Ethanol-dependent toxicity in zebrafish is partially attenuated by antioxidants

Ethanol is a well-established developmental toxicant; however, the molecular and cellular mechanism(s) of toxicity remains unclear. It has been suggested that ethanol metabolism leads to oxidative stress resulting in an increase in cell death. Alcohol developmental toxicity has not been well studied in zebrafish; however, zebrafish represent an excellent vertebrate model for investigating and understanding normal and aberrant development. To evaluate ethanol metabolism dependent toxicity, chemical inhibitors of the ethanol metabolizing enzymes were utilized. Embryos co-exposed to ethanol and a combination of ethanol metabolism inhibitors led to a significant increase in the occurrence of pericardial edema. Further, in the presence of the inhibitor mixture there was an increase in developmental malformations at lower ethanol concentrations. Cell death has been implicated as a potential explanation for ethanol-dependent toxicity. Using cell death assays, ethanol significantly increased embryonic cell death. To determine if oxidative stress underlies cardiovascular dysfunction, embryos were co-exposed to ethanol and several antioxidants. The antioxidants, glutathione and lipoic acid, partially attenuated the incidence of pericardial edema. The effectiveness of the antioxidants to protect the embryos from ethanol-induced cell death was also evaluated. The antioxidants provided no protection against cell death. Thus, ethanol-mediated pericardial edema and cell death appear to be mechanistically distinct.     

乙醇对胚胎发育与卵黄囊超微结构的影响

为研究发育敏感期暴露于乙醇不同时间对胚胎发育和胚胎卵黄囊超微结构的影响 .以体外全胚胎培养和电镜技术研究 0 .2g·L- 1乙醇不同时间作用时对胚胎发育影响及胚胎卵黄囊超微结构改变 .结果表明发育异常与乙醇作用存在时间 效应关系 .0 .2g·L- 18h对胚胎发育和形态分化无明显影响 ,随时间延长除胚胎头长 ,体长 ,卵黄囊直径 ,蛋白质和DNA含量等主要发育指标进一步受抑制外 ,胚胎畸形 ,死亡率明显升高 .卵黄囊超微结构变化程度与染毒时间和胚胎发育状况相一致 ,乙醇可导致卵黄囊细胞内微绒毛和溶酶体数量减少 ,线粒体等部分细胞器内膜肿胀 .上述结果提示卵黄囊结构损伤和破坏在乙醇所致的发育异常中起重要作用     

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.     

Ethanol exposure alters zebrafish development: a novel model of fetal alcohol syndrome

Prenatal exposure to alcohol has been shown to produce the overt physical and behavioral symptoms known as fetal alcohol syndrome (FAS) in humans. Also, it is believed that low concentrations and/or short durations of alcohol exposure can produce more subtle effects. The purpose of this study was to investigate the effects of embryonic ethanol exposure on the zebrafish (Danio rerio) in order to determine whether this species is a viable animal model for studying FAS. Fertilized embryos were reared in varying concentrations of ethanol (1.5% and 2.9%) and exposure times (e.g., 0–8, 6–24, 12–24, and 48–72 h postfertilization; hpf); anatomical measures including eye diameter and heart rate were compared across groups. Results found that at the highest concentration of ethanol (2.9%), there were more abnormal physical distortions and significantly higher mortality rates than any other group. Embryos exposed to ethanol for a shorter duration period (0–8 hpf) at a concentration of 1.5% exhibited more subtle effects such as significantly smaller eye diameter and lower heart rate than controls. These results indicate that embryonic alcohol exposure affects external and internal physical development and that the severity of these effects is a function of both the amount of ethanol and the timing of ethanol exposure. Thus, the zebrafish represents a useful model for examining basic questions about the effects of embryonic exposure to ethanol on development.     

Zebrafish developmental toxicity assay: A fishy solution to reproductive toxicity screening, or just a red herring?

The zebrafish embryotoxicity/teratogenicity assay is described as a useful alternative screening model to evaluate the effect of drugs on embryofoetal development.Fertilized eggs were exposed to different concentrations of 15 compounds with teratogenic (8) and non-teratogenic (7) potential until 96 h post-fertilization when 28 morphological endpoints and the level of compound uptake was assessed.The majority of drugs testing positive in mammals was also positive in zebrafish (75% sensitivity), while a relative high number of false positives were noted (43% specificity). Compound uptake determination appears useful for clarifying classifications as teratogenic or potential overdose although assay sensitivity could be improved to 71% if the exposure threshold, previously suggested as ∼50 ng/larvae, is reconsidered.The zebrafish assay shows some potential, though limited in its current form, as a screening tool for developmental toxicity within Janssen drug development. Further assay refinement with respect to endpoints and body burden threshold is required.     

