Developmental toxicity,EROD, and CYP1A mRNA expression in zebrafish embryos exposed to dioxin‐like PCB126

Dioxin‐like PCB126 is a persistent organic pollutant that causes a range of syndromes including developmental toxicity. Dioxins have a high affinity for aryl hydrocarbon receptor (AhR) and induce cytochrome P4501A (CYP1A). However, the role of CYP1A activity in developmental toxicity is less clear. To better understand dioxin induced developmental toxicity, we exposed zebrafish (Danio rerio) embryos to PCB126 at concentrations of 0, 16, 32, 64, and 128 μg L^?1 from 3‐h post‐fertilization (hpf) to 168 hpf. The embryonic survival rate decreased at 144 and 168 hpf. The fry at 96 hpf displayed gross developmental malformations, including pericardial and yolk sac edema, spinal curvature, abnormal lower jaw growth, and non‐inflated swim bladder. The pericardial and yolk sac edema rate significantly increased and the heart rate declined from 96 hpf compared with the controls. PCB126 did not alter the hatching rate. To elucidate the mechanism of PCB126‐induced developmental toxicity, we conducted ethoxyresorufin‐O‐deethylase (EROD) in vivo assay to determine CYP1A enzyme activity, and real‐time PCR to study the induction of CYP1A mRNA gene expression in embryo/larval zebrafish at 24, 72, 96, and 132 hpf. In vivo EROD activity was induced by PCB126 at 16 μg L^?1 concentration as early as 72 hpf but significant increases were observed only in zebrafish exposed to 64 and 128 μg L^?1 doses (p < 0.005) at 72, 96, and 132 hpf. Induction of CYP1A mRNA expression was significantly upregulated in zebrafish exposed to 32 and 64 μg L^?1 at 24, 72, 96, and 132 hpf. Overall, the severe pericardial and yolk sac edema and reduced heart rate suggest that heart defects are a sensitive endpoint, and the general trend of dose‐dependent increase in EROD activity and induction of CYP1A mRNA gene expression provide evidence that the developmental toxicity of PCB126 to zebrafish embryos is mediated by activation of AhR. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 201–210, 2016.     

Metabolomics comparison of metabolites and functional pathways in the gills of Chlamys farreri under cadmium exposure

The biological processes of Chlamys farreri (C. farreri), an economically important shellfish, are affected when exposed to Cd²⁺. In this study, changes to biological processes and metabolite levels in C. farreri were examined when exposed to Cd²⁺. Ultra-performance liquid chromatography-tandem TOF mass spectrometry (UPLC-TOF/MS)-based untargeted metabolomics was used to examine changes in the metabolism of C. farreri gill tissue exposed to 0.050 mg/L Cd²⁺ for 96 h in a natural environment. Sixty-eight metabolites with significant differences were screened by multivariate statistical analysis. Eleven enriched functional pathways displayed significant changes in inactivity. Differential metabolites, mainly C00157 and C00350, have a significant impact on functional pathways and can be used as potential major biomarkers. Lipid phosphorylation, disruption of signal transduction, and autophagy activation were observed to change in C. farreri when exposed to Cd. The metabolome information supplements research on C. farreri exposure to heavy metals and provides a platform for further multi-omics analysis.     

Toxicological assessment and developmental abnormalities induced by butylparaben and ethylparaben exposure in zebrafish early-life stages

Toxicological effects of butylparaben (BuP) and ethylparaben (EtP) on zebrafish (Danio rerio) early-life stages are not well established. The present study evaluated, using zebrafish embryos and larvae, the toxicity of BuP and EtP through benchmark dose (BMD) approach. BuP was more toxic than EtP to zebrafish larvae. In fact, Lethal Concentration 50 (LC50) values at 96 h post-fertilization (hpf) for BuP and EtP were 2.34 mg/L and 20.86 mg/L, respectively. Indeed, BMD confidence interval (lower bound (BMDL) - upper bound (BMDU) was 0.91–1.92 mg/L for BuP and 10.8–17.4 mg/L for EtP. Zebrafish embryos exposed to 1 mg/L, 2.5 mg/L of BuP and 5 mg/L, 10 mg/L, 20 mg/L, 30 mg/L of EtP showed several developmental abnormalities and teratological effects compared to negative control. Exposed zebrafish developed reduced heartbeat, reduction in blood circulation, blood stasis, pericardial edema, deformed notochord and misshaped yolk sac. Embryos exposed to the highest concentrations of the chemicals (2.5 mg/L of BuP, 10 mg/L, 20 mg/L and 30 mg/L of EtP) showed the developmental abnormalities at 48 hpf while those treated with 1 mg/L of BuP and 10 mg/L of EtP reported behavioral changes at 72 hpf, including trembling of head, pectoral fins and spinal cord. This research identified the lethal and sublethal effects of BuP and EtP in zebrafish early-life stages and could be helpful to elucidate the developmental pathways of toxicity of parabens.     

