First-Generation Synthetic Cathinones Produce Arrhythmia in Zebrafish Eleutheroembryos: A New Approach Methodology for New Psychoactive Substances Cardiotoxicity Evaluation.

The increasing number of new psychoactive substances (NPS) entering the illicit drug market, especially synthetic cathinones, as well as the risk of cardiovascular complications, is intensifying the need to quickly assess their cardiotoxic potential. The present study aims to evaluate the cardiovascular toxicity and lethality induced by first-generation synthetic cathinones (mephedrone, methylone, and MDPV) and more classical psychostimulants (cocaine and MDMA) in zebrafish embryos using a new approach methodology (NAM). Zebrafish embryos at 4 dpf were exposed to the test drugs for 24 h to identify drug lethality. Drug-induced effects on ventricular and atrial heart rate after 2 h exposure were evaluated, and video recordings were properly analyzed. All illicit drugs displayed similar 24 h LC50 values. Our results indicate that all drugs are able to induce bradycardia, arrhythmia, and atrial-ventricular block (AV block), signs of QT interval prolongation. However, only MDPV induced a different rhythmicity change depending on the chamber and was the most potent bradycardia and AV block-inducing drug compared to the other tested compounds. In summary, our results strongly suggest that the NAM presented in this study can be used for screening NPS for their cardiotoxic effect and especially for their ability to prolong the QT intervals.

Developmental neurotoxicity evaluation of three Chinese herbal medicines in zebrafish larvae by means of two behavioral assays: Touch-evoked response and light/dark transition.

Chinese herbal medicine (CHM) is used among pregnant women. However, the question of its safety during pregnancy remains unclear. The use of these products relies on history of use data but there are specific toxicities like developmental neurotoxicity that are clearly understudied. Here we use the zebfrafish embryo developmental toxicity assay (ZEDTA) in combination with two behavioral assays: touch-evoked response and Light/Dark (L/D) transition assay to evaluate the neuro/developmental toxicity of three herbal products commonly used in CHM [Chinese name (abbreviation; part of the plant and Scientific name]: tian ma (TM; tuber form Gastrodia elata Blume), lei gong teng (LGT; root and rhizome of Tripterygium wilfordii Hook.f) and cha ye (green tea, leaves from Camellia sinensis (L.) Kuntze). In case significant alterations were detected, single components with potential exposure during pregnancy were identified in the literature and further tested. TM had no neurodevelopmental toxic potential in zebrafish embryos, while LGT and its main compounds triptolide and celastrol induced significant alterations in behavior. Developmental exposure to EGCG, the main catechin of green tea, also produced significant alterations in zebrafish embryos behavior after developmental exposure. A combination of ZEDTA with L/D Transition assay is proposed as a useful combination of alternative methods for DNT assessment of CHM products together with other New Approach Methodologies (NAMs).

Application of the adverse outcome pathway to identify molecular changes in prenatal brain programming induced by IUGR: Discoveries after EGCG exposure.

Following a multi-disciplinary approach integrating information from several experimental models we have collected new evidence supporting, expanding and redesigning the AOP "Disrupted laminin/int-ß1 interaction leading to decreased cognitive function". Investigations in vitro in rabbit and rat neurospheres and in vivo in mice exposed to EGCG (epigallocatechin-gallate) during neurodevelopment are combined with in vitro evaluations in neural progenitor cells overexpressing int-ß1 and literature information from int-ß1 deficiency models. We have discovered for the first time that neural progenitor cells from intrauterine growth restricted (IUGR) animals overexpress int-ß1 at gene and protein level and due to this change in prenatal brain programming they respond differently than control neurospheres to the exposure of EGCG, a compound triggering neural progenitor cell migration alterations. We have also identified that EGCG developmental exposure has deleterious effects on neuronal branching and arborization in vitro and in vivo. Our results warn that a thorough developmental neurotoxicity characterization of this and other catechin-based food supplements is needed before recommending their consumption during pregnancy.

Developmental exposure to MDMA (ecstasy) in zebrafish embryos reproduces the neurotoxicity adverse outcome 'lower motor activity' described in humans.