Strain-dependent effects of developmental ethanol exposure in zebrafish

Developmental ethanol exposure from maternal consumption of alcoholic beverages and many other consumer products has been linked to developmental abnormalities in humans and animal models. The sensitivity of an individual to ethanol-induced perturbation of developmental processes is strongly influenced by genetic factors. In this study, we show that there are strain- and dose-dependent differences in sensitivity to developmental ethanol exposure in zebrafish (Danio rerio), suggesting that genetic variation within regulatory factors, influencing critical developmental pathways, is responsible for these differences. Embryos/larvae from genetically distinct strains of zebrafish [Ekkwill (EK), AB, and Tuebingen (TU)] were treated with different concentrations of ethanol. Embryo/larval survival, neurocranial and craniofacial skeletal development, and CNS cell death were analyzed. EK was the most resistant strain to the embryolethal effects of ethanol exposure but had the greatest increase in ethanol-induced cell death. AB survival was affected moderately, as were the neurocranial and craniofacial skeletal structures and ethanol-induced cell death. TU had the lowest survival rate but was the most resistant to alterations in neurocranial and craniofacial skeletal elements. No single strain is the most sensitive or the most resistant to any of the phenotypes examined, suggesting that alcohol influences each of these pathways independently. Further analysis of the molecular and biochemical pathways underlying the strain-dependent differences reported herein could lead to a significant advancement in our mechanistic understanding of the teratogenic effects of ethanol in humans.     

Uptake and elimination of ethanol by young zebrafish embryos

Among animal models being explored to understand ethanol-induced teratogenesis, the zebrafish (Danio rerio) is attracting attention because its embryonic development is well characterized and readily visualized. Despite the potential of the zebrafish embryo in research on developmental anomalies produced by ethanol exposure, little is known about the relationship between embryonic ethanol content and the nature/severity of ethanol-mediated deficits. Here, using gas chromatography and radiometry of labeled ethanol carbon, we examine accumulation and clearance of ethanol by dechorionated zebrafish embryos during blastulation/gastrulation. Our data indicate that: (a) rates of uptake and loss of ethanol are directly proportional to the extra-/intra-embryonic ethanol concentration gradient and (b) ethanol in the water fraction of embryos reaches near equimolarity with ethanol in the exposure medium. It appears that, within a wide range of exposure concentrations, embryonic ethanol content can be predicted accurately according to exposure time. Furthermore, it appears that embryonic ethanol can be adjusted rapidly to and maintained at a targeted concentration.     

Evaluation of the developmental toxicities of ethanol,acetaldehyde, and thioacetamide using FETAX

Potential mechanisms of the developmental toxicities of ethanol, acetaldehyde, and thioacetamide were evaluated using frog embryo teratogenesis assay-Xenopus (FETAX). Early X. laevis embryos were exposed to ethanol and thioacetamide in two separate definitive concentration-response tests with and without differentially induced exogenous metabolic activation systems (MAS) or selectively inhibited MAS. Two concentration-response tests were also performed with ethanol metabolites, acetaldehyde and acetic acid. The MAS was treated with 3,4-amino-1,2,4-triazole to modulate CYP2E1 activity, and heat to inactivate flavin containing monooxygenases (FMO) activity. Results from these studies suggested that thioacetamide may be bioactivated by both CYP2E1 and the FMO systems. Ethanol also appeared to be bioactivated by CYP2E1. Acetaldehyde was markedly more potent as a developmental toxicant than ethanol or acetic acid. Binary joint mixture studies conducted with ethanol and acetaldehyde indicated that the parent compound and metabolite acetaldehyde acted in a response additive manner. These results warrant the continued use of FETAX as a means of evaluating mechanisms of developmental toxicity in vitro.     