The effect of salinity on cadmium toxicity to the estuarine mysid Mysidopsis bahia: role of chemical speciation

The effect of salinity on the toxicity of cadmium to the bay mysid, Mysidopsis bahia Molenock, was studied. When toxicity was expressed in terms of free cadmium ion (Cd²⁺) rather than total cadmium (Cd_T), CdCl⁺ or CdCl₂ only a slight salinity effect was apparent, suggesting that Cd²⁺ is the primary toxic species. Mysids were more tolerant of Cd²⁺ at an intermediate salinity of 22‰ and less tolerant at low and high salinity extremes (6 and 38‰). Alteration of cadmium speciation by use of an artificial ligand, nitrilotriacetic acid (NTA), over a range of salinities (14–34‰) produced conflicting results. Toxicity of Cd²⁺ was independent of Cd_T concentration at a given salinity but increased with increasing salinity and/or NTA concentration and was determined to be the result of salinity—Cd²⁺ and NTA-Cd²⁺ interactions. Possible mechanisms of the salinity effect and NTA interaction with cadmium toxicity are discussed.     

Cadmium resistance and content of cadmium-binding protein in two enzyme-deficient mutants of mouse fibroblasts (L-cells)

The toxic Cd²⁺ ion accumulates in mammalian organisms, the main storage organs are apparently the liver and the kidney. In these organs Cd²⁺ is bound to low molecular weight proteins (thioneins) as metallothionein. We describe here the development of resistance to otherwise lethal concentrations of Cd²⁺ by two non-epithelial cell lines, both derived from mouse fibroblasts (L-cells). One of the cell lines (clone ID) is deficient in thymidine kinase and resistant to 5-bromodeoxyuridine, the other (A9) deficient in hypoxanthine-guanine phosphoribosyl transferase and resistant to 8-azaguanine. After stepwise increase in Cd²⁺ concentration, clone 1D cells had apparently normal growth rate in the presence of 100 micromolar Cd²⁺ after 6 months of Cd treatment. The A9 cells were apparently more sensitive to Cd²⁺, after about one year's Cd treatment they had apparently normal growth in the presence of 100 micromolar Cd²⁺. This concentration of Cd²⁺ would kill cells of both cell lines not previously exposed to Cd. In the resistant A9 cells about 40 per cent of the cadmium were bound to a cadmiumbinding protein (Cd-BP) of molecular weight of about 12,000, most probably metallothionein, in the resistant clone 1D cells the corresponding figure was 60 per cent. The non-resistant cell lines had apparently no metallothionein. We have thus found that also non-epithelial cells can synthesize low molecular weight Cd-BP and that there apparently is a good correlation between cadmium resistance and content of Cd-BP.     

Toxicity effects of captan on different life stages of zebrafish (Danio rerio)

The objective of this study was to evaluate the toxicity and developmental effects of captan on different life stages (embryo and adult) of zebrafish (Danio rerio). The results showed that the 96-h lethal concentration 50 (LC₅₀) values of embryo and adult zebrafish (exposed to captan) were 0.81(0.75−0.87) mg/L and 0.65(0.62−0.68) mg/L, respectively. The results of developmental effect experiment showed that captan can significantly decrease the heartbeats and inhibit the hatching rate and growth of zebrafish embryos. Moreover, captan exposure can induce a series of deformities, including pericardial edema, yolk sac edema, spine curvature, and tail bending, in zebrafish embryos during the developmental period. Among these, the most significant were tail bending and spine curvature.     

