s43440-020-00136-9

Folic acid reduces the ethanol-induced morphological and behavioral defects in embryonic and larval zebrafish (Danio rerio) as a model for fetal alcohol spectrum disorder (FASD)

Reproductive Toxicology

2020-08-04

Highlights

 

• Embryonic EtOH exposure induced morphological defects in the zebrafish FASD model.
• FA prevented defects on morphology, swimming, thigmotaxis, and optomotor response in larvae.
• EtOH exposure affected the sleep pattern, inducing several arousal periods in zebrafish.
• EtOH produced developmental defects, and FA reduced partially these defects.

 Abstract

The objective of this work was to determine whether folic acid (FA) reduces the embryonic ethanol (EtOH) exposure induced behavioral and morphological defects in our zebrafish fetal alcohol spectrum disorder (FASD) model. Teratogenic effects, mortality, the excitatory light-dark locomotion (ELD), sleep (SL), thigmotaxis (TH), touch sensitivity (TS), and optomotor response (OMR) tests were evaluated in larvae (6–7 days post-fertilization) using four treatment conditions: Untreated, FA, EtOH and EtOH + FA. FA reduced morphological defects on heart, eyes and swim bladder inflation seen in EtOH exposed fish. The larvae were more active in the dark than in light conditions, and EtOH reduced the swimming activity in the ELD test. EtOH affected the sleep pattern, inducing several arousal periods and increasing inactivity in zebrafish. FA reduces these toxic effects and produced more consistent inactivity during the night, reducing the arousal periods. FA also prevented the EtOH-induced defects in thigmotaxis and optomotor response of the larvae. We conclude that in this FASD model, EtOH exposure produced several teratogenic and behavioral defects, FA reduced, but did not totally prevent, these defects. Understanding of EtOH-induced behavioral defects could help to identify new therapeutic or prevention strategies for FASD.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0890623820301878

Folic acid reduces the ethanol-induced morphological and behavioral defects in embryonic and larval zebrafish (Danio rerio) as a model for fetal alcohol spectrum disorder (FASD)

Reproductive Toxicology

2020-08-04

Highlights

 

• Embryonic EtOH exposure induced morphological defects in the zebrafish FASD model.
• FA prevented defects on morphology, swimming, thigmotaxis, and optomotor response in larvae.
• EtOH exposure affected the sleep pattern, inducing several arousal periods in zebrafish.
• EtOH produced developmental defects, and FA reduced partially these defects.

 

Abstract

The objective of this work was to determine whether folic acid (FA) reduces the embryonic ethanol (EtOH) exposure induced behavioral and morphological defects in our zebrafish fetal alcohol spectrum disorder (FASD) model. Teratogenic effects, mortality, the excitatory light-dark locomotion (ELD), sleep (SL), thigmotaxis (TH), touch sensitivity (TS), and optomotor response (OMR) tests were evaluated in larvae (6–7 days post-fertilization) using four treatment conditions: Untreated, FA, EtOH and EtOH + FA. FA reduced morphological defects on heart, eyes and swim bladder inflation seen in EtOH exposed fish. The larvae were more active in the dark than in light conditions, and EtOH reduced the swimming activity in the ELD test. EtOH affected the sleep pattern, inducing several arousal periods and increasing inactivity in zebrafish. FA reduces these toxic effects and produced more consistent inactivity during the night, reducing the arousal periods. FA also prevented the EtOH-induced defects in thigmotaxis and optomotor response of the larvae. We conclude that in this FASD model, EtOH exposure produced several teratogenic and behavioral defects, FA reduced, but did not totally prevent, these defects. Understanding of EtOH-induced behavioral defects could help to identify new therapeutic or prevention strategies for FASD.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0890623820301878

Parental exposure to environmental concentrations of tris(1,3-dichloro-2-propyl)phosphate induces abnormal DNA methylation and behavioral changes in F1 zebrafish larvaeParental exposure to environmental concentrations of tris(1,3-dichloro-2-propyl)phosphate induces abnormal DNA methylation and behavioral changes in F1 zebrafish larvae

Environmental Pollution

2020-08-11

Highlights

 

• Residues of TDCIPP were detected in F1 eggs after exposure of parents.
• Parental exposure had effect on motor behaviors of F1 individuals.
• Parental exposure induced DNA methylation alterations in F1 larvae.
• Parental exposure downregulated the expression of genes related to neuronal cell body.
• Downregulation of slc1a2b was positively correlated with reduced swimming speed of F1 larvae.

 

Abstract

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) has been demonstrated to be transferred from parental animals to their offspring. However, whether parental exposure to environmental concentrations of TDCIPP show neurodevelopmental toxicity in the F1 generation and the possible underlying mechanism remain unclear. Therefore, in this study, zebrafish embryos were exposed to environmental concentrations of TDCIPP (3, 30 and 300 ng·L-1) for 120 days. The effects of exposure on motor behaviors, neurotransmitter levels, DNA methylation, and gene expression of F1 larvae were investigated. Parental exposure left TDCIPP residues in F1 eggs as well as reduced body length of F1 larvae. Moreover, parental exposure significantly reduced swimming activity in F1 5 dpf larvae, although it did not significantly alter serotonin, dopamine, 3,4-dihydroxyphenylacetic acid, γ-aminobutyrate, and acetylcholine levels. Genes encoding DNA methylation transferases (dnmt3aa and dnmt1) were downregulated in F1 larvae. Reduced representation bisulfite sequencing analysis revealed 446 differentially methylated regions and enriched neuronal cell body Gene Ontology term in F1 generation. Correlation analysis between the expression of genes related to neural cell body and swimming speed indicated that solute carrier family 1 member 2b (slc1a2b) downregulation might be responsible for the inhibition of motor behaviors. Furthermore, bisulfite amplicon sequencing analysis confirmed hypermethylation of the promoter region of slc1a2b in F1 larvae following parental exposure to 300 ng·L-1 TDCIPP, which might have led to significant downregulation of gene expression and, in turn, influenced the motor behaviors. These results indicate that parental exposure to environmental concentrations of TDCIPP alters DNA methylation, downregulates gene expressions and, thus inducing developmental neurotoxicity, in F1 larvae.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0269749120359935

Parental exposure to environmental concentrations of tris(1,3-dichloro-2- propyl)phosphate induces abnormal DNA methylation and behavioral changes in F1 zebrafish larvae

Environmental Pollution

2020-08-11

Highlights

 

Residues of TDCIPP were detected in F1 eggs after exposure of parents.

Parental exposure had effect on motor behaviors of F1 individuals.

Parental exposure induced DNA methylation alterations in F1 larvae.

Parental exposure downregulated the expression of genes related to neuronal cell body.

Downregulation of slc1a2b was positively correlated with reduced swimming speed of F1 larvae.

 

Abstract

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) has been demonstrated to be transferred from parental animals to their offspring. However, whether parental exposure to environmental concentrations of TDCIPP show neurodevelopmental toxicity in the F1 generation and the possible underlying mechanism remain unclear. Therefore, in this study, zebrafish embryos were exposed to environmental concentrations of TDCIPP (3, 30 and 300 ng L−1) for 120 days. The effects of exposure on motor behaviors, neurotransmitter levels, DNA methylation, and gene expression of F1 larvae were investigated. Parental exposure left TDCIPP residues in F1 eggs as well as reduced body length of F1 larvae. Moreover, parental exposure significantly reduced swimming activity in F1 5 dpf larvae, although it did not significantly alter serotonin, dopamine, 3,4-dihydroxyphenylacetic acid, γ-aminobutyrate, and acetylcholine levels. Genes encoding DNA methylation transferases (dnmt3aa and dnmt1) were downregulated in F1 larvae. Reduced representation bisulfite sequencing analysis revealed 446 differentially methylated regions and enriched neuronal cell body Gene Ontology term in F1 generation. Correlation analysis between the expression of genes related to neural cell body and swimming speed indicated that solute carrier family 1 member 2b (slc1a2b) downregulation might be responsible for the inhibition of motor behaviors. Furthermore, bisulfite amplicon sequencing analysis confirmed hypermethylation of the promoter region of slc1a2b in F1 larvae following parental exposure to 300 ng L−1 TDCIPP, which might have led to significant downregulation of gene expression and, in turn, influenced the motor behaviors. These results indicate that parental exposure to environmental concentrations of TDCIPP alters DNA methylation, downregulates gene expressions and, thus inducing developmental neurotoxicity, in F1 larvae.

Link to the publication :

https://www.sciencedirect.com/science/article/abs/pii/S0269749120359935

A multiorganism pipeline for antiseizure drug discovery: Identification of chlorothymol as a novel γ‐aminobutyric acidergic anticonvulsant

Epilepsia

2020-08-14

Abstract

Objective:

Current medicines are ineffective in approximately one-third of people with epilepsy. Therefore, new antiseizure drugs are urgently needed to address this problem of pharmacoresistance. However, traditional rodent seizure and epilepsy models are poorly suited to high-throughput compound screening. Furthermore, testing in a single species increases the chance that therapeutic compounds act on molecular targets that may not be conserved in humans. To address these issues, we developed a pipeline approach using four different organisms.

