In toxicology, standard sigmoidal concentration-response curves are used to predict effects concentrations and set chemical regulations. However, current literature also establishes the existence of complex, bimodal concentration-response curves, as is the case for arsenic toxicity. This bimodal response has been observed at the molecular level, but not characterized at the whole organism level. This study investigated the effect of arsenic (sodium arsenite) on post-gastrulated zebrafish embryos and elucidated effects of bimodal concentration-responses on different phenotypic perturbations.Six hour post fertilized (hpf) zebrafish embryos were exposed to arsenic to 96 hpf. Hatching success, mortality, and morphometric endpoints were evaluated both in embryos with chorions and dechorionated embryos. Zebrafish embryos exhibited a bimodal response to arsenic exposure. Concentration-response curves for exposed embryos with intact chorions had an initial peak in mortality (88%) at 1.33 mM arsenic, followed by a decrease in toxicity (~20% mortality) at 1.75 mM, and subsequently peaked to 100% mortality at higher concentrations. To account for the bimodal response, two distinct concentration-response curves were generated with estimated LC10 values (and 95% CI) of 0.462 (0.415, 0.508) mM and 1.69 (1.58, 1.78) mM for the ‘low concentration’ and ‘high concentration’ peaks, respectively. Other phenotypic analyses, including embryo length, yolk and pericardial edema all produced similar concentration-response patterns. Tests with dechorionated embryos also resulted in a bimodal toxicity response but with lower LC10 values of 0.170 (0.120, 0.220) mM and 0.800 (0.60, 0842) mM, respectively. Similarities in bimodal concentration-responses between with-chorion and dechorionated embryos indicate that the observed effect was not caused by the chorion limiting arsenic availability, thus lending support to other studies such as those that hypothesized a conserved bimodal mechanism of arsenic interference with nuclear receptor activation.

Several studies highlighted the ubiquitous presence of ibuprofen and aluminum in the aquatic environment around the world and demonstrated their potential to induce embryotoxic and teratogenic defects on aquatic species individually. Although studies that evaluate developmental alterations induced by mixtures of these pollutants are scarce; and, since environmental contamination presented in the form of a mixture of toxicants with different chemical properties and toxicity mechanisms capable of generating interactions; the objective of this study was to evaluate the developmental defects, teratogenic alterations, and oxidative stress induced by individual forms and the mixture of ibuprofen (IBU) and aluminum (Al) on zebrafish embryos. Oocytes exposed to environmentally relevant concentrations of IBU (0.1–20 μg L-1) and Al (0.01–8 mg L-1) and one binary mixture. The LC50 and EC50 were obtained to calculate the teratogenic index (TI). The IBU LC50, EC50, and TI were 8.06 μg L-1, 2.85 μg L-1 and 2.82. In contrast, Al LC50 was 5.0 mg L-1with an EC50 of 3.58 mg L-1 and TI of 1.39. The main alterations observed for individual compounds were hatching alterations, head malformation, skeletal deformities, hypopigmentation, pericardial edema, and heart rate impairment. The mixture also showed significant delays to embryonic development. Moreover, oxidative stress biomarkers of cellular oxidation and antioxidant defenses at 72 and 96 hpf significantly increased. Results show that environmentally relevant concentrations of ibuprofen (IBU), aluminum (Al), and their mixture promote a series of developmental defects, teratogenic effects, and oxidative disruption on D. rerio embryos, and the interaction of both substances altered the response. In conclusion, morphological and biochemical tests are suitable tools for assessing the health risk of aquatic wildlife by exposure to individual and mixed pollutants in freshwater bodies.

