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.     

斑马鱼胚胎发育毒性评价方法的建立

目的以致胚胎毒性阳性药物全反式维甲酸（ATRA）及丙戊酸钠进行斑马鱼胚胎发育毒性试验,建立有效的斑马鱼胚胎发育毒性评价方法。方法采用水浴染毒法,将受精后2h（2hpf）的斑马鱼胚胎暴露于不同浓度梯度的阳性约物。分别在24、48、72和144hpf观察并记录畸形及死亡的胚胎数目。统计阳性药物的EC50和LG50,计算致畸指数（TI=LC50／EG50）。结果两种阳性药物所致斑马鱼胚胎发育早期（24～48hpf）与发育后期（72～144hpf）的畸形表现不同。在72hpf,两种阳性药物对胚胎孵化率均有明显抑制作甩144hpf可见ATRA（≥1．6×10^-3mg／L）和丙戊酸钠（≥1．25×10^2mg／L）严晕致畸作用,T1分别为10.35和5．72。两种阳性药物胚胎敛畸率及死亡率均呈明显的浓度依赖关系,且与已有动物试验及体外试验结果相符。结论以两种阳性药物建立有效的斑马鱼胚胎发育毒性评价方法,可进行进一步的验证。     

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.     

Functional expressions of adenosine triphosphate‐binding cassette transporters during the development of zebrafish embryos and their effects on the detoxification of cadmium chloride and β‐naphthoflavone

Adenosine triphosphate‐binding cassette (ABC) transporters, including ABCB, ABCC and ABCG families represent general biological defenses against environmental toxicants in varieties of marine and freshwater organisms, but their physiological functions at differential developmental stages of zebrafish embryos remain undefined. In this work, functional expressions of typical ABC transporters including P‐glycoprotein (Pgp), multiresistance associated protein 1 (Mrp1) and Mrp2 were studied in zebrafish embryos at 4, 24, 48 and 72 h post‐fertilization (hpf). As a result, both the gene expressions and activities of Pgp and Mrps increased with the development of embryos. Correspondingly, 4–72 hpf embryos exhibited an increased tolerance to the toxicity caused by cadmium chloride (CdCl₂) and β‐naphthoflavone (BNF) with time. Such a correlation was assumed caused by the involvement of ABC transporters in the detoxification of chemicals. In addition, the assumption was supported by the fact that model efflux inhibitors of Pgp and Mrps such as reversine 205 and MK571 significantly inhibited the efflux of toxicants and increased the toxicity of Cd and BNF in zebrafish embryos. Moreover, exposure to CdCl₂ and BNF induced the gene expressions of Pgp and Mrp1 in 72 hpf embryos. Thus, functional expressions of Pgp and Mrps increased with the development of zebrafish embryos, which could cause an increasing tolerance of zebrafish embryos to CdCl₂ and BNF. Copyright © 2015 John Wiley & Sons, Ltd.     

Toxicity evaluation of β‐diketone antibiotics on the development of embryo‐larval zebrafish (Danio rerio)

This study evaluated the effects of β‐diketone antibiotics (DKAs) on the development of embryo‐larval zebrafish (Danio rerio). When exposure to DKAs, developmental malformations, such as hatching delay, curved body axis, pericardial edema, uninflated swim bladder and yolk sac edema, were observed at 120 h postfertilization (hpf). The estimated 120 hpf nominal concentrations of no observed effect concentration and lowest observed effect concentration for DKAs were 18.75 and 37.50 mg/L, respectively, suggesting that DKAs have much lower toxicity than other persistent pollutants. Following DKA exposure, embryonic heart rates were significantly reduced as compared to the controls at 48 and 60 hpf. The peak bending motion frequency appeared 1 h earlier than in control embryos. The 2.34 and 9.38‐mg/L treatment groups had a higher basal swim rate than control groups at 120 hpf in both light and light‐to‐dark photoperiod experiments. The occurrence of high speed swim rates was enhanced approximately threefold to sevenfold in the 2.34 and 9.38 mg/L treatments compared to the control. Glutathione (GSH) concentrations in the 2.34 and 9.38‐mg/L treatments were significantly higher than the control at 72 hpf, suggesting that GSH production was induced at the end of the hatching period. When exposed to DKAs, zebrafish superoxide dismutase enzyme (SOD) activities were significantly inhibited in the early embryonic period, demonstrating that the clearing ability in zebrafish was lower than the generation rate of free radicals. In summary, the combined DKAs were developmentally toxic to zebrafish in their early life stages and had the ability to impair individual behaviors that are of great importance in the assessment of their ecological fitness. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1134–1146, 2014.     