Methods:

We sequentially employed compound library screening in the zebrafish, Danio rerio, chemical genetics in the worm, Caenorhabditis elegans, electrophysiological analysis in mouse and human brain slices, and preclinical validation in mouse seizure models to identify novel antiseizure drugs and their molecular mechanism of action.

Results:

Initially, a library of 1690 compounds was screened in an acute pentylenetetrazol seizure model using D rerio. From this screen, the compound chlorothymol was identified as an effective anticonvulsant not only in fish, but also in worms. A subsequent genetic screen in C elegans revealed the molecular target of chlorothymol to be LGC-37, a worm γ-aminobutyric acid type A (GABAA) receptor subunit. This GABAergic effect was confirmed using in vitro brain slice preparations from both mice and humans, as chlorothymol was shown to enhance tonic and phasic inhibition and this action was reversed by the GABAA receptor antagonist, bicuculline. Finally, chlorothymol exhibited in vivo anticonvulsant efficacy in several mouse seizure assays, including the 6-Hz 44-mA model of pharmacoresistant seizures.

 

Link to the publication : 

https://onlinelibrary.wiley.com/doi/full/10.1111/epi.16644

Acute and chronic effects of environmental realistic concentrations of clofibric acid in Danio rerio: Behaviour, oxidative stress, biotransformation and lipid peroxidation endpoints

Environmental Toxicology and Pharmacology

2020-08-14

Highlights

• Clofibric acid exposure in zebrafish affected behaviour through hypoactivity.
• Clofibric acid properties caused an increase in all biochemical biomarkers.
• The metabolism of this antihyperlipidemic seems to have modulated behavioral and biochemical results.
• No evidences were found in terms of histology endpoints being altered by clofibric acid.

Abstract

Due to their widespread use, pharmaceuticals can be metabolized, excreted and ultimately discarded in the environment, thereby affecting aquatic organisms. Lipid-regulating drugs are one of the most prescribed medications around the world, controlling human cholesterol levels, in more than 20 million patients. Despite this growing use of lipid-regulating drugs, particularly those whose active metabolite is clofibric acid, the potential toxicological effects of these pharmaceuticals in the environment is not fully characterized. This work intended to characterize the toxicity of an acute (120 hours post-fertilization) and chronic (60 days post-fertilization) exposures to clofibric acid in concentrations of 10.35, 20.7, 41.4, 82.8 and 165.6 µg L-1 in zebrafish (Danio rerio). The concentrations which were implemented in both exposures were based on predicted environmental concentrations for Portuguese surface waters. The acute effects were analysed focusing on behavioural endpoints (small and large distance travelled, swimming time and total distance travelled), biomarkers of oxidative stress (activity of the enzymes superoxide dismutase, Cu/Zn- and Mn SOD; catalase, CAT; glutathione peroxidase, Se- and total GPx), biotransformation (activity of glutathione S-transferases, GSTs) and lipid peroxidation (thiobarbituric acid reactive substances, TBARS). Chronically exposed individuals were also histologically analysed for sex determination and gonadal developmental stages. In terms of acute exposure, significant alterations were reported, in terms of behavioural alterations (hypoactivity), followed by an overall increase in all tested biomarkers. Chronically exposed organisms did not show alterations in terms of sex ratio and maturation stages, suggesting that clofibric acid did not act as an endocrine disruptor. Moreover, the metabolism of clofibric acid resulted in increased levels of both forms of SOD activity, especially for animals exposed to higher levels of this drug. An increase of CAT activity was observed in fish exposed to low levels, and a decrease in those exposed to higher amounts of clofibric acid. Both GPx forms had their activities increased. The enzyme of biotransformation GSTs were increased at low levels of clofibric acid but inhibited at higher amounts of this substance. Lipid peroxidation levels were also changed, with an induction of this parameter with increasing amounts of clofibric acid. Changes also occurred in behavioural endpoints and patterns for control organisms and for those exposed to clofibric acid were significantly distinct, for all types (light and darkness) of exposure, and for the two analysed endpoints (small and large distance). Results from this assay allow inferring that clofibric acid can have an ecologically relevant impact in living organisms exposed to this substance, with putative impact on the metabolism of individuals, affecting their behaviour and ultimately their survival.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/pii/S1382668920301447

Developmental neurotoxicity fingerprint of silica nanoparticles at environmentally relevant level on larval zebrafish using a neurobehavioral-phenomics-based biological warning method

Science of the total environment

2020-08-21

Highlights

 

• Silica NPs at environmental level induced the negligible embryonic teratogenicity.
• Silica NPs at environmental level altered behavioral profiles determined by NBP.
• NBP are more sensitive and effective than FET to determine sub-teratogenic toxicity.

Abstract

Background

Larval zebrafish (Danio rerio) is not only an ideal vertebrate applied in Fish Embryos Toxicity (FET) test but also a well-accepted model in behavioral neurotoxicity research. By applying the commercial standard behavioral tracking system (Zebrabox), the locomotion profiles (neurobehavioral-phenomics) of larval zebrafish can be comprehensively monitored and systematically analyzed to probe ecotoxicological neurotoxicity of nano-pollutants at environmental relevant concentration level.

 

Link to the publication : 

https://www.sciencedirect.com/science/article/abs/pii/S0048969720354073

Multiple Screening of Pesticides Toxicity in Zebrafish and Daphnia Based on Locomotor Activity Alterations

Biomolecules

2020-08-23

Abstract:

Pesticides are widely used to eradicate insects, weed species, and fungi in agriculture. The half-lives of some pesticides are relatively long and may have the dire potential to induce adverse effects when released into the soil, terrestrial and aquatic systems. To assess the potential adverse effects of pesticide pollution in the aquatic environment, zebrafish (Danio rerio) and Daphnia magna are two excellent animal models because of their transparent bodies, relatively short development processes, and well-established genetic information. Moreover, they are also suitable for performing high-throughput toxicity assays. In this study, we used both zebrafish larvae and water flea daphnia neonates as a model system to explore and compare the potential toxicity by monitoring locomotor activity. Tested animals were exposed to 12 various types of pesticides (three fungicides and 9 insecticides) for 24 h and their corresponding locomotor activities, in terms of distance traveled, burst movement, and rotation were quantified. By adapting principal component analysis (PCA) and hierarchical clustering analysis, we were able to minimize data complexity and compare pesticide toxicity based on locomotor activity for zebrafish and daphnia. Results showed distinct locomotor activity alteration patterns between zebrafish and daphnia towards pesticide exposure. The majority of pesticides tested in this study induced locomotor hypo-activity in daphnia neonates but triggered locomotor hyper-activity in zebrafish larvae. According to our PCA and clustering results, the toxicity for 12 pesticides was grouped into two major groups based on all locomotor activity endpoints collected from both zebrafish and daphnia. In conclusion, all pesticides resulted in swimming alterations in both animal models by either producing hypo-activity, hyperactivity, or other changes in swimming patterns. In addition, zebrafish and daphnia displayed distinct sensitivity and response against different pesticides, and the combinational analysis approach by using a phenomic approach to combine data collected from zebrafish and daphnia provided better resolution for toxicological assessment.

Vape flavourants dull sensory perception and cause hyperactivity in developing zebrafish embryos

Biology Letters

2020-08-24

E-cigarette use (vaping) during pregnancy has been increasing, and the potential exists for the developing brain in utero to be exposed to chemical constituents in the vape. Vapes come in over 7000 unique flavours with and without nicotine, and while nicotine is a known neurotoxicant, the effects of vape flavouring alone, in the absence of nicotine, on brain function are not well understood. Here, we performed a screen of vape aerosol extracts (VAEs) to determine the potential for prenatal neurotoxicity using the zebrafish embryo photomotor response (PMR)—a translational biosensor of neurobehavioural effects. We screened three commonly used aerosolized vape liquids (flavoured and flavourless) either with or without nicotine. No neurobehavioural effects were detected in flavourless, nicotine-free VAEs, while the addition of nicotine to this VAE dulled sensory perception. Flavoured nicotine-free VAEs also dulled sensory perception and caused hyperactivity in zebrafish embryos. The combination of flavour and nicotine produced largely additive effects. Flavoured VAEs without nicotine had similar neuroactive potency to nicotine. Together, using zebrafish PMR as a high throughput translational behavioural model for prenatal exposure, our results demonstrate that e-cigarette flavourants that we screened elicit neurobehavioural effects worthy of further investigation for long-term neurotoxic potential and also have the potential to modulate nicotine impact on the developing brain.

 

Link to the publication : 

https://royalsocietypublishing.org/doi/abs/10.1098/rsbl.2020.0361

Lobetyolin Efficiently Promotes Angiogenesis and Neuronal Development in Transgenic Zebrafish

Natural Product Communications

2020-08-31

Studies have shown that lobetyolin (LBT), a component of traditional Chinese herbal medicine, has many very good biological activities and functions. However, its side effects and toxicities have not been evaluated adequately. In this work, we investigated the effects of LBT in transgenic zebrafish. LBT treatments promoted angiogenesis and led to vascular morphological malformation in zebrafish embryos, although they were normal in appearance. Interestingly, our results indicated that LBT has a function of promoting nerve growth in the embryonic stage of zebrafish. We also treated the zebrafish with combretastatin A-4 (which resulted in neuronal apoptosis) and LBT simultaneously and found that LBT promoted nerve growth at higher concentrations. Taken together, our findings clearly display that LBT efficiently promotes angiogenesis, leading to vascular morphological malformation, has low toxicity, and notably promotes neuronal development in zebrafish.