乙醇及乙醛对胚胎发育的联合作用

为探讨乙醇及其代谢产物乙醛在胎儿酒精综合征 (FAS)中的作用 ,应用植入后全胚胎培养研究不同浓度的乙醇 ,乙醛组合对 9.5- 1 1 .5d大鼠胚胎发育的联合作用 .结果显示在卵黄囊直径 ,头长 ,体长 ,心脏 ,前脑 ,DNA含量等指标乙醇与乙醛存在交互作用 .进一步分析发现 0 .1 g·L-1乙醇与不同剂量乙醛组合可导致拮抗 ,协同和独立等多种联合作用类型 ,但对头长和脑均表现为独立作用 . 0 .2 g· L-1乙醇与不同剂量乙醛组合除心脏呈协同作用外其余均为乙醇独立作用 .上述结果提示 FAS中的脑发育异常是乙醇独立作用的结果 ,乙醛则引起FAS中的辅助畸形     

Embryonic ethanol exposure alters synaptic properties at zebrafish neuromuscular junctions

Pre-natal alcohol exposure induces delays in fine and gross motor skills, and deficiencies in reflex development via mechanisms that remain to be elucidated. The purpose of the present study was to investigate the effect of embryonic ethanol exposure (16-hour exposure window with1.5%, 2% or 2.5% EtOH) on synaptic properties at the neuromuscular junction (NMJ) in 3 day post fertilization (dpf) zebrafish larvae. Immunohistochemical studies show that exposure of embryos to 2.5% ethanol for 16 h results in motor neuron axons that display abnormal branching patterns. Co-labelling embryos with pre-synaptic markers such as SV-2 or 3A10, and the post-synaptic marker, α-bungarotoxin, which irreversibly binds to nicotinic acetylcholine receptors (nAChRs), indicates that pre- and post-synaptic sites are properly aligned even when motor neuron axons display abnormal morphology. Miniature endplate currents (mEPCs) recorded from muscle fibers revealed the presence of two types of mEPCs that we dubbed fast and slow. Ethanol treated fish experienced significant changes in the frequencies of fast and slow mEPCs, and an increase in the rise time of slow mEPCs recorded from red muscle fibers. Additionally, embryonic exposure to ethanol resulted in a significant increase in the decay time of fast mEPCs recorded from white fibers. Mean mEPC amplitude was unaffected by ethanol treatment. Together, these results indicate that zebrafish embryos exposed to ethanol may experience altered synaptic properties at the NMJ.     

The role of development and duration of exposure to the embryotoxicity of diazinon

Medaka, Oryzias latipes, were used as a laboratory surrogate for species of concern to define the effects of diazinon exposure on teleost embryogenesis. Medaka embryos were placed in a static, non-renewal system and exposures initiated on days 1, 3, or 5 of development. Following initiation of exposure, replicates (n=5) remained in diazinon for a total of 4 days or from the day of initiation to day 9 of development. This exposure scenario was designed to elucidate sensitive periods in development for diazinon-induced toxicity but also shows the effect of added exposure duration on the degree of toxicity. Embryos were observed daily and endpoints recorded included: edema formation, total hatch, mean day of hatch, percentage of larvae with swim bladder inflation, and total length of larvae on day 14, when observations were terminated. Diazinon exposure resulted in decreases in hatch success, swim bladder inflation and the total length of larvae. In addition, dose-response increases in the incidence of edemas of the pericardial sac and vitelline veins were recorded. As expected, severity of embryotoxicity was positively correlated with duration of exposure. While no developmental period was the most sensitive for all toxic effects, for certain endpoints the severity of effects was dependent on exposure timing. Total hatch was greatly affected in embryos exposed from day 1 until day 5 whereas edema was more prevalent in embryos exposed later in development. Finally, among endpoints recorded, total length of larvae was the most sensitive indicator of exposure with all exposure groups showing significant (P<0.05) decreases in length at 5 ppm.     

Arsenic impairs embryo development via down-regulating Dvr1 expression in zebrafish

Exposure to environmental inorganic arsenic compounds has serious health effects on humans, including cancer, cardiovascular disease, developmental and reproductive problems. Our previous study showed that arsenic exposure caused various signs of toxicity in early stages of zebrafish development, including cardiac and neural system, such as pericardium edema, circulation failure, heart malformation. However, how arsenic exerts these effects is little known. Here, real-time quantitative RT-PCR and whole-mount in situ hybridization data showed that zebrafish Dvr1, a mammalian homolog of GDF1 related to the formation of left-right axis, was significantly down-regulated in embryos exposed to arsenite. Embryos with Dvr1 knockdown by antisense morpholino displayed abnormal development, including pericardium edema and failed looping, which is similar to the defects phenotype with arsenic treatment. Furthermore, overexpression of GDF1 significantly rescued development anomalies caused by morpholino or arsenite. Taken together, our results indicated for the first time that Dvr1 played an important role in the left-right asymmetry establishment of zebrafish embryo, and Dvr1 was involved in arsenic-mediated embryo toxicity, which gives novel insight into molecular mechanism of arsenic-mediated embryo toxicity.     