Diclofop-methyl (DM) is one of the most widely used herbicides in agriculture production and has been frequently detected in both freshwater and environments, even agricultural products. However, the potential toxic effects of DM on organisms and the underlying mechanisms are still poorly understood. In this study, we utilized zebrafish to evaluate the toxicity of DM during the cardiovascular developmental process. Exposure of zebrafish embryos to 0.75, 1.0 and 1.25 mg/L DM induced cardiac defects, such as pericardial edema, slow heart rate and long SV-BA distance but the vascular development in zebrafish larvae was not influenced by DM treatment. The expression of cardiac-related genes were disordered and DM exposure initiated disordering cardiogenesis from the period of precardiac mesoderm formation. Moreover, the apoptosis and proliferation of cardiomyocytes were not influenced but the levels of oxidative stress were upregulated by DM exposure. Fullerenes and astaxanthin was able to rescue cardiac defects caused by DM via downregulating oxidative stress. Wnt signaling was downregulated after DM treatment and activation of Wnt signaling could rescue cardiac defects. Therefore, our results suggest that DM has the potential to induce cardiac developmental toxicity through upregulation of Wnt-Mediated (reactive oxygen species) ROS generation in zebrafish larvae.

As a new type of pollutant, fluoroquinolones (FQs) antibiotics are ubiquitous in environment and have some threat to human health and ecological environment. Their ecological toxicity to the environment urgently need to be assessed. Therefore, we firstly explored the toxic effects and possible mechanism of cardiovascular toxicity induced by gatifloxacin (GTFX) and ciprofloxacin (CPFX) using zebrafish model. After 24 h exposure, the zebrafish treated with GTFX showed pericardial edema which was further investigated by histopathological examination, while CPFX exposure did not induce morphological abnormalities. However, both of them induced cardiac dysfunction, such as decreased heart rate and cardiac output which was showed a positive correlation with the concentration. To better understand the possible molecular mechanisms underlying cardiovascular toxicity in zebrafish, we investigated the transcriptional level of genes related to calcium signaling pathway and cardiac muscle contraction. The results indicated that the expression of ATPase (atp2a1l) and cardiac troponin C (tnnc1a) genes were significantly inhibited, the expression of calcium channel (cacna1ab) gene showed slight promoted trend after CPFX exposure. For zebrafish treated with GTFX, the expression of atp2a1l genes was also significantly inhibited, while the expression of tnnc1a genes was slightly inhibited and cacna1ab genes expression had no obvious effect. The present study firstly revealed that GTFX exposure can induce morphological and functional abnormalities on the cardiovascular system of zebrafish. Though CPFX exposure did not induce morphological abnormalities, the function of cardiovascular system was still damaged. Mechanistically, this toxicity might result from the pressure of down-regulation of genes associated with calcium signaling pathway and cardiac muscle contraction. The results of this study can provide a valuable theoretical basis for the establishment of FQs environmental quality standards in water environment, environmental drug regulation and risk management.

The health risk of triadimefon (TF) to cardiovascular system of human is still unclear, especially to pesticide suicides population, occupational population (farmers, retailers and pharmaceutical workers), and special population (young children and infants, pregnant women, older people, and those with compromised immune systems) who are at a greater risk. Therefore, firstly we explored the toxic effects and possible mechanism of cardiovascular toxicity induced by TF using zebrafish model. Zebrafish at stage of 48 h post fertilization (hpf) exposed to TF for 24 h exhibited morphological malformations which were further confirmed by histopathologic examination, including pericardial edema, circulation abnormalities, serious venous thrombosis and increased distance between the sinus venosus (SV) and bulbus arteriosus (BA) regions of the heart. In addition to morphological changes, TF induced functional deficits in the heart of zebrafish, including bradycardia and a significant reduced cardiac output that became more serious at higher concentrations. To better understand the possible molecular mechanisms underlying cardiovascular toxicity in zebrafish, we investigated the transcriptional level of genes related to calcium signaling pathway and cardiac muscle contraction. Q-PCR (quantitative real-time polymerase chain reaction) results demonstrated that the expression level of genes related to ATPase (atp2a1l, atp1b2b, atp1a3b), calcium channel (cacna1ab, cacna1da) and cardiac troponin C (tnnc1a) were significantly decreased after TF exposure. For the first time, the present study revealed that TF exposure had observable morphological and functional negative impacts on cardiovascular system of zebrafish. Mechanistically, this toxicity might result from the pressure of down-regulation of genes associated with calcium signaling pathway and cardiac muscle contraction following TF exposure. These findings generated here can provide information for better pesticide poisoning treatments, occupational disease prevention, and providing theoretical foundation for risk management measures.

