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.     

A representative retinoid X receptor antagonist UVI3003 induced teratogenesis in zebrafish embryos

Retinoid X receptor (RXR) interfering activity has been detected in different water resources. To study RXR disruptor‐induced toxicological effects on vertebrates, embryos of zebrafish (Danio rerio) were exposed to a representative RXR antagonist UVI3003. Results showed that the teratogenic index (LC₅₀/EC₅₀) of UVI3003 was as high as 5.4. UVI3003 induced multiple malformations of embryos, including deformed fins, reduced brains, small jaws, bent tails and edema in hearts, the degree of which became more severe with increasing exposure concentration. Although no significant difference was observed in the hatching rates between the exposure group and control, the whole body length was significantly reduced by 6.5% and 8.9% when exposed to 200 and 300 µg l^?1 of UVI3003, respectively. The heart rate also significantly decreased by 8.8–50.2% during exposure. Further experiments revealed that the pharyngula stage was the most sensitive development phase in terms of embryo response to UVI3003. The results demonstrated severe teratogenicity of RXR antagonist in zebrafish embryos and provided important data for ecotoxicological evaluation of RXR antagonists. Copyright © 2014 John Wiley & Sons, Ltd.     

Detection of exposure effects of mixtures of heavy polycyclic aromatic hydrocarbons in zebrafish embryos

In this study we evaluated the exposure effects of mixtures of five polycyclic aromatic hydrocarbons (PAHs); namely, benzo[a ]anthracene, benzo[a ]pyrene, benzo[b ]fluoranthene, benzo[k ]fluoranthene and chrysene on zebrafish embryos. Supplementation of the exposure media with 0.45% dimethyl sulfoxide and 50 ppm of Tween 20 could guarantee the solubilization and stabilization of the PAHs up to 24 h without affecting the embryos development. The exposure effects were tested by detecting the differential expression of a number of genes related to the aryl hydrocarbon receptor gene battery. Effects were detectable already after 6 h of exposure. After 24 h of exposure, all PAHs, except for benzo[a ]anthracene, acted as potent inducers of the gene cyp1a1 . Benzo[k ]fluoranthene was the major inducer; the effect caused by the mixture at the lower concentration tested (1 ng ml⁻¹) was dominated by its presence. However, in the mixture at the highest concentration tested (10 ng ml⁻¹) it caused less induction and was not dominant. No significant bioaccumulation values were detected on embryos exposed to the PAHs tested in this study; however, the results obtained, indicated that PAHs undergo a very rapid metabolization inside the embryos, and that those biotransformation products yield changes on the expression of genes involved in the aryl hydrocarbon receptor pathway. Future work should focus on identification of the PAH metabolization products and on the effect of these metabolites on toxicity. Copyright © 2016 John Wiley & Sons, Ltd.     

Particle size-dependent and surface charge-dependent biodistribution of gold nanoparticles after intravenous administration

Gold nanoparticles (GNP) provide many opportunities in imaging, diagnostics, and therapies of nanomedicine. Hence, their biokinetics in the body are prerequisites for specific tailoring of nanomedicinal applications and for a comprehensive risk assessment.We administered ¹⁹⁸Au-radio-labelled monodisperse, negatively charged GNP of five different sizes (1.4, 5, 18, 80, and 200 nm) and 2.8 nm GNP with opposite surface charges by intravenous injection into rats. After 24 h, the biodistribution of the GNP was quantitatively measured by gamma-spectrometry.The size and surface charge of GNP strongly determine the biodistribution. Most GNP accumulated in the liver increased from 50% of 1.4 nm GNP to >99% of 200 nm GNP. In contrast, there was little size-dependent accumulation of 18-200 nm GNP in most other organs. However, for GNP between 1.4 nm and 5 nm, the accumulation increased sharply with decreasing size; i.e. a linear increase with the volumetric specific surface area. The differently charged 2.8 nm GNP led to significantly different accumulations in several organs.We conclude that the alterations of accumulation in the various organs and tissues, depending on GNP size and surface charge, are mediated by dynamic protein binding and exchange. A better understanding of these mechanisms will improve drug delivery and dose estimates used in risk assessment.     