Cadmium induced oxidative damage and apoptosis in the hepatopancreas of <Emphasis Type="Italic">Meretrix meretrix</Emphasis>

Even trace amounts of cadmium (Cd), a non-essential metal, are known to be toxic to aquatic organisms. Here we investigated the relationship between cadmium ion (Cd²⁺) exposure and oxidative damage and apoptosis in the hepatopancreas of the clam Meretrix meretrix. Clams were exposed to different concentrations of Cd²⁺ (0, 1.5, 3, 6 and 12 mg L^?1) for 5 days. We monitored both antioxidant enzyme activity, including that of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx), and levels of malondialdehyde (MDA), glutathione (GSH) and glutathione disulfide (GSSG). Apoptosis of hepatopancreatic cells was detected by DNA laddering and AO/EB double fluorescent staining. The results show that the rate of apoptotis, MDA levels, and caspase-3 activity, increased with Cd²⁺ concentration, whereas GPx activity and the ratio of GSH/GSSG, decreased. SOD and CAT enzyme activity first increased, then decreased, with increasing Cd²⁺ concentration; peak activity of these enzymes was recorded in the 3 mg L^?1 Cd²⁺-treatment group. These results show that Cd-induced oxidative damage can both induce, and aggravate, apoptosis in the hepatopancreatic cells of clams, even at Cd²⁺ concentrations far below the semi-lethal dose for adult clams. The observed changes in caspase-3 activity enhanced significantly at lower Cd²⁺ concentrations, indicating that caspase-3 is a suitable biomarker for heavy metal pollution, especially cadmium pollution, in marine organisms.     

Metals (Ag+, Cd2+, Cr6+) affect ATPase activity in the gill,kidney, and muscle of freshwater fish Oreochromis niloticus following acute and chronic exposures

Freshwater fish Oreochromis niloticus were individually acutely exposed to different concentrations (0, 0.1, 0.5, 1.0, and 1.5 μg/mL) of Cd²⁺, Cr⁶⁺, and Ag⁺ for 96 h and 0.05 μg/mL concentration of the same metals for different periods (0, 5, 10, 20, and 30 days) chronically. Following each experimental protocol, Na⁺/K⁺‐ATPase, Mg²⁺‐ATPase, and Ca²⁺‐ATPase activities were measured in the gill, kidney, and muscle of O. niloticus. In vitro experiments were also performed to determine the direct effects of metal ions (0, 0.1, 0.5, 1.0, and 1.5 μg/mL) on ATPases. Except Ag⁺, none of the metals caused fish mortality within 30 days. Silver killed all the fishes within 16 days. Metal exposures generally decreased Na⁺/K⁺‐ATPase and Ca²⁺‐ATPase activities in the tissues of O. niloticus, although there were some fluctuations in Mg²⁺‐ATPase activity. Ag⁺ and Cd²⁺ were found to be more toxic to ATPase activities than Cr⁶⁺. It was also observed that metal efficiency was higher in the gill than in the other tissues. Results indicated that the response of ATPases varied depending on metals, exposure types, and tissues. Because ATPases are sensitive to metal toxicity, their activity can give valuable data about fish physiology. Therefore, they may be used as a sensitive biomarker in environmental monitoring in contaminated waters. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 707–717, 2013.     

Effects of embryonic exposure to polychlorinated biphenyls on zebrafish (Danio rerio) retinal development