Hydraulic fracturing to extract oil and natural gas reserves is an increasing practice in many international energy sectors. Hydraulic fracturing flowback and produced water (FPW) is a hyper saline wastewater returned to the surface from a fractured well containing chemical species present in the initial fracturing fluid, geogenic contaminants, and potentially newly synthesized chemicals formed in the fracturing well environment. However, information on FPW toxicological mechanisms of action remain largely unknown. Both cardiotoxic and respirometric responses were explored in zebrafish (Danio rerio) embryos after either an acute sediment-free (FPW-SF) or raw/sediment containing (FPW-S) fraction exposure of 24 and 48 h at 2.5% and 5% dilutions. A 48 h exposure to either FPW fraction in 24–72 h post fertilization zebrafish embryos significantly increased occurrences of pericardial edema, yolk-sac edema, and tail/spine curvature. In contrast, larval heart rates significantly decreased after FPW fraction exposures. FPW-S, but not FPW-SF, at 2.5% doses significantly reduced embryonic respiration/metabolic rates (MO2), while for 5% FPW, both fractions reduced MO2. Expression of select cardiac genes were also significantly altered in each FPW exposure group, implicating a cardiovascular system compromise as the potential cause for reduced embryonic MO2. Collectively, these results support our hypothesis that organics are major contributors to cardiac and respiratory responses to FPW exposure in zebrafish embryos. Our study is the first to investigate cardiac and respiratory sub-lethal effects of FPW exposure, demonstrating that FPW effects extend beyond initial osmotic stressors and verifies the use of respirometry as a potential marker for FPW exposure.

During hydraulic fracturing, wastewaters - termed flowback and produced water (FPW) - are created as a by-product during hydrocarbon extraction. Given the large volumes of FPW that a single well can produce, and the history of FPW release to surface water bodies, it is imperative to understand the hazards that hydraulic fracturing and FPW pose to aquatic biota. Using rainbow trout embryos as model organisms, we investigated impacts to cardio-respiratory system development and function following acute (48 h) and sub-chronic (28-day) FPW exposure by examining occurrences of developmental deformities, rates of embryonic respiration (MO₂), and changes in expression of critical cardiac-specific genes. FPW-exposed embryos had significantly increased rates of pericardial edema, yolk-sac edema, and tail/trunk curvatures at hatch. Furthermore, when exposed at three days post-fertilization (dpf), acute 5% FPW exposures significantly increased embryonic MO₂ through development until 15 dpf, where a switch to significantly reduced MO₂ rates was subsequently recorded. A similar trend was observed during sub-chronic 1% FPW exposures. Interestingly, at certain specific developmental timepoints, previous salinity exposure seemed to affect embryonic MO₂; a result not previously observed. Following acute FPW exposures, embryonic genes for cardiac development and function were significantly altered, although at termination of sub-chronic exposures, significant changes to these same genes were not found. Together, our evidence of induced developmental deformities, modified embryonic MO₂, and altered cardiac transcript expression suggest that cardio-respiratory tissues are toxicologically targeted following FPW exposure in developing rainbow trout. These results may be helpful to regulatory bodies when developing hazard identification and risk management protocols concerning hydraulic fracturing activities.