Effects of quillaja saponin (Quillaja saponaria) on early embryonic zebrafish (Danio rerio) development

Although much attention has focused on environmental contamination by heavy metals, pesticides, and polychlorinated biphenyls, potential deleterious effects of naturally occurring organic compounds have received much less consideration. Saponins, which are glycosides found in many plants, are important, environmentally ubiquitous organic compounds. Saponins have both beneficial and deleterious effects in adults, but little is known about how saponins effect early vertebrate embryonic development. The authors tested the toxicity of quillaja saponin using a zebrafish embryo assay. Quillaja saponin, extracted from bark of the tree, Quillaja saponaria, is a common foaming agent used in foods and beverages. At 6 h post fertilization, zebrafish embryos were exposed to five concentrations (0 [negative control], 1, 5, 10 or 20 micro g) of quillaja saponin per milliliter of medium. Zebrafish embryos exposed to 2% ethanol were positive controls (100% embryonic death). Embryos were assessed at 30, 54, and 72 h post fertilization for changes in embryonic development, mortality, time of hatching, and morphological deformities. Embryos exposed to 1 and 5 micro g saponin were healthy, showed no obvious deformities, but exhibited shrinkage of the chorion. Hatching time for zebrafish embryos exposed to 1 and 5 micro g/ml saponin decreased by 18 h compared to unexposed embryos. Zebrafish embryos treated with 5 micro g/ml saponin responded less to touch than embryos treated with 1 micro g/ml saponin or controls. Zebrafish embryos exposed to more than 5 micro g/ml saponin exhibited 100% embryonic mortality. These results indicate that exposure to 5 micro g/ml or less of quillaja saponin acts as a growth promoter, whereas concentrations of 10 micro g/ml or greater are lethal.     

Ethanol effects on the developing zebrafish: neurobehavior and skeletal morphogenesis

Exposure to ethanol during development can lead to a constellation of congenital anomalies, resulting in prenatal and postnatal failure to thrive, central nervous system (CNS) deficits, and a number of patterning defects that lead to defects in the cardiovascular system, facial structures, and limbs. The cellular, biochemical, and molecular mechanisms by which ethanol exerts its developmental toxicity and the genes that influence sensitivity to developmental ethanol exposure have yet to be discovered, despite being one of the more common nongenetic causes of birth defects. The zebrafish undergoes much the same patterning and morphogenesis as other vertebrate embryos do—including humans—that are distinct and cannot be studied in invertebrates. Developmental processes in zebrafish are affected by ethanol exposure in a dose-dependent manner, resulting in learning and memory deficits, cell death in the CNS, skeletal dysmorphogenesis, and alterations in startle reflex responses. Interestingly, significant ethanol effects on learning and behavioral endpoints occurred at concentrations well below those that induced cell death in the CNS. This work provides the foundation for identifying genes and pathways involved in developmental alcohol toxicity in vertebrates, leading to a more complete mechanistic understanding of fetal alcohol disorders in humans.     

Ethanol and acetaldehyde disturb TNF-alpha and IL-6 production in cultured astrocytes

Ethanol disturbs astroglial growth and differentiation and causes functional alterations. Furthermore, many signalling molecules produced by astrocytes contribute to these processes. The aim of the present study was to investigate the influence of ethanol and its primary metabolite, acetaldehyde, on TNF-alpha and IL-6 production in a rat cortical astrocyte primary culture. We are the first to report that both ethanol and acetaldehyde can modulate TNF-alpha and IL-6 secretion from cultured astrocytes. Long-term exposure (7 days) to ethanol and acetaldehyde was more toxic than an acute (24 hours) exposure. However, both compounds showed a biphasic, hormestic effect on the IL-6 secretion after the acute as well as the long-term exposure, and the maximum stimulation was reached for 50-mM ethanol and 1-mM acetaldehyde after 7-day exposure. In contrast, both compounds reduced the TNF-alpha secretion, where the effect was concentration-dependent. The catalase inhibitor 2-amino-1,2,4 triazole significantly reduced the ethanol toxicity in the cultured astrocytes after the acute as well as the long-term exposure. In conclusion, both ethanol and acetaldehyde affect the production of IL-6 and TNF-alpha in cultured astrocytes. The effect depends on the concentration of the compounds and the duration of the exposure. Acetaldehyde is a more potent toxin than ethanol, and ethanol's toxicity in the brain is at least partially due to its primary metabolite, acetaldehyde.     