The recreational use of MDMA (ecstasy) by pregnant women is associated with impaired neuromotor function in infants, but the Adverse Outcome Pathway behind this effect is not clear yet. We present for the first time the evaluation of developmental neurotoxic (DNT) effects of MDMA in zebrafish embryos. The aim of the study was to determine whether the zebrafish model reproduces the adverse outcome occurring in humans. We have studied the DNT effects of MDMA in zebrafish within a range of 5-250 µM performing different behavioural tests: spontaneous tail-coiling and light-dark locomotor response; after exposing the embryos to 4 different scenarios combining changes in pH, in starting exposure time and exposure duration. In these scenarios we evaluated the effects of MDMA in general embryonic development and compared the concentrations producing them with those inducing specific DNT effects. As a result, we have established the experimental conditions leading to the adverse outcome "lower motor activity" in zebrafish without producing general developmental delay or general toxicity. The experimental condition chosen opens the door to use this model in future mechanistic investigations to better characterize the Adverse Outcome Pathway associated with the adverse effects caused by MDMA prenatal exposure in humans.

The Zebrafish Embryo as a Model to Test Protective Effects of Food Antioxidant Compounds.

The antioxidant activity of food compounds is one of the properties generating the most interest, due to its health benefits and correlation with the prevention of chronic disease. This activity is usually measured using in vitro assays, which cannot predict in vivo effects or mechanisms of action. The objective of this study was to evaluate the in vivo protective effects of six phenolic compounds (naringenin, apigenin, rutin, oleuropein, chlorogenic acid, and curcumin) and three carotenoids (lycopene B, ß-carotene, and astaxanthin) naturally present in foods using a zebrafish embryo model. The zebrafish embryo was pretreated with each of the nine antioxidant compounds and then exposed to tert-butyl hydroperoxide (tBOOH), a known inducer of oxidative stress in zebrafish. Significant differences were determined by comparing the concentration-response of the tBOOH induced lethality and dysmorphogenesis against the pretreated embryos with the antioxidant compounds. A protective effect of each compound, except ß-carotene, against oxidative-stress-induced lethality was found. Furthermore, apigenin, rutin, and curcumin also showed protective effects against dysmorphogenesis. On the other hand, ß-carotene exhibited increased lethality and dysmorphogenesis compared to the tBOOH treatment alone.

Automated measurement of the spontaneous tail coiling of zebrafish embryos as a sensitive behavior endpoint using a workflow in KNIME.

Neuroactive substances are the largest group of chemicals detected in European surface waters. Mixtures of neuroactive substances occurring at low concentrations can induce adverse neurological effects in humans and organisms in the environment. Therefore, there is a need to develop new screening tools to detect these chemicals. Measurement of behavior or motor effects in rodents and fish are usually performed to assess potential neurotoxicity for risk assessment. However, due to pain and stress inflicted on these animals, the scientific community is advocating for new alternative methods based on the 3R principle (reduce, replace and refine). As a result, the behavior measurement of early stages of zebrafish embryos such as locomotor response, photomotor response and spontaneous tail coiling are considered as a valid alternative to adult animal testing. In this study, we developed a workflow to investigate the spontaneous tail coiling (STC) of zebrafish embryos and to accurately measure the STC effect in the KNIME software. We validated the STC protocol with 3 substances (abamectin, chlorpyrifos-oxon and pyracostrobin) which have different mechanisms of action. The KNIME workflow combined with easy and cost-effective method of video acquisition makes this STC protocol a valuable method for neurotoxicity testing.•Video acquisition duration of 60 s at 25 ± 1 hpf was used•20 embryos exposed per dish and acclimatized for 30 min before video acquisition•Capability to inspect and correct errors for high accuracy.

Modulation and Protection Effects of Antioxidant Compounds against Oxidant Induced Developmental Toxicity in Zebrafish.