Developmental lead acetate exposure induces embryonic toxicity and memory deficit in adult zebrafish

Lead is a persistent metal and commonly present in our living environment. The present study was aimed to investigate lead-induced embryonic toxicity, behavioral responses, and adult learning/memory deficit in zebrafish. Lead acetate (PbAc) induced malformations such as uninflated swim bladder, bent spine and yolk-sac edema with an EC₅₀ of 0.29 mg/L at 120 h post fertilization (hpf). Spontaneous movement as characterized by tail bend frequency was significantly altered in zebrafish embryos following exposure to PbAc. Behavior assessment demonstrated that lead exposure changed behavioral responses in zebrafish larvae, as hyperactivity was detected within the first minute of light-to-dark transition in the fish exposed to PbAc from 6 to 96 hpf, and a different dose-dependent change was found in swimming speeds in the dark and in the light at 120 hpf following lead exposure. Learning/memory task assay showed that embryos exposed to PbAc from 6 to 120 hpf developed learning/memory deficit at adulthood as exhibited by a significant decrease in accuracy rate to find the food and a significant increase in finding time. Overall, our results suggested that low dose of developmental lead exposure resulted in embryonic toxicity, behavioral alteration, and adult learning/memory deficit in zebrafish.     

核酸染料Goodview、Gelred和Gelsafe对斑马鱼胚胎发育的毒性作用

目的研究核酸染料Goodview、Gelred和Gelsafe对斑马鱼胚胎发育的影响。方法将受精后时间（hpf）为3hpf的斑马鱼胚胎随机分组,分别加入0、0.01%、0.02%、0.04%、0.08%、0.16%和0.32%浓度的Goodview、Gelred和Gelsafe 3种核酸染料,观察3种核酸染料对斑马鱼胚胎发育的影响,记录胚胎48 hpf心率,计算72和96 hpf孵化率,96 hpf死亡率及畸形率;同时观察了4、24和48 hpf 3种染料穿透胚胎膜的情况。结果 Goodview、Gelred和Gelsafe 3种核酸染料半数致死浓度（LC50）分别为0.037%、0.092%和0.130%。3种核酸染料0.04%～0.32%浓度组的胚胎48 hpf心率、72和96 hpf孵化率、96hpf死亡率和畸形率与对照组相比都存在显著差异,3种核酸染料96 hpf死亡率、72和96 hpf孵化率还存在剂量-效应关系。Gelsafe 0.08%～0.32%浓度组部分幼鱼出现心囊水肿和脊柱弯曲,0.32%Gelred组染毒后还出现眼点发育不全,Goodview0.04～0.16%浓度组部分幼鱼除心囊水肿和脊柱弯曲外,还出现尾部发育不全等畸形表征。0.01%Goodview 4 hpf能穿透胚胎膜进入胚胎内,随着浓度和时间增加,胚胎内荧光增强。而Gelred和Gelsafe 0.32%浓度下4 hpf未发现明显荧光蓄积点,24hpf和48 hpf仍未发现。结论 3种核酸染料对斑马鱼胚胎发育有毒性,随着使用浓度的增加毒性增加。     

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

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.     

Effects of embryonic propofol exposure on axonal growth and locomotor activity in zebrafish

Prenatal propofol exposure induced neurotoxicity in the developing brains and led to persistent learning deficits in the offspring. Our goal was to use zebrafish to explore whether the decline in learning and memory was correlated with inhibition of neuronal growth after propofol exposure. Zebrafish embryos at 6 hours postfertilization (hpf) were exposed to control or 1, 2 or 4 μg/mL propofol until 48 hpf. Spontaneous locomotor activity and swimming behavior in response to dark-to-light photoperiod stimulation were studied in zebrafish larvae at 6 days postfertilization (dpf). The adaptability to repeated stimulation was used to indicate learning and memory function of larvae. Transgenic NBT line zebrafish was used to quantitate the effect of propofol on motor neuronal growth of embryos in vivo. Six dpf transgenic zebrafish larvae went through photoperiod stimulation after their neuronal length had been analyzed during the embryonic period. Our data indicate that embryonic exposure to 1, 2 and 4 μg/mL propofol had no adverse effect on spontaneous movement in zebrafish larvae, but 2 and 4 μg/mL propofol significantly impaired the learning and memory function of larvae. Moreover, propofol significantly inhibited axonal growth of motor neurons during the embryonic stage, which was correlated with learning and memory deficiency in larvae. Our findings demonstrate that the neuronal growth was correlated with learning and memory function, indicating the relevance of zebrafish as a new model to explore the mechanisms through which propofol induces long-term learning and memory impairment.     