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that affect embryonic development. The purpose of this study was to examine the effects of embryonic exposure to PCBs on early retinal development in zebrafish, Danio rerio. Zebrafish embryos were immediately exposed to different concentrations (0, 0.125, 0.25, 0.5, 1.0 and 2.0 mg) of PCBs per liter of medium at 28.5 °C. Embryos were assessed at 30, 48, 72 and 96 h post‐fertilization (hpf) for changes in embryonic survival rate, development, larval retinal morphology and ultrastructure of the retina. The results show that PCB exposure decreased the survival rate of embryos in a time‐ and dose‐dependent manner. Embryos exposed to the higher concentrations of PCBs (0.5, 1.0 and 2.0 mg l^?1) displayed obvious gross morphological deformities. At 72 hpf, the retinal layer development of zebrafish was delayed at higher PCB concentrations (1.0 mg l^?1). At 96 hpf, irregularity of photoreceptor cells arrangement and thickening of photoreceptor and ganglionic layers were observed in PCB‐treated larvae at concentrations of 0.25–1 mg l^?1. Ultrastructural examination showed signs of growth inhibition of the photoreceptor outer segment at 0.25–1 mg l^?1 PCB exposure at 72 hpf, as well as the appearance of massive vacuoles and holes inside the outer segments in the PCB exposure group at 96 hpf. These results suggest that embryonic exposure to moderate and high levels of PCBs induced developmental deficits in zebrafish retinas, particularly in photoreceptor cells. Copyright © 2011 John Wiley & Sons, Ltd.     

The effect of methylmercury exposure on early central nervous system development in the zebrafish (Danio rerio) embryo

Much attention is focused on environmental contamination by heavy metals. The heavy metal mercury is found worldwide and is ranked number 3 on the Comprehensive Environmental Response, Compensation and Liability Act substance list. We examined the effect of low‐level methylmercury exposure on central nervous system development of wild‐type zebrafish embryos (ZFEs) of the AB strain because methylmercury is the most common form of mercury to which humans are exposed in the environment. ZFEs were exposed to nine different concentrations of methylmercury [0 (negative control), 5, 10, 50, 80, 100, 200, 500 and 1000 parts per billion (μg l^?1)] starting at 6 h post‐fertilization, which is the time the neural tube is first beginning to form. ZFEs were exposed to 2% ethanol as positive controls (100% embryonic death). ZFEs were assessed at 30, 54, 72 and 96 h post‐fertilization for changes in embryonic development, mortality, time of hatching and morphological deformities. No abnormalities were observed in ZFEs exposed to 5 μg l^?1 methylmercury. The time of hatching from the chorion was delayed in ZFEs exposed to methylmercury concentrations of 50 μg l^?1 or higher. Significantly more ZFEs exposed to 0, 5 or 10 μg l^?1 methylmercury successfully completed hatching compared with ZFEs exposed to 50 μg l^?1 or higher methylmercury. ZFEs exposed to more than 200 μg l^?1 methylmercury exhibited 100% embryonic mortality. The rate of cell proliferation within the neural tube was significantly decreased in embryos exposed to 10, 50 and 80 μg l^?1 methylmercury and there were no differences between these doses. Copyright © 2011 John Wiley & Sons, Ltd.     

Effects of soluble sulfide on zebrafish (Danio rerio) embryonic development

Zebrafish embryos were used to investigate the developmental effects of sulfide. Mortality, teratogenic effects, and developmental parameters of early developmental embryos were recorded. The biodistribution of sulfide in developing zebrafish embryos and larvae were measured through fluorescence imaging. The influences of sulfide on the cardiac function and development velocity of zebrafish embryos were dependent on sulfide concentration. Heart rate and development velocity increased with exposure to lower sulfide concentrations, which may be attributed to the cardioprotective properties of H₂S. Meanwhile, heart rate and development velocity decreased, whereas pericardial edema, yolk sac edema, and trunk abnormalities occurred with exposure to higher sulfide concentrations. Sulfide accumulated in the blastoderm of early developmental embryos and was then transported to the yolk sac and yolk extension with the embryonic development. Finally, sulfide was transferred from the yolk to the eyes of zebrafish larvae. The details of mechanism of sulfide toxicity require further research.     

Assessment of sediment quality of Yangtze River estuary using zebrafish (Danio rerio) embryos