The antioxidant effect of compounds is regularly evaluated by in vitro assays that do not have the capability to predict in vivo protective activity or to determine their underlying mechanisms of action. The aim of this study was to develop an experimental system to evaluate the in vivo protective effects of different antioxidant compounds, based on the zebrafish embryo test. Zebrafish embryos were exposed to tert-butyl hydroperoxide (tBOOH), tetrachlorohydroquinone (TCHQ) and lipopolysaccharides from Escherichia coli (LPS), chemicals that are known inducers of oxidative stress in zebrafish. The developmental toxic effects (lethality or dysmorphogenesis) induced by these chemicals were modulated with n-acetyl l-cysteine and Nω-nitro l-arginine methyl ester hydrochloride, dimethyl maleate and dl-buthionine sulfoximine in order to validate the oxidant mechanism of oxidative stress inducers. The oxidant effects of tBOOH, TCHQ, and LPS were confirmed by the determination of significant differences in the comparison between the concentration-response curves of the oxidative stress inducers and of the modulators of antioxidant status. This concept was also applied to the study of the effects of well-known antioxidants, such as vitamin E, quercetin, and lipoic acid. Our results confirm the zebrafish model as an in vivo useful tool to test the protective effects of antioxidant compounds.

Optimization of the spontaneous tail coiling test for fast assessment of neurotoxic effects in the zebrafish embryo using an automated workflow in KNIME®.

Neuroactive chemicals are frequently detected in the environment. At sufficiently high concentrations or within mixtures, they could provoke neurotoxic effects and neurological diseases to organisms and humans. Fast identification of such neuroactive compounds in the environment could help in hazard assessment and risk mitigation. Behavior change is considered as an important endpoint and might be directly or indirectly connected to a neuroactive mode of action. For a fast evaluation of environmental samples and pure substances, we optimized the measurement of a behavioral endpoint in zebrafish embryos - the spontaneous tail coiling (STC). Evaluation of results is automated via the use of a workflow established with the KNIME® software. Analysis duration and developmental stage were optimized to 1 min and 25 ± 1 hpf respectively during measurement. Exposing the embryos in a group of 10 or 20 and acclimatizing for 30 min at room temperature proved to be reliable. The optimized method was used to investigate neurotoxic effects of 18 substances with different modes of action (MoA). The STC test accurately detected the effect of 8 out of 11 neuroactive substances (chlorpyrifos, chlorpyrifos-oxon, diazinon, paraoxon-methyl, abamectin, carbamazepine, propafenone and diazepam). Aldicarb and nicotine showed subtle effects which were considered to be conditional and imidacloprid showed no effect. For substances with unknown neuroactive MoA, 3 substances did not provoke any effect on the STC (pyraclostrobin, diuron and daunorubicin-hydrochloride) while 4 other substances provoked an increased STC (hexaconazole, aniline, dimethyl-sulfoxide and 3,4-dichloroaniline). Such unexpected effects indicate possible neuroactive side effects or unknown mechanisms of action that impact on the STC. In conclusion, the optimized STC parameters and the automated analysis in KNIME® indicate opportunities for the harmonization of the STC test and further development for prospective and diagnostic testing.

Comparative Assessment of the Sensitivity of Fish Early-Life Stage, Daphnia, and Algae Tests to the Chronic Ecotoxicity of Xenobiotics: Perspectives for Alternatives to Animal Testing.

No-observed-effect concentrations (NOECs) are used in environmental hazard classification and labeling of chemicals and their environmental risk assessment. They are typically obtained using standard tests such as the fish early-life stage (FELS) toxicity test, the chronic Daphnia reproduction test, and the algae growth inhibition test. Given the demand to replace and reduce animal tests, we explored the impact of the FELS toxicity test on the determination of effect concentrations by comparing the FELS toxicity test and the Daphnia and algae acute or chronic toxicity tests. Lowest-observed-effect concentrations (LOECs) were used instead of NOECs for better comparison with median lethal or effect concentration data. A database of FELS toxicity data for 223 compounds was established. Corresponding Daphnia and algae toxicity tests were identified using established databases (US Environmental Protection Agency ECOTOX, Organisation for Economic Co-operation and Development QSAR Toolbox, eChemPortal, EnviroTox, and OpenFoodTox). Approximately 9.5% of the investigated compounds showed a 10-fold higher sensitivity with the FELS toxicity test in comparison with the lowest effect concentrations obtained with any of the other tests. Some of these compounds have been known or considered as endocrine disrupting, or are other non-narcotic chemicals, indicating that the higher sensitivity in the FELS toxicity test is related to a specific mechanism of action. Targeting these mechanisms by alternative test systems or endpoints, using fish embryos for instance, may allow reduction or replacement of the FELS toxicity test or may allow us to prioritize compounds for conduction of the FELS toxicity test. Environ Toxicol Chem 2019;39:30-41. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Cardiovascular Effects of PCB 126 (3,3',4,4',5-Pentachlorobiphenyl) in Zebrafish Embryos and Impact of Co-Exposure to Redox Modulating Chemicals.