Developmental toxicity of auranofin in zebrafish embryos

Auranofin (AF) is used in clinic for the treatment of rheumatoid arthritis, repurposing of AF as an anticancer drug has just finished a phase I/II clinical trial, but the developmental toxicity of AF remains obscure. This study focused on its developmental toxicity by using zebrafish embryos. Zebrafish embryos were exposed to different concentrations (1, 2.5, 5, 10 μm ) of AF from 2 h post‐fertilization (hpf) to 72 hpf. At 72 hpf, two major developmental defects caused by AF were found, namely severe pericardial edema and hypopigmentation, when embryos were exposed to concentrations higher than 2.5 μm . Biochemical detection of oxidative stress enzyme combined with expressions of a series of genes related to oxidative stress, cardiac, metal stress and pigment formation were subsequently tested. The superoxide dismutase activity was decreased while malondialdehyde content was accumulated by AF treatment. The expression of oxidative stress‐related genes (sod1 , gpx1a , gst ), pigment‐related genes (mitfb , trp‐1a ) and one metal stress‐related gene ctr1 were all decreased by AF exposure. The expressions of cardiac‐related genes (amhc , vmhc ) and one metal‐related gene hsp70 were found to be significantly upregulated by AF exposure. These findings indicated the potential developmental toxicity of AF on zebrafish early development. Copyright © 2016 John Wiley & Sons, Ltd.     

Exposure time to caffeine affects heartbeat and cell damage‐related gene expression of zebrafish Danio rerio embryos at early developmental stages

Caffeine is white crystalline xanthine alkaloid that is naturally found in some plants and can be produced synthetically. It has various biological effects, especially during pregnancy and lactation. We studied the effect of caffeine on heartbeat, survival and the expression of cell damage related genes, including oxidative stress (HSP70), mitochondrial metabolism (Cyclin G1) and apoptosis (Bax and Bcl2), at early developmental stages of zebrafish embryos. We used 100 µm concentration based on the absence of locomotor effects. Neither significant mortality nor morphological changes were detected. We monitored hatching at 48 h post‐fertilization (hpf) to 96 hpf. At 60 and 72 hpf, hatching decreased significantly (P < 0.05); however, the overall hatching rate at 96 hpf was 94% in control and 93% in caffeine treatment with no significant difference (P > 0.05). Heartbeats per minute were 110, 110 and 112 in control at 48, 72 and 96 hpf, respectively. Caffeine significantly increased heartbeat – 122 and 136 at 72 and 96 hpf, respectively. Quantitative RT‐PCR showed significant up‐regulation after caffeine exposure in HSP70 at 72 hpf; in Cyclin G1 at 24, 48 and 72 hpf; and in Bax at 48 and 72 hpf. Significant down‐regulation was found in Bcl2 at 48 and 72 hpf. The Bax/Bcl2 ratio increased significantly at 48 and 72 hpf. We conclude that increasing exposure time to caffeine stimulates oxidative stress and may trigger apoptosis via a mitochondrial‐dependent pathway. Also caffeine increases heartbeat from early phases of development without affecting the morphology and survival but delays hatching. Use of caffeine during pregnancy and lactation may harm the fetus by affecting the expression of cell‐damage related genes. Copyright © 2012 John Wiley & Sons, Ltd.     

Gold nanorods induce early embryonic developmental delay and lethality in zebrafish (Danio rerio)

ABSTRACT

Due to their unique electronic and optical features, gold nanoparticles (AuNP) have received a great deal of attention for application in different fields such as catalysis, electronics, and biomedicine. The large-volume manufacturing predicted for future decades and the inevitable release of these substances into the environment necessitated an assessment of potential adverse human and ecological risks due to exposure to AuNP. Accordingly, this study aimed to examine the acute and developmental toxicity attributed to a commercial suspension of Au nanorods stabilized with cetyltrimethylammonium bromide (CTAB-AuNR) using early embryonic stages of zebrafish (Danio rerio), a well-established model in ecotoxicology. Zebrafish embryos were exposed to CTAB-AuNR (0–150 µg/L) to determine for developmental assessment until 96 hr post fertilization (hpf) and lethality. Uptake of CTAB-AuNR by embryos and nanoparticles potential to induce DNA damage was also measured at 48 and 96 hpf. Analysis of the concentration-response curves with cumulative mortality at 96 hpf revealed a median lethal concentration (LC_50,96h) of 110.2 μg/L. At sublethal concentrations, CTAB-AuNR suspensions were found to produce developmental abnormalities such as tail deformities, pericardial edema, decreased body length, and delayed eye, head, and tail elongation development. Further, less than 1% of the initial concentration of CTAB-AuNR present in the exposure media was internalized by zebrafish embryos prior to (48 hpf) and after hatching (96 hpf). In addition, no marked DNA damage was detected in embryos after exposure to CTAB-AuNR. Overall, CTAB-AuNR suspensions produced lethal and sublethal effects on zebrafish embryos with possible repercussions in fitness of adult stages. However, these results foresee a low risk for fish since the observed effects occurred at concentrations above the levels expected to find in the aquatic environment.     