 

Link to the publication : 

https://journals.sagepub.com/doi/full/10.1177/1934578X20937174

Genetic Risk Factors in prediction and treatment of Chronic Post-Surgical Pain

Universiteit Maastricht

2020-09-01

Concluding remarks :

This dissertation aimed to study the role of genetics in prediction and treatment of chronic postsurgical pain. In view of this, we started genetic screening and provided a genome-wide insight into the genetics of CPSP. This initial step identified the first potential risk loci and candidates for follow up studies. Furthermore, we have shown the important role of genetic variations related to dopaminergic neurotransmission in CPSP and preformed first steps in genetic and pharmacological screening in a zebrafish nociception model. With this dissertation a framework is provided for future studies and a combined and focussed clinical /pre-clinical approach aimed to develop personalized medicine in treatment of CPSP.

 

Link to the publication : 

https://www.researchgate.net/profile/Roel_Van_Reij/publication/343850803_Genetic_Risk_Factors_in_prediction_and_treatment_of_Chronic_Post-Surgical_Pain/links/5f44bf9992851cd302283738/Genetic-Risk-Factors-in-prediction-and-treatment-of-Chronic-Post-Surgical-Pain.pdf#page=137

Loss-of-function of the NOMO1 gene causes neuropsychiatric disorder-related phenotypes

Research Square

2020-09-01

Abstract Background:

Clinical genome-wide analysis identified NOMO1 in human chromosome 16p13.11 as a candidate gene associated with neuropsychiatric disorders such as autism, schizophrenia and epilepsy. However, the important contributions underlying NOMO1 deficiency resulting in neuropsychiatric disorders is not understand, and the molecular and pathogenesis mechanisms of nomo1 gene are unclear. Therefore, it is necessary to construct animal models to systematically study the effects of nomo1 deficiency on neuropsychiatric system and explore pathogenic molecular mechanism of diseases.

Methods:

We developed a viable vertebrate model of loss-of-function of nomo1 using CRISPR/Cas9 and studied the characterization of nomo1 mutant zebrafish. Phenotypic research was performed in developing nomo1 mutant zebrafish, including morphological measurements, behavioral tests, and functional mechanism analyses.

 

Link to the publication : 

https://assets.researchsquare.com/files/rs-54088/v1_stamped.pdf

Neurobehavioral effects of cyanobacterial biomass field extracts on zebrafish embryos and potential role of retinoids

Aquatic Toxicology

2020-09-06

Highlights

 

• Field cyanobacterial biomass extracts cause behavioural changes in zebrafish.
• The effects were localized in fractions with retinoid compounds content.
• The behavioural effect increased after combination of retinoids containing fractions.
• The effects on behaviour did not occur after exposure to the ATRA alone.
• Several genes related to the retinoid pathway were affected by the biomass extracts.

 

Abstract

Cyanobacteria are known for their ability to produce and release mixtures of up to thousands of compounds into the environment. Recently, the production of novel metabolites, retinoids, was reported for some cyanobacterial species along with teratogenic effects of samples containing these compounds. Retinoids are natural endogenous substances derived from vitamin A that play a crucial role in early vertebrate development. Disruption of retinoid signalling- especially during the early development of the nervous system- might lead to major malfunctions and malformations. In this study, the toxicity of cyanobacterial biomass samples from the field containing retinoids was characterized by in vivo and in vitro bioassays with a focus on the potential hazards towards nervous system development and function. Additionally, in order to identify the compounds responsible for the observed in vitro and in vivo effects the complex cyanobacterial extracts were fractionated (C18 column, water-methanol gradient) and the twelve obtained fractions were tested in bioassays. In all bioassays, all-trans retinoic acid (ATRA) was tested along with the environmental samples as a positive control. Retinoid-like activity (mediated via the retinoic acid receptor, RAR) was measured in the transgenic cell line p19/A15. The in vitro assay showed retinoid-like activity by specific interaction with RAR for the biomass samples. Neurotoxic effects of selected samples were studied on zebrafish (Danio rerio) embryos using the light/dark transition test (Viewpoint, ZebraLab system) with 120 hpf larvae. In the behavioural assay, the cyanobacterial extracts caused significant hyperactivity in zebrafish at 120 hpf after acute exposure (3 h prior to the measurement) at concentrations below the teratogenicity LOEC (0.2 g dw L−1). Similar effect was observed after exposure to fractions of the extracts with detected retinoid-like activity and additive effect was observed after combining the fractions. However, the effect on behaviour was not observed after exposure to ATRA only. To provide additional insight into the behavioural effects and describe the underlying mechanism gene expression of selected biomarkers was measured. We evaluated an array of 28 genes related to general toxicity, neurodevelopment, retinoid and thyroid signalling. We detected several affected genes, most notably, the Cyp26 enzymes that control endogenous ATRA concentration, which documents an effect on retinoid signalling.

 

Link to the publication : https://www.sciencedirect.com/science/article/abs/pii/S0166445X20303635

Biomass-derivedcellulosenanoparticlesdisplayconsiderableneurotoxicity in zebrafish

International Journal of Biological MacroMolecules

2020-10-05

Abstract

The widespread use of nanomaterials poses a great threat to human living environments. Among them, biomass-derived cellulose nanoparticle (CN) is one of the widely used nanomaterials. To date, the toxicity of CNs during embryonic development remains undetermined. In this study, we exposed zebrafish embryos to cellulose nanofibrils (CNFs) and cellulose nanocrystals (CNCs) to evaluate the toxicity of these CNs. Exposure to CNFs or CNCs below 30 mg/ml exhibited no dose-dependent increases in malformation and mortality in zebrafish embryos. Then we demonstrated that CNs were highly enriched in zebrafish embryo via imaging analyses of embryos treated with FITC-coupled CNCs. In addition, we found that CNF or CNC exposure resulted in compromised motor ability of zebrafish larva. Furthermore, it was revealed that the differentiation and the morphogenesis of motor neurons were significantly interrupted. While, blood vessels were normally patterned, suggesting the specific neurotoxicity of these nanomaterials. Transcriptome sequencing assay showed that the neurotoxicity of CNs in the motor neurons might be attributed to the expression alteration of neural genes. In summary, we discovered the neurotoxicity of CNs for the first time.

 

Link to the publication :

https://www.sciencedirect.com/science/article/abs/pii/S0141813020346559

Zebrafish and mouse models for anxiety evaluation – A comparative study with xanthotoxin as a model compound

Brain Research Bulleting

2020-10-10

Highlights

• Xanthotoxin modifies anxiety-related behavior in two animal models: zebrafish and mice.
• Xanthotoxin exerted reversed U-shape effect on anxiety behaviors in both models.
• The predictive power of zebrafish for behavioral research of natural compounds was proven.

Abstract

The ever-present trend for introducing new drugs of natural origin with anxiolytic properties meets healthcare needs of the population, whose almost 34 % struggles with anxiety-related disorders. At the same time, animal assays that could serve as fast and reliable models of anxiety-like behaviors are of great interest to scientists. Thus, the aim of the present study was to evaluate the utility of the zebrafish model for assessing the influence of natural compounds on anxiety in comparison with the well-known mouse model. Secondly, this study is also the first attempt to investigate the influence of a naturally occurring metabolite, i.e. xanthotoxin, on anxiety-related behaviors. The anxiety level in zebrafish was assessed by measuring thigmotaxis, a specific animal behavior to move closer to the boundaries of an open area and to avoid its center. In mice, the elevated plus maze test was chosen to study anxiety-related behaviors. Our results show that xanthotoxin exerted reversed U-shape effect on anxiety behaviors in both models. The similar pattern of xanthotoxin-induced anxiety-related behaviors in both animal models not only confirms the pharmacological properties of xanthotoxin but also proves the predictive power of the zebrafish model for behavioral research of natural compounds.

 

Link to the publication :

https://www.sciencedirect.com/science/article/abs/pii/S0361923020306596

Antidepressant exposure causes a nonmonotonic reduction in anxiety-related behaviour in female mosquitofish

Journal of Hazardous Materials Letters

2020-10-14

Highlights

• Antidepressants, such as fluoxetine, are routinely detected in aquatic ecosystems.
• Fluoxetine, and antidepressants more generally, could disrupt wildlife behaviour.
• We report fluoxetine exposure (18 ng/L) altered anxiety-related behaviour of fish.
• Behavioural effects were nonmonotonic, with stronger effects at the lower dosage.
• Changes in anxiety-related behaviour could impact the viability of exposed populations.