The developing cardiovascular system of zebrafish is a sensitive target for many environmental pollutants, including dioxin-like compounds and pesticides. Some polychlorinated biphenyls (PCBs) can compromise the cardiovascular endothelial function by activating oxidative stress-sensitive signaling pathways. Therefore, we exposed zebrafish embryos to PCB126 or to several redox-modulating chemicals to study their ability to modulate the dysmorphogenesis produced by PCB126. PCB126 produced a concentration-dependent induction of pericardial edema and circulatory failure, and a concentration-dependent reduction of cardiac output and body length at 80 hours post fertilization (hpf). Among several modulators tested, the effects of PCB126 could be both positively and negatively modulated by different compounds; co-treatment with α-tocopherol (vitamin E liposoluble) prevented the adverse effects of PCB126 in pericardial edema, whereas co-treatment with sodium nitroprusside (a vasodilator compound) significantly worsened PCB126 effects. Gene expression analysis showed an up-regulation of cyp1a, hsp70, and gstp1, indicative of PCB126 interaction with the aryl hydrocarbon receptor (AhR), while the transcription of antioxidant genes (sod1, sod2; cat and gpx1a) was not affected. Further studies are necessary to understand the role of oxidative stress in the developmental toxicity of low concentrations of PCB126 (25 nM). Our results give insights into the use of zebrafish embryos for exploring mechanisms underlying the oxidative potential of environmental pollutants.

pH-Dependent Uptake and Sublethal Effects of Antihistamines in Zebrafish (Danio rerio) Embryos.

Reported off-target effects of antihistamines in humans draw interest in ecotoxicity testing of first- and second-generation antihistamines, the latter of which have fewer reported side effects in humans. Because antihistamines are ionizable compounds, the pH influences uptake and toxicity and thus is highly relevant when conducting toxicity experiments. Zebrafish embryo toxicity tests were performed with the 3 first-generation antihistamines ketotifen, doxylamine, and dimethindene and the 2 second-generation antihistamines cetirizine and levocabastine at pH 5.5, 7.0, and 8.0. We detected effects on survival, phenotype, swimming activity, and heart rate for 4 antihistamines with the exception of levocabastine, which did not show any lethal or sublethal effects. When compared to lethal concentrations, effect concentrations neither of phenotype malformation nor of swimming activity or heart rate deviated by more than a factor of 10 from lethal concentrations, indicating that all sublethal effects were fairly nonspecific. First-generation antihistamines are weak bases and showed decreasing external effect concentrations with increasing neutral fraction, accompanied by increased uptake in the fish embryo. As a result, internal effect concentrations were independent from external pH. The pH-dependent toxicity originates from speciation-dependent uptake, with neutral species taken up in higher amounts than the corresponding ionic species. Cetirizine, which shifts from a zwitterionic to an anionic state in the measured pH range, did not show any pH-dependent uptake or toxicity. Environ Toxicol Chem 2019;00:1-11. © 2019 SETAC.

Automated Morphological Feature Assessment for Zebrafish Embryo Developmental Toxicity Screens.

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 3 participating laboratories using 3 known developmental toxicants (methotrexate, dexamethasone, and topiramate) and 2 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.