Acute and sub‐lethal exposure to copper oxide nanoparticles causes oxidative stress and teratogenicity in zebrafish embryos

Nano‐copper oxides are a versatile inorganic material. As a result of their versatility, the immense applications and usage end up in the environment causing a concern for the lifespan of various beings. The ambiguities surround globally on the toxic effects of copper oxide nanoparticles (CuO‐NPs). Hence, the present study endeavored to study the sub‐lethal acute exposure effects on the developing zebrafish embryos. The 48 hpf LC₅₀ value was about 64 ppm. Therefore, we have chosen the sub‐lethal dose of 40 and 60 ppm for the study. Accumulation of CuO‐NPs was evidenced from the SEM‐EDS and AAS analyzes. The alterations in the AChE and Na⁺/K⁺‐ATPase activities disrupted the development process. An increment in the levels of oxidants with a concomitant decrease in the antioxidant enzymes confirmed the induction of oxidative stress. Oxidative stress triggered apoptosis in the exposed embryos. Developmental anomalies were observed with CuO‐NPs exposure in addition to oxidative stress in the developing embryos. Decreased heart rate and hatching delay hindered the normal developmental processes. Our work has offered valuable data on the connection between oxidative stress and teratogenicity leading to lethality caused by CuO‐NPs. A further molecular mechanism unraveling the uncharted connection between oxidative stress and teratogenicity will aid in the safe use of CuO‐NPs. Copyright © 2015 John Wiley & Sons, Ltd.     

Systematic determination of the relationship between nanoparticle core diameter and toxicity for a series of structurally analogous gold nanoparticles in zebrafish

Abstract

Predictive models for the impact of nanomaterials on biological systems remain elusive. Although there is agreement that physicochemical properties (particle diameter, shape, surface chemistry, and core material) influence toxicity, there are limited and often contradictory, data relating structure to toxicity, even for core diameter. Given the importance of size in determining nanoscale properties, we aimed to address this data gap by examining the biological effects of a defined series of gold nanoparticles (AuNPs) on zebrafish embryos. Five AuNPs samples with narrowly spaced core diameters (0.8–5.8?nm) were synthesized and functionalized with positively charged N,N,N-trimethylammonium ethanethiol (TMAT) ligands. We assessed the bioactivity of these NPs in a high-throughput developmental zebrafish assay at eight concentrations (0.5–50?µg/mL) and observed core diameter-dependent bioactivity. The smaller diameter AuNPs were the most toxic when expressing exposures based on an equal mass. However, when expressing exposures based on total surface area, toxicity was independent of the core diameter. When holding the number of nanoparticles per volume constant (at 6.71?×?10¹³/mL) in the exposure medium across AuNPs diameters, only the 5.8?nm AuNPs exhibited toxic effects. Under these exposure conditions, the uptake of AuNPs in zebrafish was only weakly associated with core diameter, suggesting that differential uptake of TMAT-AuNPs was not responsible for toxicity associated with the 5.8?nm core diameter. Our results indicate that larger NPs may be the most toxic on a per particle basis and highlight the importance of using particle number and surface area, in addition to mass, when evaluating the size-dependent bioactivity of NPs.     

Proteomic analysis of ametryn toxicity in zebrafish embryos

Ametrym (AMT) is the most widely used herbicide and frequently detected in the aquatic environment. AMT also represent a potential health risk to aquatic organisms and animals, including humans. However, little data are available on their toxicity to zebrafish (Danio rerio). The aim of the present study was to evaluate the toxicological effects of AMT exposure on zebrafish embryos. In the acute toxicity test, 6 hpf embryos were exposed to various concentrations of AMT for 24 or 48 h. The results indicated that AMT induced malformation in larvae. To investigate the toxicological mechanism on the protein expression level. A proteomic approach was employed to investigate the proteome alterations of zebra fish embryos exposed to 20 mg/L AMT for 48 h. Among 2925 unique proteins identified, 298 differential proteins (> or <1.3‐fold, P < 0.05) were detected in the treated embryos as compared to the corresponding proteins in the untreated embryos. Gene ontology analysis showed that these up‐regulated proteins were most involved in glycolysis, lipid transport, protein polymerization, and nucleotide binding, and the down‐regulated proteins were related to microtubule‐based process, protein polymerization, oxygen transport. Moreover, KEGG pathway analysis indicated that tight junction, ribosome, and oxidative phosphorylation were inhibited in the treated embryos. These findings provide new insight into the mechanisms of toxicity induced by AMT.     

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.     