Abstract

Worldwide, biologically active pharmaceuticals, such as psychoactive drugs, are routinely detected in aquatic ecosystems. In this regard, selective serotonin reuptake inhibitors (SSRIs), a class of antidepressant, are of major environmental concern. Through targeted action on evolutionarily conserved physiological pathways, SSRIs could alter ecologically important behaviours in exposed organisms. Here, using two field-realistic dosages (measured concentrations: 18 and 215 ng/L) of the SSRI fluoxetine (Prozac), we examined the effects of exposure on anxiety-related behaviours in wild-caught female mosquitofish, Gambusia holbrooki. Anxiety-related behaviour was assessed using a light/dark transition test, with the swimming activity of fish recorded under two alternating light conditions, complete darkness and bright light, with the shift in light condition used to induce an anxiety-like response. Fluoxetine exposure resulted in a nonmonotonic decrease in anxiety-related behaviour (i.e. nonlinear with dose), with fish in the low-fluoxetine treatment being less responsive to shifts in light condition compared to unexposed fish. There was no such difference between unexposed and high-exposed fish. Further, we detected a significant interaction between exposure treatment and fish weight on general swimming activity, suggesting the presence of a mass-specific effect of fluoxetine. More broadly, contaminant-induced disruption of animal behaviour—as documented here—could have wide-reaching effects on population-level fitness.

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S2666911020300046

Systematic Assessment of Exposure Variations onObserved Bioactivity in Zebrafish Chemical Screening

Toxics

2020-10-14

Abstract:

The embryonic zebrafish is a powerful tool for high-throughput screening of chemicals.While this model has significant potential for use in safety assessments and chemical prioritization,a lack of exposure protocol harmonized across laboratories has limited full model adoption. To assessthe potential that exposure protocols alter chemical bioactivity, we screened a set of eight chemicalsand one 2D nanomaterial across four different regimens: (1) the current Tanguay laboratory’sstandard protocol of dechorionated embryos and static exposure in darkness; (2) exposure withchorion intact; (3) exposure under a 14 h light: 10 h dark cycle; and (4) exposure with daily chemicalrenewal. The latter three regimens altered the concentrations, resulting in bioactivity of the testagents compared to that observed with the Tanguay laboratory’s standard regimen, though notdirectionally the same for each chemical. The results of this study indicate that with the exceptionfor the 2D nanomaterial, the screening design did not change the conclusion regarding chemicalbioactivity, just the nominal concentrations producing the observed activity. Since the goal of tierone chemical screening often is to differentiate active from non-active chemicals, researchers couldconsider the trade-offs regarding cost, labor, and sensitivity in their study design without alteringhit rates. Taken further, these results suggest that it is reasonably feasible to reach agreement on astandardized exposure regiment, which will promote data sharing without sacrificing data content

 

See Publication : https://www.mdpi.com/2305-6304/8/4/87/htm

Backbone Cyclization Turns a Venom Peptide into a Stable andEquipotent Ligand at Both Muscle and Neuronal Nicotinic Receptors

Journal of Medicinal Chemistry

2020-10-16

Abstract

Abstract Image

Venom peptides are promising drug leads, but their therapeutic use is often limited by stability and bioavailability issues. In this study, we designed cyclic analogues of α-conotoxin CIA, a potent muscle nicotinic acetylcholine receptor (nAChR) blocker with a significantly lower affinity at the neuronal α3β2 subtype. Remarkably, all analogues retained the low nanomolar activity of native CIA toward muscle-type nAChRs but showed greatly improved resistance to degradation in human serum and, surprisingly, displayed up to 52-fold higher potency for the α3β2 neuronal nAChR subtype (IC50 1.3 nM). Comparison of nuclear magnetic resonance-derived structures revealed some differences that might explain the gain of potency at α3β2 nAChRs. All peptides were highly paralytic when injected into adult zebrafish and bath-applied to zebrafish larvae, suggesting barrier-crossing capabilities and efficient uptake. Finally, these cyclic CIA analogues were shown to be unique pharmacological tools to investigate the contribution of the presynaptic α3β2 nAChR subtype to the train-of-four fade.

 

Link to the publication :

https://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.0c00957#

Bioconcentration and developmental neurotoxicity of novelbrominatedflame retardants, hexabromobenzene andpentabromobenzene in zebrafish

Environmental Pollution

2020-10-21

abstract

Theflame retardants hexabromobenzene (HBB) and pentabromobenzene (PBB) have been extensivelyused and become ubiquitous pollutants in the aquatic environment and biota, but their potential toxiceffects on wildlife remained unknown. In this study, by using zebrafish (Danio rerio) as a model, thebioconcentration and developmental neurotoxicity were investigated. Zebrafish embryos were exposedto HBB and PBB (0, 30, 100 and 300mg/L) from 2 until 144 h post-fertilization (hpf). Chemical analysisshowed bioconcentrations of both chemicals, while HBB is readily metabolized to PBB in zebrafish larvae.Embryonic exposure to both chemicals did not cause developmental toxicity, but induced locomotorbehavioral anomalies in larvae. Molecular docking results indicated that both chemicals could bind tozebrafish acetylcholinesterase (AChE). Furthermore, HBB and PBB significantly inhibited AChE activities,accompanied by increased contents of acetylcholine and decreased choline in larvae. Downregulation ofthe genes associated with central nervous system (CNS) development (e.g.,mbp,a1-tubulin,gfap,shha)aswell as the corresponding proteins (e.g., Mbp,a1-Tubulin) was observed, but gap-43 was upregulated atboth gene and protein levels. Together, our results indicate that both HBB and PBB exhibit developmentalneurotoxicity by affecting various parameters related to CNS development and indications for futuretoxicological research and risk assessment of the novel brominatedflame retardants.

 

Link to the publication :

https://www.sciencedirect.com/science/article/abs/pii/S0269749120365842

The oxidative stress responses caused by phthalate acid esters increases mRNA abundance of base excision repair (BER) genes in vivo and in vitro

Ecotoxicology and Environmental Safety

2020-10-22

Highlights

• PAEs effects the development of zebrafish embryos.
• PAEs decreases cell viability in HEK293T cells.
• PAEs increases the production of ROS and decreases SOD activity in vivo and in vitro.
• PAEs increases pro-apoptotic gene expression and decreases anti-apoptotic gene expression.
• BER pathway were activated in response to PAEs exposure (with the exceptions of larvae at 50 μM MEHP) in vivo and vitro.

Abstract

The base excision repair (BER) pathway is an important defense response to oxidative DNA damage. It is known that exposures to phthalate esters (PAEs), including Dibutyl phthalate (DBP), Mono-(2-ethylhexyl) phthalate (MEHP), and Di-(2-ethylhexyl) phthalate (DEHP), cause reactive oxygen species-induced DNA damage and oxidative stress. Here, we determined the mRNA levels of BER pathway-related genes (ogg1, nthl1, apex1, parp1, xrcc1, lig3, ung, pcna, polb, pold, fen1, and lig1), pro-apoptotic gene (bax), and apoptotic suppressor gene (bcl2) in different PAEs-exposed zebrafish larvae and HEK293T cells. Further investigations were performed to examine reactive oxygen species (ROS) accumulation, superoxide dismutase (SOD) activity, developmental toxicity, and cell viability after PAEs exposure in vivo and in vitro. The results showed that PAEs exposure can induce developmental abnormalities in zebrafish larvae, and inhibit cell viability in HEK293T cells. Additionally, we found that PAEs exposure results in the accumulation of ROS and the inhibition of SOD activation in vivo and in vitro. Notably, the mRNA levels of BER pathway-related genes (OGG1, NTHL1, APEX1, XRCC1, UNG, POLB, POLD, FEN1) were significantly upregulated after DBP or MEHP exposure, whereas the mRNA levels of NTHL1, UNG, POLB, POLD, and FEN1 were significantly altered in DEHP-treated HEK293T cells. In zebrafish, the mRNA levels of ogg1, pcna, fen1 and lig1 genes were increased after DBP or DEHP exposure, whereas the mRNA levels of nthl1, apex1, parp1, lig3, pcna and polb were decreased after MEHP exposure, respectively. Thus, our findings indicated that PAEs exposure can induce developmental toxicity, cytotoxicity, and oxidative stress, as well as activate BER pathway in vivo and in vitro, suggesting that BER pathway might play critical roles in PAEs-induced oxidative stress through repairing oxidative DNA damage.

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0147651320313622

Assessing the effects of textile leachates in fish using multiple testing methods: From gene expression to behavior

Ecotoxicology and Environmental Safety

2020-10-24

ABSTRACT

The textile industry, while of major importance in the world economy, is a toxic industry utilizing and emitting thousands of chemical substances into the aquatic environment. The aim of this project was to study the potentially harmful effects associated with the leaching of chemical residues from three different types of textiles: sportswear, childrens bath towels, and denim using different fish models (cell lines, fish larvae and juvenile fish). A combination of in vitro and in vivo test systems was used. Numerous biomarkers, ranging from gene expression, cytotoxicity and biochemical analysis to behavior, were measured to detect effects of leached chemicals. Principle findings indicate that leachates from all three types of textiles induced cytotoxicity on fish cell lines (RTgill-W1). Leachates from sportswear and towels induced mortality in zebrafish embryos, and chemical residues from sportswear reduced locomotion responses in developing larval fish. Sportswear leachate increased Cyp1a mRNA expression and EROD activity in liver of exposed brown trout. Leachates from towels induced EROD activity and VTG in rainbow trout, and these effects were mitigated by the temperature of the extraction process. All indicators of toxicity tested showed that exposure to textile leachate can cause adverse reactions in fish. These findings suggested that chemical leaching from textiles from domestic households could pose an ecotoxicological threat to the health of the aquatic environment.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0147651320313609

Waterborne uranium causes toxic effect and thyroid disruption in zebrafish larvae

Ecotoxicology and Environmental Safety

2020-11-09

Highlights

• The effects of uranium on development and thyroid system of zebrafish were assessed.
• Uranium could bioaccumulate in zebrafish bodies.
• Uranium affected the development and locomotor behavior of zebrafish.
• The levels of thyroid hormones in zebrafish were changed after uranium exposure.
• Negative feedback loops through HPT axis-related genes regulated thyroid hormones.