Neurochemical and Behavioral Consequences of Ethanol and/or Caffeine Exposure: Effects in Zebrafish and Rodents

Zebrafish are increasingly being utilized to model the behavioral and neurochemical effects of pharmaceuticals and, more recently, pharmaceutical interactions. Zebrafish models of stress establish that both caffeine and ethanol influence anxiety, though few studies have implemented co-administration to assess the interaction of anxiety and reward-seeking. Caffeine exposure in zebrafish is teratogenic, causing developmental abnormalities in the cardiovascular, neuromuscular, and nervous systems of embryos and larvae. Ethanol is also a teratogen and, as an anxiolytic substance, may be able to offset the anxiogenic effects of caffeine. Co-exposure to caffeine and alcohol impacts neuroanatomy and behavior in adolescent animal models, suggesting stimulant substances may moderate the impact of alcohol on neural circuit development. Here, we review the literature describing neuropharmacological and behavioral consequences of caffeine and/or alcohol exposure in the zebrafish model, focusing on neurochemistry, locomotor effects, and behavioral assessments of stress/anxiety as reported in adolescent/juvenile and adult animals. The purpose of this review is twofold: (1) describe the work in zebrafish documenting the effects of ethanol and/or caffeine exposure and (2) compare these zebrafish studies with comparable experiments in rodents. We focus on specific neurochemical pathways (dopamine, serotonin, adenosine, GABA), anxiety-type behaviors (assessed with a novel tank, thigmotaxis, shoaling), and locomotor changes resulting from both individual and co-exposure. We compare findings in zebrafish with those in rodent models, revealing similarities across species and identifying conservation of mechanisms that potentially reinforce co-addiction.     

Food colorant Sunset Yellow (E110) intervenes developmental profile of zebrafish (Danio rerio)

In this study, we tested the teratogenic/embryotoxic potentials of food colorant, Sunset Yellow (E110) using zebrafish embryos as a model. Laboratory‐raised developing embryos of Danio rerio were exposed to graded concentrations (00, 0.1, 1.0, 2.0, 3.0, 4.0, 5.0, 10, 20, 30, 40, 50 and 100 mm ) of E110 from gastrulation stage (~6 hours post‐fertilization [hpf]) up until hatching. The developmental trajectory of each embryo and post‐hatched larva was traced from 24 to 168 hpf. The no observed effect concentration (NOEC), median effective concentration (EC₅₀), median lethal concentration (LC₅₀) and teratogenic index were determined. In the 0.1 mm E110‐exposed embryos, the development proceeded as in controls (NOEC), while, exposure of embryos to 1‐5 mm of E110 led to a decrease in body size, dry body mass of resultant larvae along with appearance of morphological deformities such as, microphthalmia, pericardial edema, yolk sac edema and spinal curvature. Larvae of 10‐50 mm E110‐exposed embryos exhibited increased cellular apoptosis in the cardiac region with significantly declined heartbeats and elevated mortality rates, in addition to the above‐mentioned abnormalities. In the 100 mm exposure group, all embryos succumbed to death within 24 hpf. The NOEC and LC₅₀ recorded were at 0.1 and 42.57 mm respectively. EC₅₀ (96 hpf) recorded for pericardial edema and yolk sac edema was 19.41 and 39.84 mm with teratogenic index quotient 2.1 and 1.06 respectively The study provides direct evidence for the developmental toxicity/teratogenic potential of E110.     

Meta-analysis of toxicity and teratogenicity of 133 chemicals from zebrafish developmental toxicity studies

Zebrafish developmental toxicity testing is an emerging field, which faces considerable challenges regarding data meta-analysis and the establishment of standardized test protocols. Here, we present an initial correlation study on toxicity of 133 chemicals based on data in the literature to ascertain predictive developmental toxicity endpoints. We found that the physical properties of chemicals (BCF or log P) did not fully predict lethality or developmental outcomes. Instead, individual outcomes such as pericardial edema and yolk sac edema were more reliable indicators of developmental toxicity. In addition, we ranked the chemicals based on toxicity with the Toxicological Priority Index (ToxPi) program and via a teratogenic ratio, and found that perfluorooctane sulfonate (PFOS) had the highest ToxPi score, triphenyltin acetate had the highest average ToxPi score (corrected for missing data and having more than 4 outcomes), and N-methyl-dithiocarbamate had the highest teratogenic ratio.     

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.