Fluoxetine is frequently detected in aquatic environment, and chronic FLX exposure exhibits adverse effects on aquatic communities. Its chirality makes the adverse effects more complicated. This study aimed at the enantioselective cardiotoxicity in developmental zebrafish induced by racemic (rac-)/S-/R-fluoxetine. The accumulation profiles demonstrated that biotransformation of fluoxetine to norfluoxetine occurred during rac-fluoxetine exposure, with a higher enrichment of S-norfluoxetine than R-norfluoxetine. Heart malformations including pericardial edema, circulation abnormalities, and thrombosis were observed, and enantioselective changes also occurred. According to H&E staining and Masson’s trichrome staining, the loose severity of cardiac structure and cardiac fibrosis in rac-norfluoxetine treated group was worse than that in fluoxetine treated groups. Results of toxicity-associated parameters in our homochiral enantiomers’ exposure also indicated that the toxicity induced by S-fluoxetine was more severe than R-fluoxetine. Enantioselective arrhythmia in developmental zebrafish after chiral fluoxetine exposure could be caused by myocardial fibrosis, abnormal developmental processes, and the biotransformation of fluoxetine to norfluoxetine could make that worse. Our findings can be used to assess the environmental risk of the two enantiomers of fluoxetine that induce cardiotoxicity in aquatic organisms.

Triclosan (TCS) and its two derivatives (2,4-dichlorophenol and 2,4,6-trichlorophenol) are priority pollutants that coexist in aquatic environments. Joint exposure of TCS, 2,4-dichlorophenol and 2,4,6-trichlorophenol, hereafter referred to as TCS-DT, contributes severe toxicity to aquatic organisms. There is currently a paucity of data regarding TCS-DT molecular toxicity, especially on cardiac diseases. We used zebrafish (Danio rerio) as a model organism, and evaluated the molecular-level cardiotoxicity induced by TCS-DT from embryonic to adult stages. TCS-DT exposure prominently led to phenotypic malformations, such as pericardial cysts, cardiac bleeding, increased SV-BA distance, decreased heart rate and reduced ejection fraction, as well as abnormal swimming behavior. Analyses of the GO and KEGG pathways revealed enrichment pathways related to cardiac development and screened for significantly down-regulated adrenaline signaling in cardiomyocytes. The cardiac marker genes (amhc, cmlc2, vmhc, and nkx2.5) were obtained through protein-protein interaction (PPI) networks, and expressed as down-regulation by WISH. After chronic exposure to TCS-DT from 30 to 90-dpf, both body mass and heart indexes prominently increased, showing myocardial hypertrophy, abnormal heart rate and histopathological injury. Heart tissue damage included disordered and ruptured myocardial fibers, broken and dissolved myofilaments, nuclear pyknosis, mitochondrial injury and inflammatory cell infiltration. Further, abnormal changes in a series of cardiac functions-related biomarkers, including superoxide dismutase, triglyceride, lactate dehydrogenase and creatinine kinase MB, provided evidence for cardiac pathological responses. These results highlight the molecular mechanisms involving TCS-DT induced cardiac toxicity, and provide theoretical data to guide prevention and treatment of pollutant-induced cardiac diseases.

The potential risks of phthalates affecting human and animal health as well as the environment are emerging as serious concerns worldwide. However, the mechanism by which phthalates induce developmental effects is under debate. Herein, we found that embryonic exposure of zebrafish to di-(2-ethylhexyl) phthalate (DEHP) and di-butyl phthalate (DBP) increased the rate of heart defects including abnormal heart rate and pericardial edema. Changes in the transcriptional profile demonstrated that genes involved in the development of the heart, such as tbx5b, nppa, ctnt, my17, cmlc1, were significantly altered by DEHP and DBP at 50 μg/L, which agreed with the abnormal cardiac outcomes. Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) further showed that significant hypomethylation of nppa and ctnt was identified after DEHP and DBP exposure, which was consistent with the up-regulation of these genes. Notably, hypermethylation on the promoter region (<1 kb) of tbx5b was found after DEHP and DBP exposure, which might be responsible for its decrease in transcription. In conclusion, phthalates have the potential to induce cardiac birth defects, which might be associated with the transcriptional regulation of the involved developmental factors such as tbx5b. These findings would contribute to understand the molecular pathways that mediated the cardiac defects caused by phthalates.