Influence of pH on the uptake and toxicity of ß-blockers in embryos of zebrafish, Danio rerio.

ß-Blockers are weak bases with acidity constants related to their secondary amine group. At environmental pH they are protonated with the tendency to shift to their neutral species at more alkaline pH. Here we studied the influence of pH from 5.5 to 8.6 on the toxicity of the four ß-blockers atenolol, metoprolol, labetalol and propranolol in zebrafish embryos, relating toxicity not only in a conventional way to external aqueous concentrations but also to measured internal concentrations. Besides lethality, we evaluated changes in swimming activity and heartbeat, using the Locomotor Response (LMR) method and the Vertebrate Automated Screening Technology (VAST) for high throughput imaging. Effects of metoprolol, labetalol and propranolol were detected on phenotype, heart rate and swimming activity. External effect concentrations decreased with increasing neutral fraction for all three pharmaceuticals, attributed by an enhanced uptake of the neutral species in comparison to the corresponding charged form. The LC50 of metoprolol decreased by a factor of 35 from 1.91 mM with almost complete cationic state at pH 7.0 to 0.054 mM with 8% neutral fraction at pH 8.6. For propranolol the LC50 of 2.42 mM at pH 5.5 was even 100 fold higher than the LC50 at pH 8 with 0.023 mM where 3% were neutral fraction. No effects were detected in the zebrafish embryo exposed to atenolol. The internal concentrations for metoprolol and propranolol were quantified at non-toxic concentrations and at the LC10. Apparent bioconcentration factors (BCF) ranged from 1.96 at pH 7.0 to 32.0 at pH 8.6 for metoprolol and from 1.86 at pH 5.5 to 169 at pH 8.0 for propranolol. The BCFs served to predict the internal effect concentrations from the measured external effect concentrations. Internal effect concentrations of metoprolol and propranolol were in a similar range for all pH-values and for all endpoints. Interestingly, the internal effect concentrations were in the internal concentration range of baseline toxicity, which suggests that the effects of the ß-blockers are rather unspecific, even for sublethal effects on heart rate. In summary, our data confirm that the pH-dependent toxicity related to external concentrations can be explained by toxicokinetic effects and that the internal effect concentrations are pH-independent.

In vitro studies on the tumorigenic potential of the halonitromethanes trichloronitromethane and bromonitromethane.

Epidemiological data indicate that chronic exposure to water disinfection by-products (DBPs) may result in increased risk of cancer. However, the real carcinogenic potential of individual DBPs is not well known. In this study, we assessed the in vitro carcinogenic potential of trichloronitromethane (TCNM) and bromonitromethane (BNM), two halonitromethanes (HNMs) commonly found in DBPs' mixtures at comparably high concentrations. Human lung BEAS-2B cells were exposed for 8weeks to TCNM and BNM, and the acquisition of different in vitro cancer-like features was evaluated. The results indicate that long-term exposure to non-cytotoxic doses of TCNM and BNM did not cause carcinogenic transformation as indicated by the absence of morphological changes, no effects on cell growth, no changes in the level of matrix metalloproteinases (MMPs) secretion, and no increased anchorage-independent cell growth capacity. Furthermore, TCNM- and BNM-exposed BEAS-2B cells were unable to enhance tumour growth directly or by indirect influence of the surrounding stroma. Our results indicate that the carcinogenic effects of DBP mixtures cannot be attributed to the evaluated HNMs. This is the first study evaluating the cell transformation effects of TCNM and BNM under a long-term exposure scenario using suitable hallmarks of the cancer process.

Textile dyes induce toxicity on zebrafish early life stages.