Comparative analysis of the toxicity of gold nanoparticles in zebrafish

The use of nanoparticles – particles that range in size from 1 to 100 nm – has become increasingly prevalent in recent years, bringing with it a variety of potential toxic effects. Zebrafish embryos were exposed during the 3 day postfertilization period to gold nanospheres (GNSs), gold nanorods (GNRs), GNRs coated with polystyrene sulphate (PSS‐GNRs) and GNRs coated with both PSS and polyallamine hydrochloride (PAH‐PSS‐GNRs). All nanorods were stabilized with cetyltrimethylammonium bromide. GNSs were the least toxic of the nanoparticles studied, with exposure resulting in no significant changes in mortality, hatching or heart rate. Exposure to GNRs and PSS‐GNRs resulted in significant increases in mortality and significant decreases in hatching and heart rate. Treatment with GNRs caused significant changes in the expression of a variety of oxidative stress genes. The toxic effects of GNRs were ameliorated by coating them with PSS and, to a more marked extent, with a double coating of PSS and polyallamine hydrochloride.     

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.     

佐匹克隆及其杂质对斑马鱼胚胎发育毒性的比较研究

目的 对佐匹克隆及其主要杂质的斑马鱼胚胎发育毒性进行比较研究.方法 以斑马鱼胚胎为实验对象,在体视光学显微镜下观察佐匹克隆及其主要杂质(杂质A、B、C和2-氨基-5-氯吡啶)在不同暴露浓度时对斑马鱼胚胎发育情况的影响,包括胚胎致畸、致死情况的检测与统计.结果 佐匹克隆及其4个杂质对斑马鱼胚胎发育的毒性表型类似;毒性作用...     

Developmental neurotoxicity and immunotoxicity induced by graphene oxide in zebrafish embryos

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

吡嗪酰胺对斑马鱼胚胎发育和氧化应激效应的影响

目的 本文研究吡嗪酰胺(PZA)对斑马鱼胚胎的发育毒性及其产生机制。方法 采用受精后4h的斑马鱼胚胎进行吡嗪酰胺不同浓度的暴露,分别于暴露后24、48、72和96h进行死亡率、孵化率的统计。给药96h后进行斑马鱼幼鱼形态评分、行为学分析、活性氧簇(ROS)和氧化应激指标检测。结果 表明吡嗪酰胺显著抑制斑马鱼胚胎孵化,导致斑马鱼幼鱼发育畸形(如卵黄囊吸收迟滞、鱼鳔缺失、心包水肿和体节模糊等),同时吡嗪酰胺的暴露造成斑马鱼幼鱼运动行为能力的降低。吡嗪酰胺的暴露能够引起斑马鱼体内ROS、总超氧化物歧化酶(T-SOD)、过氧化氢酶(CAT)、谷胱甘肽(GSH)和丙二醛(MDA)水平的变化。结论 吡嗪酰胺对斑马鱼胚胎有明显的发育毒性,并呈剂量和时间依赖性,氧化应激在吡嗪酰胺发育毒性中起着重要的作用。     

斑马鱼皮肤损伤模型的建立及分子机制

目的以皮肤荧光转基因斑马鱼(ktr4:NTR-h Kik GR)为实验动物,研究长春新碱对皮肤细胞的损伤作用及其分子机制,建立皮肤损伤模型。方法取受精后24 h皮肤荧光斑马鱼胚胎,将脱膜后的胚胎分别置于0.01~0.04 mmol·L^-1不同浓度的长春新碱中,24 h后采用荧光显像的方法对斑马鱼皮肤荧光斑点进行计数和统计处理;同时对鱼体细胞死亡情况进行TUNEL试剂盒检测,确定其皮肤细胞死亡的水平;利用蛋白免疫印迹的方法对各组斑马鱼胚胎的caspase-3、KRT4及p53表达水平进行检测。结果在长春新碱与斑马鱼胚胎共同孵育24 h后,0.02、0.04 mmol·L^-1长春新碱明显引起斑马鱼荧光皮肤细胞减少(P<0.01);TUNEL检测表明,长春新碱与对照组比较能明显引起斑马鱼皮肤细胞凋亡;Western bolt分析显示,0.02、0.04 mmol·L^-1长春新碱能诱导斑马鱼胚胎caspase-3和p53表达增加(P<0.01),同时高浓度的长春新碱可使皮肤角蛋白KRT4表达明显减少(P<0.01)。结论利用长春新碱引起斑马鱼皮肤损伤,该局部毒性作用可作为皮肤损伤的造模新方法。     