Abstract

Uranium is a radioactive element that is widely present in aquatic environment. However, limited knowledge is available about the effect of uranium on thyroid system, which plays a key role in the development of animals. In this study, zebrafish embryos were exposed to different environmentally relevant concentrations of uranium (2, 20 and 100 μg/L) for 120 h. The bioaccumulation, developmental toxicities, changes of thyroid hormones (THs) and key genes related to the hypothalamic–pituitary–thyroid (HPT) axis in larvae were analyzed after exposure. Results showed that uranium could bioaccumulate in zebrafish larvae, with the bioconcentration factors ranging from 49.6 to 523. Consequently, significant developmental toxicities and changes in locomotor activities were observed with a concentration-dependent manner. The levels of triiodothyronine (T3) levels in larvae were substantially decreased, whereas those of thyroxine (T4) were increased in fish bodies. The levels of THs were regulated by the negative feedback loops through HPT axis related genes, most of which (NIS, Deio1, Deio2, TRα, TSHβ and UGT1ab) were significantly depressed after exposure to uranium. Our results suggest the potential toxicities and thyroid disruption of uranium on zebrafish, which would provide baseline data set for better understanding the impact of waterborne uranium on aquatic organisms and the associated mechanisms. This study also highlights the key role of thyroid disruption in the ecological risk assessment of uranium pollution.

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0147651320314226

Developmental toxicity in zebrafish (Danio rerio) exposed to uranium: A comparison with lead, cadmium, and iron

Environmental Pollution

2020-11-18

Highlights

• Uranyl acetate (UA) does not cause morphology malformations or mortality in developing zebrafish.
• 3 μg/L UA exposure alters 120-h post fertilization (hpf) zebrafish swimming behavior.
• First study to detect behavioral malformations in developing zebrafish exposed to UA.
• Lead, cadmium, and iron impacted larval zebrafish behavior with same exposure paradigm as UA.
• Our study sets the stage for future metal mixture studies using zebrafish.

Abstract

Populations of plants and animals, including humans, living in close proximity to abandoned uranium mine sites are vulnerable to uranium exposure through drainage into nearby waterways, soil accumulation, and blowing dust from surface soils. Little is known about how the environmental impact of uranium exposure alters the health of human populations in proximity to mine sites, so we used developmental zebrafish (Danio rerio) to investigate uranium toxicity. Fish are a sensitive target for modeling uranium toxicity, and previous studies report altered reproductive capacity, enhanced DNA damage, and gene expression changes in fish exposed to uranium. In our study, dechorionated zebrafish embryos were exposed to a concentration range of uranyl acetate (UA) from 0 to 3000 μg/L for body burden measurements and developmental toxicity assessments. Uranium was taken up in a concentration-dependent manner by 48 and 120 h post fertilization (hpf)-zebrafish without evidence of bioaccumulation. Exposure to UA was not associated with teratogenic outcomes or 24 hpf behavioral effects, but larvae at 120 hpf exhibited a significant hypoactive photomotor response associated with exposure to 3 μg/L UA which suggested potential neurotoxicity. To our knowledge, this is the first time that uranium has been associated with behavioral effects in an aquatic organism. These results were compared to potential metal co-contaminants using the same exposure paradigm. Similar to uranium exposure, lead, cadmium, and iron significantly altered neurobehavioral outcomes in 120-hpf zebrafish without inducing significant teratogenicity. Our study informs concerns about the potential impacts of developmental exposure to uranium on childhood neurobehavioral outcomes. This work also sets the stage for future, environmentally relevant metal mixture studies. Summary Uranium exposure to developing zebrafish causes hypoactive larval swimming behavior similar to the effect of other commonly occurring metals in uranium mine sites. This is the first time that uranium exposure has been associated with altered neurobehavioral effects in any aquatic organism.

 

Link to the publication :

 

 https://www.sciencedirect.com/science/article/abs/pii/S0269749120367865

Involvement of dopamine signaling pathway in neurodevelopmental toxicity induced by isoniazid in zebrafish

Chemosphere

2020-11-28

Highlights

INH impaired the development of vasculature and reduced the behavior of zebrafish.

INH induced brain apoptosis and increased the expression of apoptosis related genes.

INH inhibited dopamine neuron development and the expression of dopamine related genes.

Abstract

Aims

This study evaluated the neurodevelopmental toxicity of isoniazid (INH) in zebrafish embryos and the underlying mechanism.

Methods

Zebrafish embryos were exposed to different concentrations (2 mM, 4 mM, 8 mM, 16 mM, 32 mM) INH for 120 hpf. During the exposure period, the percentage of embryo/larva mortality, hatching, and morphological malformation were checked every 24 h until 120 hpf. The development of blood vessels in the brain was observed at 72 hpf and 120 hpf, and behavioral capacity and acridine orange (AO) staining were measured at 120 hpf. Alterations in the mRNA expression of apoptosis and dopamine signaling pathway related genes were assessed by real-time quantitative PCR (qPCR).

Results

INH considerably inhibited zebrafish embryo hatching and caused zebrafish larval malformation (such as brain malformation, delayed yolk sac absorption, spinal curvature, pericardial edema, and swim bladder defects). High concentration of INH (16 mM, 32 mM) even induced death of zebrafish. In addition, INH exposure markedly restrained the ability of the zebrafish autonomous movement, shortened the length of dopamine neurons and inhibited vascular development in the brain. No obvious apoptotic cells were observed in the control group, whereas considerable numbers of apoptotic cells appeared in the head of INH-treated larvae at 120 hpf. PCR results indicated that INH significantly raised the transcription levels of caspase-3, -8, -9, and bax and significantly decreased bcl-2 and bcl-2/bax in the zebrafish apoptotic signaling pathway. INH also markedly decreased the genes related to dopamine signaling pathway (th1, dat, drd1, drd2a, drd3, and drd4b).

Conclusions

Experimental results indicated that INH had obvious neurodevelopmental toxicity in zebrafish. Persistent exposure to INH for 120 h caused apoptosis, decreased dopaminergic gene expression, altered vasculature, and reduced behaviors.

 

Link to the publication :

https://www.sciencedirect.com/science/article/abs/pii/S0045653520333063

Polystyrene nanoparticles trigger the activation of p38 MAPK and apoptosis via inducing oxidative stress in zebrafish and macrophage cells

Environmental Pollution

2020-12-01

Highlights

• Nanoplastics pose great potential risks to environment and health.
• Nanoplastics could induce significant alterations in MAPK pathway in zebrafish larval.
• The induction of oxidative stress and inflammation were correlated with cell apoptosis.
• MAPK signaling pathway involved in the toxicity of nanoplastics to macrophage cells.

Abstract

Polystyrene nanoparticles (PS NPs), originated from weathering breakdown of large plastic wastes, have already caused much concern for their environmental risks on health. This current study was aimed to reveal the toxicological mechanism of PS NPs on developing zebrafish and macrophage cells. To fulfill this purpose, 42 nm PS NPs were exposed to earlier development stage of zebrafish for 5 days, the decreased heart rate and locomotor activity of larval zebrafish were observed. The fluorescent PS NPs were used to precisely assess the accumulation of PS NPs in zebrafish, and the results indicated that PS NPs not only accumulated in digestive system, but also infiltrated into the liver. More importantly, the transcriptomic analysis revealed that a total of 356 genes were differentially expressed and the KEGG class map showed significant differences in the MAPK pathway upon PS NPs treatment. Meanwhile, the induction of oxidative stress and inflammation were also observed in larval zebrafish. Furthermore, RAW264.7 cells were exposed to different concentrations of PS NPs and the results confirmed that PS NPs exposure could result in up-regulation of p38 protein expression, enhance cell apoptosis and oxidative damage. Our study provides a new understanding of MAPK signaling pathway involved in toxicity mechanism.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0269749120367646

Ethanol affects behavior and HPA axis activity during development in zebrafish larvae

Scientific Reports

2020-12-08

Abstract

Recent studies have shown that long-term alcohol intake from food can lead to numerous mental disorders in humans, and the social and health effects of excessive intake of alcohol currently represent serious problems for governments and families worldwide. However, to date, it has not been determined how alcohol affects the hypothalamic–pituitary–adrenal (HPA) axis. The zebrafish offers a good model for studying the toxicology of food-grade ethanol. In the present study, using zebrafish larvae exposed to 1% ethanol, we performed zebrafish behavioral analysis. Samples were collected for enzyme-linked immunosorbent assay (ELISA) and quantitative real time-polymerase chain reaction (qRT-PCR) experiments, and statistical analysis was performed. We found that ethanol decreased the locomotor activity of zebrafish larvae, which showed a more intense reaction to external stimuli. Ethanol also increased the level of HPA axis hormones in zebrafish larvae, influenced the level of neurotransmitters, and altered the expression of key genes in neurotransmitter metabolism. Ethanol exposure affects zebrafish behavior, increases the level of HPA axis hormones in zebrafish larvae, affects the level of neurotransmitters, and affects the expression of key genes in dopamine and serotonin metabolism. These findings may help to elucidate the effects of ethanol on HPA axis activity.