Yangtze River estuary is one of the largest estuaries worldwide. In this study, the sediment quality of Yangtze River estuary was evaluated using zebrafish (Danio rerio) embryos. Freshly fertilized zebrafish eggs (2 h after fertilization) were exposed to the whole sediment and its organic phase of extract, respectively. The parameters, including survival rate, abnormality, hatching rate, and heart rate of the zebrafish embryos, were recorded during the 96‐h exposure. The results demonstrated that the concentrations of heavy metals (Zn, Cu, Cd, Ni, Cr, and As) and low‐molecular weight PAHs (Fluorene) in the sediment of Yangtze River estuary exceeded their corresponding effects range low values. The maximum concentrations of Zn and Fluorene in the sediment samples were 239.6 μg/g and 45.9 ng/g, respectively. In both whole sediment test and organic extract test, the survival rate and heart rate of zebrafish embryos were reduced, as well as abnormalities and delayed hatching were induced. For example, the highest mortality of the embryos was 39% in the whole sediment exposure. Overall, the occurrence of toxic compounds in the sediment of Yangtze River estuary may have potentially teratogenic effect on biota. The sediment from the upstream of Yangtze River estuary have more observed toxic effects on zebrafish embryos than that form the downstream. Therefore, more attention should be paid to control these pollutants, especially heavy metals in the Yangtze River estuary. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

Effect of sodium isopropylxanthate,potassium amylxanthate and sodium diethyldithiocarbamate on the uptake and distribution of cadmium in the brown trout (Salmo trutta)

Brown trout, Salmo trutta, were exposed to water containing 1 μg/l of ¹⁰⁹Cd²⁺, alone or with sodium isopropylxanthate, potassium amylxanthate or sodium diethyldithiocarbamate, respectively. After one week the uptake and distribution of the ¹⁰⁹Cd²⁺ in the fish were examined by whole-body autoradiography and gamma spectrometry. Sodium diethyldithiocarbamate was found to enhance the uptake of the ¹⁰⁹Cd²⁺ in several tissues of the fish and this effect increased with increasing concentration of the carbamate. Potassium amylxanthate induced increase in the levels of ¹⁰⁹Cd²⁺ in several tissues, whereas the brain was the only tissue with increased concentration of ¹⁰⁹Cd²⁺ in the presence of sodium isopropyl-xanthate. A likely mechanism for the enhanced uptake of the ¹⁰⁹Cd²⁺ may be a facilitated penetration over the gill membranes of the lipophilic complexes formed between the studied compounds and the cadmium. A facilitated passage through cellular membranes may also be important for the increased uptake of the metal in other tissues. An elevated uptake of cadmium by the xanthates or the diethyldithiocarbamate may constitute an increased risk for noxious effects of the metal.     

Side-specific effects by cadmium exposure: Apical and basolateral treatment in a coculture model of the blood-air barrier

Cadmium (Cd²⁺) is a widespread environmental pollutant, which is associated with a wide variety of cytotoxic and metabolic effects. Recent studies showed that intoxication with the heavy metal most importantly targets the integrity of the epithelial barrier.In our study, the lung epithelial cell line, NCI H441, was cultured with the endothelial cell line, ISO-HAS-1, as a bilayer on a 24-well HTS-Transwell® filter plate. This coculture model was exposed to various concentrations of CdCl₂.The transepithelial electrical resistance decreased on the apical side only after treatment with high Cd²⁺ concentrations after 48 h. By contrast, a breakdown of TER to less than 5% of baseline could be observed much earlier (after 24 h) when Cd²⁺ was administered from the basal side. Observations of cell layer fragmentation and widening of intercellular spaces confirmed the barrier breakdown only for the basolaterally treated samples. Furthermore, the cytotoxicity and release of proinflammatory markers was enhanced if samples were exposed to Cd²⁺ from the basal side compared to treatment from the apical side. Moreover, we could demonstrate that a high concentration of Ca²⁺ could prevent the barrier-disrupting effect of Cd²⁺.In conclusion, the exposure of Cd²⁺ to cocultures of lung cells caused a decrease in TER, major morphological changes, a reduction of cell viability and an increase of cytokine release, but the effects markedly differed between the two modes of exposure. Therefore, our results suggest that intact epithelial TJs may play a major role in protecting the air-blood barrier from inhaled Cd²⁺.     