Using gold nanorods core/silver shell nanostructures as model material to probe biodistribution and toxic effects of silver nanoparticles in mice

The aim of this work was to probe the biodistribution and toxic effects of silver nanoparticles (NPs) with powerful anti-bacterial and anti-virus activities. For this purpose, novel silver NPs with gold nanorod (NR) core and silver shell (Au@Ag NRs) were developed and employed as a model material. The inner gold core provided an excellent internal reference for tracking the NRs in vivo. After subcutaneous injection of Au@Ag NRs, silver and gold contents in the subcutis and organs were examined by inductively coupled plasma mass spectrometry at different time points within 28 days. Histological analysis, physiological function and complement faction 3 (C3) and 5a (C5a) measurement were performed over time to reveal the toxic effect of Au@Ag NRs in vivo. Experimental results showed that majority of the Au@Ag NRs remained in the injection site except for a small amount migrating into the lymph nodes. The silver shell was dissolved in the subcutaneous tissue and released silver ions rapidly, which resulted in detectable silver accumulation in most of the organs. The accumulated silver ions in the kidney not only interacted with the kidney cells membrane but also induced a rapid increase of complement fraction C3 followed by a significant consumption and C3a and C5a production significantly in the serum, which resulted in kidney oxidative damage and eventually led to the morphological changes and filtration function impairment of the glomerulus. The released silver ions also caused oxidative injury of subcutaneous tissue in the injection site.     

Dissolution characteristics of cylindrical particles and tablets

In this paper, dissolution characteristics of primary-particles and compressed tablets were investigated by experiments using a mathematical model. For the primary-particle, it was found that the dissolution rate increased with a decrease in the particle size. Assuming that primary-particles of size distribution were of cylindrical shape and that the dissolution occurs from the total external surface, an extended Nernst–Noyes–Whitney equation fitted to the experimental data well. As the influences of particle size and shape on thickness of a diffusion-boundary film were found to be quite low, the dissolution rate was considered to be affected by the specific surface area dominantly. Furthermore, the same model was applied to a compressed tablet and fitted to the data well. Though the rate constant obtained were not affected by the properties of primary-particles forming the tablet, it was found to increase with the apparent voidage which occupies the inter-particle volume of tablet diluent among less soluble particles. Consequently, an increase in the apparent voidage is presumed to accelerate penetration of water into the internal voids of the tablet. Thus, the dissolution going, the effective surface area inside the tablet is considered to be extended.     

Evaluation of toxicological and teratogenic effects of carbosilane glucose glycodendrimers in zebrafish embryos and model rodent cell lines

Abstract

Glycodendrimers (Glyco-DDMs) represent a rapidly growing class of nanoparticles with promising properties for biomedical applications but concerns regarding the impact on human health and environment are still justified. Here we report, for the first time, the comparative study of in vivo developmental toxicity of carbosilane Glyco-DDMs and their cytotoxicity in vitro. Carbosilane Glyco-DDMs (generation 1–3) containing 4, 8, and 16 β-d-glucopyranosyl units at the periphery (DDM₁Glu, DDM₂Glu, and DDM₃Glu) were synthesized and characterized by ¹H, ¹³C and ²⁹Si NMR, mass spectrometry, dynamic light scattering, atomic force microscopy (AFM), and computer modeling. In vitro cytotoxicity assay (MTT) of DDM_1–3Glu was performed on three different rodent cell lines (Cricetulus griseus) – B14 (ATCC, CCL-14.1), BRL 3A (ATCC, CRL-1442), and NRK 52E (ATCC, CRL-1571). Overall, very low cytotoxicity was observed with calculated IC₅₀ in mM range with slight difference between each cell line and DDM generation investigated. Modified fish embryo test (FET) was further used for DDM₃Glu developmental toxicity testing on zebrafish (Danio rerio) embryos. While seemingly harmless to intact embryos, adverse effects of DDMs on the embryonic development become evident after chorion removal (LD₅₀=2.78?µM at 96 hpe). We summarized that the modified FET test showed a two to three orders of magnitude difference between the in vitro cytotoxicity and in vivo developmental toxicity of DDM₃Glu. While, in general, the Glyco-DDMs show great promises as efficient vectors in targeted drug delivery or as therapeutic molecules itself, we suggest that their developmental toxicity should be thoroughly investigated to exclude safety risks associated with their potential biomedical use.