Investigation into the sub‐lethal effects of the triazole fungicide triticonazole in zebrafish (Danio rerio) embryos/larvae

Global use of azole fungicides is expected to increase over the next several years. Triticonazole is a triazole fungicide that is used for turf protection, residential, and other commercial applications. As such, it can enter local rural and urban water systems via run‐off and rain events. Early life stages of aquatic organisms can be susceptible to pesticides that enter the water, but in the case of triticonazole, data on the potential for subacute toxicity are lacking. Here, we determined the effects of triticonazole on development, oxygen consumption rates, and locomotor activity in zebrafish to address this knowledge gap. Wild‐type zebrafish (ABTu strain) embryos and larvae were exposed to triticonazole (1‐100 μM) in early development for different lengths of time depending on the assay conducted. Triticonazole did not affect survival nor induce significant deformity (pericardial edema, skeletal defects) in zebrafish at doses up to 100 μM. Oxygen consumption rate was measured in embryos after 24 and 48 hour exposure to triticonazole beginning at ～6 hpf using the XF_e flux analyzer. Triticonazole did not affect basal respiration, oligomycin‐induced ATP linked respiration, FCCP‐induced maximum respiration, proton leak, spare capacity, nor non‐mitochondrial respiration at doses up to 100 μM for 24 hours, even for exposure up to 250 μM for 48 hours. To determine whether the fungicide affected larval swimming activity, the visual motor response test was conducted following triticonazole exposure for 6 days. Larval zebrafish exposed to triticonazole showed hypoactivity in the dark following a 100 μM treatment, suggesting that the fungicide can affect the locomotor activity of zebrafish, albeit at relatively high levels. Given the fact that sublethal biological responses were absent at lower environmentally relevant concentrations, we conclude that triticonazole, relative to other triazole fungicides and types of pesticides, exhibits a relatively low risk of toxicity to the early life stages of fish.     

MicroRNA expression changes during zebrafish development induced by perfluorooctane sulfonate

Perfluorooctane sulfonate (PFOS), a kind of widely distributed environmentally organic compound, has been found to cause developmental toxicity. Although microRNAs (miRNAs) play an important role in many metabolic tasks, whether and how they are involved in the process of PFOS‐induced toxicity is largely unknown. To address this problem, PFOS‐induced changes in miRNAs and target gene expression in zebrafish embryos, and the potential mechanism of PFOS‐induced toxic action were studied in this research. Zebrafish embryos were exposed to 1 µg ml^?1 PFOS or DMSO control from 6 h post‐fertilization (hpf) to 24 or 120 hpf. Subsequently, RNA was isolated from the embryo pool and the expression profiles of 219 known zebrafish miRNAs were analyzed using microarray. Finally, quantitative real‐time polymerase chain reaction was used to validate several miRNAs expression of microarray data. The analysis revealed that PFOS exposure induced significant changes in miRNA expression profiles. A total of 39 and 81 miRNAs showed significantly altered expression patterns after PFOS exposure 24 and 120 hpf. Of the changed miRNAs, 20 were significantly up‐regulated and 19 were significantly down‐regulated (p < 0.01) at 24 hpf, whereas 41 were significantly up‐regulated and 40 were significantly down‐regulated (p < 0.01) at 120 hpf. These miRNAs were involved in development, apoptosis and cell signal pathway, cell cycle progression and proliferation, oncogenesis, adipose metabolism and hormone secretion, whereas there is still little functional information available for 32 miRNAs. Our results demonstrate that PFOS exposure alters the expression of a suite of miRNAs and may induce developmental toxicity. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

Toxic effects of celastrol on embryonic development of zebrafish (Danio rerio)