 

Link to the publication :

https://www.nature.com/articles/s41598-020-78573-y

 

Ethanol affects behavior and HPA axis activity during development in zebrafish larvae

Nature Scientific Report

2020-12-08

Recent studies have shown that long-term alcohol intake from food can lead to numerous mental disorders in humans, and the social and health effects of excessive intake of alcohol currently represent serious problems for governments and families worldwide. However, to date, it has not been determined how alcohol affects the hypothalamic–pituitary–adrenal (HPA) axis. The zebrafish offers a good model for studying the toxicology of food-grade ethanol. In the present study, using zebrafish larvae exposed to 1% ethanol, we performed zebrafish behavioral analysis. Samples were collected for enzyme-linked immunosorbent assay (ELISA) and quantitative real time-polymerase chain reaction (qRT-PCR) experiments, and statistical analysis was performed. We found that ethanol decreased the locomotor activity of zebrafish larvae, which showed a more intense reaction to external stimuli. Ethanol also increased the level of HPA axis hormones in zebrafish larvae, influenced the level of neurotransmitters, and altered the expression of key genes in neurotransmitter metabolism. Ethanol exposure affects zebrafish behavior, increases the level of HPA axis hormones in zebrafish larvae, affects the level of neurotransmitters, and affects the expression of key genes in dopamine and serotonin metabolism. These findings may help to elucidate the effects of ethanol on HPA axis activity.

 

Link to the publication :

https://search.proquest.com/openview/70f94a193c47b305f2f5ab1666838871/1?pq-origsite=gscholar&cbl=2041939

In Vivo Screening of Xanthones from Garcinia oligantha Identified Oliganthin H as a Novel Natural Inhibitor of Convulsions

Journal of Natural Products

2020-12-09

Abstract

Abstract Image

Epilepsy is a chronic neurological disorder, characterized by recurrent, spontaneous, and transient seizures, and affects more than 70 million people worldwide. Although two dozen antiepileptic drugs (AEDs) are approved and available in the market, seizures remain poorly controlled in one-third of epileptic patients who are suffering from drug resistance or various adverse effects. Recently, the xanthone skeleton has been regarded as an attractive scaffold for the discovery and development of emerging anticonvulsants. We had isolated several dihydroxanthone derivatives previously, including oliganthin H, oliganthin I, and oliganthin N, whose structures were similar and delicately elucidated by spectrum analysis or X-ray crystallographic data, from extracts of leaves of Garcinia oligantha. These xanthone analogues were evaluated for anticonvulsant activity, and a novel xanthone, oliganthin H, has been identified as a sound and effective natural inhibitor of convulsions in zebrafish in vivo. A preliminary structure–activity relationship analysis on the relationship between structures of the xanthone analogues and their activities was also conducted. Oliganthin H significantly suppressed convulsant behavior and reduced to about 25% and 50% of PTZ-induced activity, in 12.5 and 25 μM treatment groups (P < 0.01 and 0.001), respectively. Meanwhile, it reduced seizure activity, velocity, seizure duration, and number of bursts in zebrafish larvae (P < 0.05). Pretreatment of oliganthin H significantly restored aberrant induction of gene expressions including npas4a, c-fos, pyya, and bdnf, as well as gabra1, gad1, glsa, and glula, upon PTZ treatment. In addition, in silico analysis revealed the stability of the oliganthin H–GABAA receptor complex and their detailed binding pattern. Therefore, direct interactions with the GABAA receptor and involvement of downstream GABA–glutamate pathways were possible mechanisms of the anticonvulsant action of oliganthin H. Our findings present the anticonvulsant activity of oliganthin H, provide a novel scaffold for further modifications, and highlight the xanthone skeleton as an attractive and reliable resource for the development of emerging AEDs.

 

Link to the publication:

https://pubs.acs.org/doi/abs/10.1021/acs.jnatprod.0c00963

Nano-titanium nitride causes developmental toxicity in zebrafish through oxidative stress

Drug and Chemical Toxicology

2020-12-09

Nano-titanium nitride (Nano-TiN) has strong resistance to wear and corrosion, good biocompatibility, and an attractive metallic luster. Nano-TiN is widely used in medical devices, such as orthopedic implants, syringe needles, coronary stents, and long-term dental implants, and also in imitation gold jewelry. Despite its widespread use, there are few reports describing safety evaluations of Nano-TiN. Here, we exposed healthy zebrafish embryos to different concentrations of Nano-TiN solution for five days, starting at about four hours post fertilization, and found that Nano-TiN caused dose- and time-dependent developmental toxicity. With increasing Nano-TiN concentration and length of exposure, mortality, and deformities gradually increased; body length shortened and hatching rate and motility were significantly reduced. We also found that exposure to Nano-TiN affected development of the heart, liver, nerves, and other organs, and led to elevated levels of reactive oxygen species and reduced antioxidant capacity. Exposure to Nano-TiN resulted in downregulation of expression of antioxidant genes, such as nrf2, gclc, gclm, ho-1, and nqo1. Our results showed that exposure to Nano-TiN caused developmental and organ toxicity in zebrafish embryos and that the toxic effects may be mediated through oxidative stress.

 

Link to the publication :

https://www.tandfonline.com/doi/full/10.1080/01480545.2020.1853765?scroll=top&needAccess=true

Exposure to tricyclic antidepressant nortriptyline affects early-life stages of zebrafish (Danio rerio)

Ecotoxicology and Environmental Safety

2021-01-08

Highlights

The toxicity of nortriptyline was investigated on early-life stages of zebrafish.

The 168 h-LC50 of nortriptyline was 2190 μg/L.

Organisms with lack of equilibrium were identifiable at concentrations ≥ 500 μg/L.

Nortriptyline impairs locomotor behaviour at environmentally relevant levels.

Nortriptyline inhibits acetylcholinesterase activity.

Abstract

Psychiatric drugs are among the leading medications prescribed for humans, with their presence in aquatic environments raising concerns relating to potentially harmful effects on non-target organisms. Nortriptyline (NTP) is a selective serotonin-norepinephrine reuptake inhibitor antidepressant, widely used in clinics and found in environmental water matrices. In this study, we evaluated the toxic effects of NTP on zebrafish (Danio rerio) embryos and early larval stages. Developmental and mortality analyses were performed on zebrafish exposed to NTP for 168 h at concentrations ranging from 500 to 46,900 µg/L. Locomotor behaviour and acetylcholinesterase (AChE) activity were evaluated by exposing embryos/larvae to lower NTP concentrations (0.006–500 µg/L). The median lethal NTP concentration after 168 h exposure was 2190 µg/L. Although we did not identify significant developmental changes in the treated groups, lack of equilibrium was already visible in surviving larvae exposed to ≥ 500 µg/L NTP. The behavioural analyses showed that NTP was capable of modifying zebrafish larvae swimming behaviour, even at extremely low (0.006 and 0.088 µg/L) environmentally relevant concentrations. We consistently observed a significant reduction in AChE activity in the animals exposed to 500 µg/L NTP. Our results highlight acute toxic effects of NTP on the early-life stages of zebrafish. Most importantly, exposure to environmentally relevant NTP concentrations may affect zebrafish larvae locomotor behaviour, which in turn could reduce the fitness of the species. More studies involving chronic exposure and sensitive endpoints are warranted to better understand the effect of NTP in a more realistic exposure scenario.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S0147651320317048

Morphological and Behavioral Effects in Zebrafish Embryos after Exposure to Smoke Dyes

Toxics

2021-01-10

Abstract:

Solvent Violet 47 (SV47) and Disperse Blue 14 (DB14) are two anthraquinone dyes thatwere previously used in different formulations for the production of violet-colored smoke. Both dyeshave shown potential for toxicity; however, there is no comprehensive understanding of their effects.Zebrafish embryos were exposed to SV47 or DB14 from 6 to 120 h post fertilization (hpf) to assessthe dyes’ potential adverse effects on developing embryos. The potential ability of both dyes tocross the blood–brain barrier was also assessed. At concentrations between 0.55 and 5.23 mg/L,SV47 showed a dose-dependent increase in mortality, jaw malformation, axis curvature, and edemas.At concentrations between 0.15 and 7.54 mg/L, DB14 did not have this same dose-dependencebut had similar morphological outcomes at the highest doses. Nevertheless, while SV47 showedsignificant mortality from 4.20 mg/L, there was no significant mortality on embryos exposed to DB14.Regardless, decreased locomotor movement was observed at all concentrations of DB14, suggestingan adverse neurodevelopmental effect. Overall, our results showed that at similar concentrations,SV47 and DB14 caused different types of phenotypic effects in zebrafish embryos.