Developmental toxicity and DNA damage to zebrafish induced by perfluorooctane sulfonate in the presence of ZnO nanoparticles

Perfluorooctane sulfonate (PFOS) and ZnO nanoparticles (ZnO–NPs) are frequently detected in the environment, but few studies have assessed their joint toxicity. In this study, the acute toxicity and chronic toxicity to zebrafish (Danio rerio) induced by PFOS in the presence of ZnO–NPs were investigated, including developmental toxicity and DNA damage. The embryos were exposed to PFOS (only) (0.4, 0.8, and 1.6 mg/L) and PFOS plus ZnO–NPs (0.4 + 50, 0.8 + 50, and 1.6 + 50 mg/L) solutions to evaluate mortality (96 h), body length (96 h), hatch rate (72 h), heart rate (48 h),and malformation rate (96 h). The results revealed that the co‐treatment could cause more severe developmental toxicity compared with the control and single‐treatments, and the toxic effects generally increased in a dose–response manner. In addition, adult zebrafish were exposed to single and mixed solutions of PFOS and ZnO–NPs (at the concentrations mentioned above) for 30 days. DNA damage to zebrafish was evaluated by the comet assay and micronucleus test. We found that the PFOS single‐treatment at all doses (0.4, 0.8, and 1.6 mg/L) could strongly induce DNA damage to peripheral blood cells and that ZnO–NPs could aggravate the formation of DNA damage in co‐treatments. Histological examination of liver, testicle, and ovary showed that the presence of ZnO–NPs (50 mg/L) could also cause more serious histological damage to adult zebrafish than PFOS alone. As a result, the synergistic effects played an important role during joint exposure. Our observations provide a basic understanding of the joint toxicity of PFOS and ZnO–NPs to aquatic organisms. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 360–371, 2016.     

Teratogenic effects and distribution of cadmium (Cd2+) administered via osmotic minipumps to gravid CF-1 mice

Studies to identify the mechanisms underlying the teratogenic effects of cadmium (Cd²⁺) have been complicated by the inherent difficulties of chronically and subchronically administering specific doses of Cd²⁺ to gravid animals under strictly controlled conditions. The objective of the present study was to develop a relatively simple animal model for examining the teratogenic effects of subchronic Cd²⁺ exposure. Cd²⁺ was administered to gravid CF-1 mice by subcutaneously implanted Alzet osmotic minipumps, which released fixed amounts of Cd²⁺ over a 14-day period between days 5 and 18 of gestation. The results showed that Cd²⁺ administered in this manner produced fetal anomalies and that the patterns of Cd²⁺ distribution and the specific developmental defects were similar to those that have been reported for other routes of Cd²⁺ administration. These findings indicate that osmotic minipumps may serve as useful tools in long-term studies of Cd²⁺ teratogenicity. They would appear to be especially useful in teratogenic evaluations where minimizing maternal stress and administering precise doses of Cd²⁺ are important.     

Axonal Transport of Cadmium in the Olfactory Nerve of the Pike

¹⁰⁹Cd²⁺ was applied in the olfactory chambers of pikes (Esox lucius) and the dynamics of the axoplasmic flow of the metal was determined in the olfactory nerves by gamma spectrometry and autoradiography. The results showed that the ¹⁰⁹Cd²⁺ is transported at a constant rate along the olfactory nerves. The profile of the ¹⁰⁹Cd²⁺ in the nerves showed a wave front of transported metal followed by a saddle region. When the nasal chambers were washed 2 hr after application of the ¹⁰⁹Cd²⁺ well-defined transport peaks for the metal were seen in the olfactory axons. The maximal velocity for the transport of ¹⁰⁹Cd²⁺, which corresponds to the movement of the wave front, was 2.38±0.10 mm hr (mean±S.E.) at the experimental temperature (10 C). The average velocity for the transport of the ¹⁰⁹Cd²⁺, which corresponds to the peak apex movement of the wave, was 2.18±0.05 mm/hr (mean±S.E.) at 10 C. The transported ¹⁰⁹Cd²⁺ was strongly accumulated in the anterior parts of the olfactory bulbs, whereas in other brain areas the levels of the metal remained low. Autoradiography of a pike exposed to ¹⁰⁹Cd²⁺ via the water showed a strong labelling in the receptor-cell-containing olfactory rosettes, whereas other structures in the olfactory chambers were only weakly labelled. The accumulation and axonal transport in the olfactory neurons may be noxious and constitute an important component in the toxicology of cadmium in fish, and this may apply also to some other heavy metals.