Celastrol is a terpenoid purified from Tripterygium wilfordii Hook F. As a natural product with pharmacological activities, this compound is a promising candidate for drug development. To provide more information about its toxicity for clinical trials, toxicity assessment of celastrol was conducted with zebrafish model in vivo. 1hour post-fertilization (hpf) embryos were treated with various concentrations of celastrol for 120h. Developmental phenotypes were observed and survival rates were recorded. The results showed that the hatching rates of embryos treated with 1.0μM or higher celastrol were significantly lower. Embryos exposed to 1.0μM celastrol had no blood flow in trunk vessels at 48hpf with a median effect concentration (EC(50)) of 0.94μM. At 72hpf serious edema in pericardial sac was observed in the surviving larvae (hatched from embryos treated with 1.5μM celastrol). Bent tails or hook-like tails were seen as 0.5μM celastrol and the EC(50) for tail malformation was 0.66 μM at 72hpf. The lethal effect of celastrol on zebrafish embryos was dose-dependent and the LC(50) values of celastrol on 1hpf embryos were approximately 1.40μM. These results indicate that celastrol affects the normal development of zebrafish embryo in μM concentrations.     

Developmental toxicity and alteration of gene expression in zebrafish embryos exposed to PFOS

Perfluorooctanesulfonate (PFOS) is a persistent organic pollutant, the potential toxicity of which is causing great concern. In the present study, we employed zebrafish embryos to investigate the developmental toxicity of this compound. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to 0.1, 0.5, 1, 3 and 5 mg/L PFOS. Hatching was delayed and hatching rates as well as larval survivorship were significantly reduced after the embryos were exposed to 1, 3 and 5 mg/L PFOS until 132 hpf. The fry displayed gross developmental malformations, including epiboly deformities, hypopigmentation, yolk sac edema, tail and heart malformations and spinal curvature upon exposure to PFOS concentrations of 1 mg/L or greater. Growth (body length) was significantly reduced in the 3 and 5 mg/L PFOS-treated groups. To test whether developmental malformation was mediated via apoptosis, flow cytometry analysis of DNA content, acridine orange staining and TUNEL assay was used. These techniques indicated that more apoptotic cells were present in the PFOS-treated embryos than in the control embryos. Certain genes related to cell apoptosis, p53 and Bax, were both significantly up-regulated upon exposure to all the concentrations tested. In addition, we investigated the effects of PFOS on marker genes related to early thyroid development (hhex and pax8) and genes regulating the balance of androgens and estrogens (cyp19a and cyp19b). For thyroid development, the expression of hhex was significantly up-regulated at all concentrations tested, whereas pax8 expression was significantly up-regulated only upon exposure to lower concentrations of PFOS (0.1, 0.5, 1 mg/L). The expression of cyp19a and of cyp19b was significantly down-regulated at all exposure concentrations. The overall results indicated that zebrafish embryos constitute a reliable model for testing the developmental toxicity of PFOS, and the gene expression patterns in the embryos were able to reveal some potential mechanisms of developmental toxicity.     

Early life stage and genetic toxicity of stannous chloride on zebrafish embryos and adults: Toxic effects of tin on zebrafish

Humans are exposed to stannous chloride (SnCl₂), known as tin chloride, present in packaged food, soft drinks, biocides, dentifrices, etc. Health effects in children exposed to tin and tin compounds have not been investigated yet. Therefore, we evaluated the possible teratogenic effects and genotoxic of SnCl₂ in zebrafish (Danio rerio) adults and their embryos. In the embryo–larval study, SnCl₂ showed embryo toxicity and developmental delay after exposure to the various concentrations of 10–250 μM for 120 h. Teratogenic effects including morphological malformations of the embryos and larvae were observed. The embryos exposed to 100 μM displayed tail deformation at 28 hpf and the larvae exposed to 50 μM showed reduced body growth, smaller head and eyes, bent trunk, mild pericardial edema, and smaller caudal fin at 96 hpf. The results of the teratological study show that SnCl₂ induced a significant decrease in the number of living embryos and larvae. Regarding the chromosome analysis, SnCl₂ induced a dose‐dependent increase in the micronucleus (MN) frequency in peripheral erythrocytes of adult zebrafish. In blood cells, the 25 μM dose of SnCl₂ caused a nonsignificant increase in the total chromosomal aberrations, but the high doses significantly increased the total number of chromosomal aberrations compared with the control groups. Overall, the results clearly indicate that SnCl₂ is teratogenic and genotoxic to zebrafish. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2011.