 

Link to the publication :

https://www.mdpi.com/2305-6304/9/1/9/htm

Morphological and Behavioral Effects in Zebrafish Embryosafter Exposure to Smoke Dyes

Toxics

2021-01-10

Abstract:

Solvent Violet 47 (SV47) and Disperse Blue 14 (DB14) are two anthraquinone dyes thatwere previously used in different formulations for the production of violet-colored smoke. Both dyeshave shown potential for toxicity; however, there is no comprehensive understanding of their effects.Zebrafish embryos were exposed to SV47 or DB14 from 6 to 120 h post fertilization (hpf) to assessthe dyes’ potential adverse effects on developing embryos. The potential ability of both dyes tocross the blood–brain barrier was also assessed. At concentrations between 0.55 and 5.23 mg/L,SV47 showed a dose-dependent increase in mortality, jaw malformation, axis curvature, and edemas.At concentrations between 0.15 and 7.54 mg/L, DB14 did not have this same dose-dependencebut had similar morphological outcomes at the highest doses. Nevertheless, while SV47 showedsignificant mortality from 4.20 mg/L, there was no significant mortality on embryos exposed to DB14.Regardless, decreased locomotor movement was observed at all concentrations of DB14, suggestingan adverse neurodevelopmental effect. Overall, our results showed that at similar concentrations,SV47 and DB14 caused different types of phenotypic effects in zebrafish embryos

Link to the publication : https://search.proquest.com/openview/0b03609d29b59b5929aa842903c37c17/1?pq-origsite=gscholar&cbl=2032322

Developmental Hazard of Environmentally Persistent Free Radicals and Protective Effect of TEMPOL in Zebrafish Model

Toxics

2021-01-16

Abstract: Environmentally persistent free radicals (EPFRs) can be detected in ambient PM2.5, ciga-rette smoke, and soils and are formed through combustion and thermal processing of organic ma-terials. The hazards of EPFRs are largely unknown. In this study, we assess the developmental tox-icity of EPFRs and the ability of TEMPOL (4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl) to pro-tect against such hazards using zebrafish embryos. Particles containing EPFRs were acquired by dosing dichlorobenzene (DCB) vapor on the Cab-o-sil/5% CuO particles at 230 °C in vacuo (referred to as DCB-230). The particles were suspended in ultrapure water to make 1 mg/mL of stock solution from which series dilution was undertaken to obtain 10, 20, 30, 40, 50, 60, 80, and 100 μg/mL final test solutions, which were then placed in individual wells with a 4 h postfertilization (hpf) zebrafish embryo. Plates were run in duplicate to obtain a sample size of 24 animals per concentration; 12 embryos were exposed per concentration per plate. Statistical analysis of the morphology endpoints was performed. We investigated overt toxicity responses to DCB-230 in a 22-endpoint battery that included developing zebrafish from 24–120 hpf. Exposure to concentrations greater than 60 μg/mL of DCB-230 induced high mortality in the developmental zebrafish model. Exposure to EPFRs in-duced developmental hazards that were closely related to the concentrations of free radicals and EPFRs. The potential protective effects of TEMPOL against EPFRs’ toxicity in zebrafish were inves-tigated. Exposure to EPFRs plus TEMPOL shifted the concentration to an induced 50% adverse ef-fect (EC50), from 23.6 to 30.8 μg/mL, which verifies TEMPOL’s protective effect against EPFRs in the early phase of zebrafish development.

 

Link tot he publication : https://www.researchgate.net/profile/Xia_Guan2/publication/348549661_Developmental_Hazard_of_Environmentally_Persistent_Free_Radicals_and_Protective_Effect_of_TEMPOL_in_Zebrafish_Model/links/60032830299bf140889ebdc6/Developmental-Hazard-of-Environmentally-Persistent-Free-Radicals-and-Protective-Effect-of-TEMPOL-in-Zebrafish-Model.pdf

Rapid Zebrafish Behavioral Profiling Assay Accelerates the Identification of Environmental Neurodevelopmental Toxicants

Environmental Science and Technology

2021-01-20

EMAnalysis A rapid zebrafish profilling assay for neurodevelopmental toxicant identificationAbstract

 

Rapid and cost-effective in vivo assays to screen potential environmental neurodevelopmental toxicants are necessary to address the limitations of in vitro platforms, such as the inability to fully recapitulate the developmental and physiological processes of whole organisms. In the present study, a rapid zebrafish behavioral profiling assay was developed to characterize the neurodevelopmental effects of environmental substances by quantitatively evaluating multiple spontaneous movement features of zebrafish embryos. This video analysis-based assay automatically segmented every embryo and thus was able to accurately quantify spontaneous movement features, including frequency, duration, intensity, interval, and the number of continuous movements. When tested with eight environmental substances known to be neurodevelopmental toxicants, such as chlorpyrifos and bisphenol A, the assay successfully captured frequency alterations that were well-documented in previous studies while also providing additional information. Using an optimized procedure, we further assessed 132 potential neurotoxins that spanned a wide range of molecular targets, many of which were previously detected in environmental waterbodies. The distinct altered behavioral barcodes indicated that the spontaneous movement was impacted by diverse neuroactive substances, and the effects could be effectively evaluated with the developed assay. The web-based tool, named EMAnalysis, is further provided at http://www.envh.sjtu.edu.cn/zebrafish_contraction.jsp. Thus, this assay provides an efficient platform to accelerate the pace of neurotoxic environmental contaminant discoveries.

 

Link to the publication : https://pubs.acs.org/doi/abs/10.1021/acs.est.0c06949

Tdp1 protects fromtopoisomerase 1–mediated chromosomal breaks inadult zebrafish but is dispensable during larval development

Science Journal - DISEASES AND DISORDERS

2021-01-29

Deficiency in the DNA end-processing enzyme, tyrosyl-DNA phosphodiesterase 1 (TDP1), causes progressive neuro-degeneration in humans. Here, we generated a tdp1 knockout zebrafish and confirmed the lack of TDP1 activity. In adulthood, homozygotes exhibit hypersensitivity to topoisomerase 1 (Top1) poisons and a very mild locomotion defect. Unexpectedly, embryonic tdp1−/− zebrafish were not hypersensitive to Top1 poisons and did not exhibit increased Top1-DNA breaks. This is in contrast to the hypersensitivity of Tdp1-deficient vertebrate models reported to date. Tdp1 is dispensable in the zebrafish embryo with transcript levels down-regulated in response to Top1-DNA damage. In contrast, apex2 and ercc4 (xpf) transcripts were up-regulated. These findings identify the tdp1−/−zebrafish embryo as the first vertebrate model that does not require Tdp1 to protect from Top1-DNA damage and identify apex2 and ercc4 (xpf) as putative players fulfilling this role. It highlights the requirement of distinct DNA repair factors across the life span of vertebrates.

 

Link to the publication : https://advances.sciencemag.org/content/advances/7/5/eabc4165.full.pdf

Ecotoxicological study on Flatfish exposed to 4-MBC

Research Square

2021-02-12

Exposure effects of the UV-lter 4-MBC to "Solea senegalensis" metamorphosis

 

Abstract

Many personal care products integrate UV-lters, such as 4-methylbenzylidene camphor (4-MBC) which has been detected in aquatic habitats. Possible effects of 4-MBC to aquatic organisms have been poorly studied. Therefore, the main objective of this work is to study the effects of 4-MBC exposure to Solea senegalensis during metamorphosis, a sensitive life stage of this atsh. To achieve this, at the beginning of metamorphosis (13 days after hatching, dah) sh were exposed to 4-MBC (0.2–2.0 mg L − 1 ) for 48 h. After this period, sh were transferred to clean medium and were fed and maintained until more than 80% of sh in control group completed the metamorphosis (24 dah). Mortality, malformations and metamorphosis progression were studied on a daily basis. In addition, growth, behavior and biochemical markers of neurotransmission (acetylcholinesterase, AChE), oxidative stress (catalase, CAT; glutathione S-transferase, GST, and lipid peroxidation, LPO) and anaerobic metabolism (lactate dehydrogenase, LDH) were determined at the end of the experiment. An acceleration of metamorphosis progression was observed during and 2 days after the 4-MBC exposure in all concentrations tested. In addition, decreased length, inhibition of CAT activity and induction of oxidative damage (LOEC= 0.928 mg L − 1 4-MBC for length, CAT and LPO) were observed. A short-term exposure to 4-MBC at the onset of metamorphosis, a critical period of development, affected S. senegalensis at several levels of organization, even after nine days in clean medium, including growth and metamorphosis progression, suggesting possible long-term adverse effects to this species.