Textile manufacturing is one of the most polluting industrial sectors because of the release of potentially toxic compounds, such as synthetic dyes, into the environment. Depending on the class of the dyes, their loss in wastewaters can range from 2% to 50% of the original dye concentration. Consequently, uncontrolled use of such dyes can negatively affect human health and the ecological balance. The present study assessed the toxicity of the textile dyes Direct Black 38 (DB38), Reactive Blue 15 (RB15), Reactive Orange 16 (RO16), and Vat Green 3 (VG3) using zebrafish (Danio rerio) embryos for 144 h postfertilization (hpf). At the tested conditions, none of the dyes caused significant mortality. The highest RO16 dose significantly delayed or inhibited the ability of zebrafish embryos to hatch from the chorion after 96 hpf. From 120 hpf to 144 hpf, all the dyes impaired the gas bladder inflation of zebrafish larvae, DB38 also induced curved tail, and VG3 led to yolk sac edema in zebrafish larvae. Based on these data, DB38, RB15, RO16, and VG3 can induce malformations during embryonic and larval development of zebrafish. Therefore, it is essential to remove these compounds from wastewater or reduce their concentrations to safe levels before discharging textile industry effluents into the aquatic environment.

Triclabendazole sulfoxide causes stage-dependent embryolethality in zebrafish and mouse in vitro.

BACKGROUND: Fascioliasis and paragonimiasis are widespread foodborne trematode diseases, affecting millions of people in more than 75 countries. The treatment of choice for these parasitic diseases is based on triclabendazole, a benzimidazole derivative which has been suggested as a promising drug to treat pregnant women and children. However, at the moment, this drug is not approved for human use in most countries. Its potential adverse effects on embryonic development have been scarcely studied, and it has not been assigned a pregnancy category by the FDA. Thus, to help in the process of risk-benefit decision making upon triclabendazole treatment during pregnancy, a better characterization of its risks during gestation is needed. METHODOLOGY: The zebrafish embryo test, a preimplantation and a postimplantation rodent whole embryo culture were used to investigate the potential embryotoxicity/teratogenicity of triclabendazole and its first metabolite triclabendazole sulfoxide. Albendazole and albendazole sulfoxide were included as positive controls. PRINCIPAL FINDINGS: Triclabendazole was between 10 and 250 times less potent than albendazole in inducing dysmorphogenic effects in zebrafish or postimplantation rodent embryos, respectively. However, during the preimplantation period, both compounds, triclabendazole and triclabendazole sulfoxide, induced a dose-dependent embryolethal effect after only 24 h of exposure in rodent embryos and zebrafish (lowest observed adverse effect concentrations = 10 µM). CONCLUSIONS/SIGNIFICANCE: In humans, after ingestion of the recommended doses of triclabendazole to treat fascioliasis and paragonimiasis (10 mg/kg), the main compound found in plasma is triclabendazole sulfoxide (maximum concentration 38.6 µM), while triclabendazole concentrations are approximately 30 times lower (1.16 µM). From our results it can be concluded that triclabendazole, at concentrations of the same order of magnitude as the clinically relevant ones, does not entail teratogenic potential in vitro during the organogenesis period, but its first metabolite triclabendazole sulfoxide has a high embryotoxic capacity in vitro during the preimplantation stage.

Developmental effects and genotoxicity of 10 water disinfection by-products in zebrafish.

Disinfection by-products are contaminants produced during drinking water disinfection. Several DBPs have been implicated in a variety of toxic effects, mainly carcinogenic and genotoxic effects. Moreover, DBPs exposure has also been associated with an increased risk of developmental effects. In this study, the developmental toxicity and genotoxicity of 10 DBPs (four trihalomethanes [THMs], five haloacetic acids [HAAs] and sodium bromate) in the zebrafish embryo model were evaluated. Embryos exposed for 72 hours were observed for different endpoints such as growth, hatching success, malformations and lethality. THMs exposure resulted in adverse developmental effects and a significant reduced tail length. Two HAAs, tribromoacetic acid and dichloroacetic acid, along with sodium bromate were found to cause a significant increase in malformation rate. Chloroform, chlorodibromomethane and sodium bromate produced a weak induction of DNA damage to whole embryos. However, developmental effects occurred at a range of concentrations (20-100 µg/mL) several orders of magnitude above the levels that can be attained in fetal blood in humans exposed to chlorinated water. In conclusion, the teratogenic and genotoxic activity observed by some DBPs in zebrafish reinforce the view that there is a weak capacity of disinfection products to cause developmental effects at environmentally relevant concentrations.