 

Link to the publication :

https://assets.researchsquare.com/files/rs-166939/v1/7636640f-e00b-496a-ab00-6680383a1a94.pdf

Coumarins fromSeseli devenyenseSimonk.: Isolation byLiquid–Liquid Chromatography and Potential AnxiolyticActivity Using an In Vivo Zebrafish Larvae Mode

International Journal of Molecular Sciences

2021-02-12

Abstract :

Different types of anxiety disorders have become the number one mental health issue indeveloped countries. The search for new, safer and effective drug-like molecules among naturallyderived substances faces two difficulties: an efficient method of isolation compounds with a high-purity and high-throughput animal model for activity assay. Thus, the aim of the present studywas to isolate by liquid–liquid chromatography high-purity rare coumarins from the fruits ofSeselidevenyenseSimonk. and evaluate their anxiolytic effect (defined as reversed thimotaxis) using a5-days post-fertilization (dpf)Danio reriolarvae model. Liquid–liquid chromatography enabled theisolation of one simple hydroxycoumarin (devenyol) and four pyranocoumarins (cis-khellactone,d-laserpitin, isolaserpitin and octanoyllomatin). The anxiolytic effect was defined as a decrease inthe time spent in the boundaries of the living space (also described as reversed thigmotaxis). Ourresults show that all isolated courmarins exerted a significant influence on the anxiety behavior(anxiolytic activity) in the zebrafish larvae model. According to our knowledge, this is the first reportof anxiolytic activity of pyranocoumarins and devenyol.

Link to the publication :

http://scholar.google.fr/scholar_url?url=https://www.mdpi.com/1422-0067/22/4/1829/pdf&hl=fr&sa=X&d=8059670310638163479&ei=f94qYL61OI-emwGh4qnQDQ&scisig=AAGBfm2rV0O9r0Cq7utywocL97sqv5YwSQ&nossl=1&oi=scholaralrt&html=

Echinacoside (ECH) promotes mitochondrial fusion progression

Signal Transduction and Targeted Therapy

2021-02-19

Small molecule induces mitochondrial fusion for neuroprotection via targeting CK2 without affecting its conventional kinase activity

 

Abstract

Mitochondrial fusion/fission dynamics plays a fundamental role in neuroprotection; however, there is still a severe lack of therapeutic targets for this biological process. Here, we found that the naturally derived small molecule echinacoside (ECH) significantly promotes mitochondrial fusion progression. ECH selectively binds to the previously uncharacterized casein kinase 2 (CK2) α′ subunit (CK2α′) as a direct cellular target, and genetic knockdown of CK2α′ abolishes ECH-mediated mitochondrial fusion. Mechanistically, ECH allosterically regulates CK2α′ conformation to recruit basic transcription factor 3 (BTF3) to form a binary protein complex. Then, the CK2α′/BTF3 complex facilitates β-catenin nuclear translocation to activate TCF/LEF transcription factors and stimulate transcription of the mitochondrial fusion gene Mfn2. Strikingly, in a mouse middle cerebral artery occlusion (MCAO) model, ECH administration was found to significantly improve cerebral injuries and behavioral deficits by enhancing Mfn2 expression in wild-type but not CK2α′+/− mice. Taken together, our findings reveal, for the first time, that CK2 is essential for promoting mitochondrial fusion in a Wnt/β-catenin-dependent manner and suggest that pharmacologically targeting CK2 is a promising therapeutic strategy for ischemic stroke.

 

Link to the publication :

https://www.nature.com/articles/s41392-020-00447-6

 

Keywords : neuroprotection, zebrafish, casein kinase 2, (CK2), oxidative stress, locomotor behavior; 

Zebrafish swimming performance

Embryonic codeine or morphine exposure in zebrafish

Neurotoxicology and Teratology

2021-02-20

 Zebrafish (Danio rerio) larvae show behavioral and embryonic development defects when exposed to opioids at embryo stage

Zebrafish Embryo exposed to morphine or codeine 

Highlights

• Embryonic opioid exposure induced behavioral defects in the zebrafish model.
• Codeine exposure caused morphological defects on eyes.
• Behavioral effects of codeine at similar experiments were milder than with morphine.

Abstract

Opioid abuse continues to plague society, and in recent years, there has been an epidemic, leading to increased addiction and death. It is poorly understood how prenatal opioid use affects the lives of children. The aim of this work was to evaluate the effect of early embryonic codeine or morphine exposure in zebrafish (Danio rerio), examining gastrulation progression (epiboly), teratogenic effects, mortality and locomotor behavior response to light/dark cycles. Zebrafish embryos were exposed to codeine or morphine (designated C or M) at 1, 5 or 10 mg/L (designated 01, 05 or 10, respectively) from 3 to 24 h postfertilization (hpf) or from 3 to 48 hpf (designated −24 or − 48 for 1 or 2 days of exposure, respectively). The C10–24, C01–48, C05–48 and C10–48 groups showed significantly smaller eyes than control larvae at 7 days postfertilization (dpf). Locomotor behavior of control larvae in light/dark cycles showed greater swimming time and distance in dark cycles. Two-day codeine exposure produced strong effects, showing no significant response due to light/dark cycles in distance moved. Morphine exposed groups showed similar effects as observed in 2-day codeine exposed groups, showing less large movement activity and also no significant difference between inactive duration in response to light/dark cycles. In conclusion, we observed low teratogenic effects and mortality effects. Animals exposed to high levels and higher exposure times of opioids were hypoactive, relative to controls, in the dark period. Future studies will be needed to understand the neural defects producing behavior changes.

 

Link to the publication : https://www.sciencedirect.com/science/article/abs/pii/S0892036221000180

Impact of microplastics on locomotor behavior of aquatic organisms

Journal of Hazardous Materials

2021-03-04

Environmentally relevant concentrations of microplastics influence the locomotor activity of aquatic biota

 

 

Microplastics ecotoxicity tested on zebrafish larvae

• Microplastics (MPs) inhibited the locomotor activity by 6% at realistic concentrations
• MPs decreased the average speed by 5% and the moved distance by 8%
• Egger’s test indicated the results were stable without obvious publication bias
• Effects presented size-, time-, concentration-dependent patterns and multi-factors interactions
• Potential mechanisms underlying MPs-induced locomotor activity decline were provided

Abstract

The occurrence of microplastics (MPs) in various marine and freshwater matrices has attracted great attention. However, the effect of MPs in natural environment on the locomotor performance of aquatic biota is still controversial. Therefore, this meta-analysis was conducted, involving 116 effect sizes from 2347 samples, to quantitatively evaluate the alteration in locomotor behavior of aquatic organisms induced by MPs at environmentally relevant concentrations (≤ 1 mg/L, median = 0.125 mg/L). It was shown that MP exposure significantly inhibited the average speed and moved distance of aquatic organisms by 5% and 8% (p < 0.05), respectively, compared with the control, resulting in an obvious reduction of locomotor ability by 6% (p < 0.05). Egger’s test indicated that the results were stable without publication bias (p > 0.05). The complex influence of MPs on the locomotor ability were characterized through random-effects meta-regression analyses, presenting size-, time-, concentration-dependent manners and multi-factors interactions. In addition, several physiological changes, including energy reserve reduction, metabolism disorder, gut microbiota dysbiosis, inflammation response, neurotoxic response, and oxidative stress, of aquatic organisms triggered by MP exposure at environmentally relevant concentrations were also provided, which might account for the MPs-induced locomotor activity decline.

 

Link to the publication :

https://www.sciencedirect.com/science/article/abs/pii/S0304389421005446

Anti Inflammatory TnP drug poses no threats to Danio Rerio brain

Toxicology Reports

2021-03-16

Early preclinical screening using zebrafish (Danio rerio) reveals the safety of the candidate anti-inflammatory therapeutic agent TnP

 

EMAnalysis A rapid zebrafish profilling assay for neurodevelopmental toxicant identification

Zebrafish as a model organism to study inflammatory diseases

 

Highlights

TnP has been indicated for chronic inflammatory diseases, multiple sclerosis
• Zebrafish is an alternative animal model for preclinical drug development
• Preclinical toxicology studies have shown that TnP has a wide therapeutic index range from 1 nM to 10 μM
TnP did not induce cardiotoxic effect or cardiac dysfunction
TnP crossed the blood-brain barrier without causing neurotoxicity

Abstract

The patented anti-inflammatory peptide TnP had its effectiveness recently confirmed in vivo in a murine model of multiple sclerosis and asthma. In this work, the safety of the TnP was evaluated in investigative toxicology tests using zebrafish (Danio rerio) as a model. We conducted the OECD #236 test to investigate effects of the TnP on the survival, hatching performance, and morphological formation of zebrafish embryos. After determining these endpoints, morphometric analysis termination of locomotion eartbeat rate in zebrafish larvae were evaluated to identify adverse effects such as neurotoxicity and cardiotoxicity. The results highlight a wide therapeutic index for TnP with non-lethal and safe doses rom 1 nM to 10 μM, without causing neurotoxicity or cardiotoxic effect. The low frequencyf abnormalities by TnP was associated with high safety of the molecule and the developing embryo's ability to process and eliminate it. TnP crossed the blood-brain barrier without disturbing the normal architecture of forebrain, midbrain and hindbrain. Our data reinforce the importance of zebrafish as an accurate investigative toxicology model to assess acute toxicity as well as cardiotoxicity and neurotoxicity of molecules in the preclinical phase of development.

 

Link to the publication :

https://www.sciencedirect.com/science/article/pii/S2214750020304467