Estrogenic and toxic effects of methoxychlor on zebrafish (Danio rerio)

Although zebrafish (Danio rerio) have been suggested as a good candidate for screening potential endocrine disruptors, little information is available on the effects of weak estrogens on this species. We investigated the sensitivity of different life stages of zebrafish toward toxic and estrogenic properties of methoxychlor (MXC). Short-term tests with adults resulted in a sex-specific 96-h lethal concentration for 50% (LC50) of the test animals of 36 microg/L for males and 129 microg/L for females. To determine the estrogenic capacity of MXC, adult zebrafish were exposed to 0, 0.5, 5, and 50 microg MXC/L for 14 d. Induction of vitellogenin ([VTG] measured with protein electrophoresis and Western blot) in males was detected at 5 and 50 microg MXC/L. Females, however, did not exhibit higher blood VTG concentrations at the tested MXC concentrations. In a second series of experiments, juvenile zebrafish were exposed to 0, 0.05, 0.5, and 5 microg MXC/L for 33 d. Survival, length, weight, and condition of larvae were examined as indicators of toxic stress and the VTG content in whole body homogenates of juveniles was measured to determine xenoestrogenic effects. No effects of the tested concentrations of MXC were observed. Finally, the effect of MXC on zebrafish eggs, exposed to 0, 1, 10, and 32 microg MXC/L, was examined. Hatching and survival of hatched zebrafish were affected at 10 and 32 microg MXC/L. This study demonstrated that adult male zebrafish are sensitive toward the estrogenic effects of MXC. However, the use of VTG induction to detect effects of (xeno)estrogens in early life stages has to be further investigated, as low concentrations of VTG were detectable in exposed as well as unexposed juvenile fish.     

Effect of carbon nanotubes on developing zebrafish (Danio rerio) embryos

The impact of carbon nanotubes (CNTs) on the aquatic environment was investigated by examining the properties of raw CNTs under several environmental conditions and using developing zebrafish (Danio rerio) embryos. The agglomerate size for single-walled CNTs (SWCNTs) was significantly larger at pH 11 or greater and was stable at temperatures from 4 to 40 degrees C and salinities from 0 to 30 ppt. Exposure to SWCNTs induced a significant hatching delay in zebrafish embryos between 52 to 72 h postfertilization (hpf) at concentrations of greater than 120 mg/L, but 99% of the exposed embryos hatched by 75 hpf. Double-walled CNTs also induced a hatching delay at concentrations of greater than 240 mg/L, but carbon black did not affect hatching at the concentrations tested. Molecular and cellular analysis showed that the embryonic development of the exposed embryos up to 96 hpf was not affected at SWCNT concentrations of up to 360 mg/L. Scanning-electron microscopic inspection showed that the size of the pores on the embryo chorion was nanoscaled and that the size of SWCNT agglomerates was microscaled or larger, indicating that the chorion of zebrafish embryos was an effective protective barrier to SWCNT agglomerates. The hatching delay observed in this study likely was induced by the Co and Ni catalysts used in the production of SWCNTs that remained at trace concentrations after purification. This study suggests that materials associated with raw SWCNTs (perhaps metal contaminants) have the potential to affect aquatic life when released into the aquatic environment.     

Control of mycobacteriosis in zebrafish (Danio rerio) mucosally vaccinated with heat-inactivated Mycobacterium bovis

BackgroundMycobacterial infections greatly affect human and animal health worldwide, and vaccines are effective, sustainable and economic interventions for the prevention and control of these infectious diseases. Recent results support the use of zebrafish as a model for studying the pathophysiology of mycobacterial infection and for the development of novel interventions for tuberculosis (TB) control. Recently, we showed that oral immunization with the heat-inactivated M. bovis vaccine (M. bovis IV) protect wild boar against TB, and suggested that this vaccine may controls mycobacterial infection in other species.MethodsIn this study we evaluated the effect of M. bovis IV on the control of mycobacteriosis in zebrafish mucosally vaccinated by immersion and challenged intraperitoneally with Mycobacterium marinum.ResultsThe results showed that the M. bovis IV administered by immersion protected zebrafish against mycobacteriosis caused by M. marinum by reduction in mycobacterial infection, the number of mycobacteria per granuloma and the number of granulomas per fish. An IgM antibody response against M. bovis antigens was developed in vaccinated fish. Evidences suggested that the protective mechanism elicited by mucosal vaccination with M. bovis IV in zebrafish was based on the activation of the innate immune response through the C3 pathway.ConclusionsThese results support the use of the M. bovis IV administered by immersion for the control of mycobacteriosis in fish.     

Reproductive effects of ethynylestradiol and 4t-octylphenol on the zebrafish (Danio rerio)

In this study, the impact of ethynylestradiol (EE2) and 4t-octylphenol (OP) on reproduction in zebrafish (Danio rerio) was evaluated using spawning and fertilization success, gonadosomatic index, and plasma vitellogenin (VTG) levels as endpoints. Adult male and female zebrafish were exposed under semi-static conditions to 5, 10, 25 and 50 ng/L EE2 and to 12.5, 25, 50 and 100 μg/L OP for 3 weeks. A dose-related reduction in the number of females capable of spawning was observed at 10 ng/L EE2 with a complete inhibition of spawning at levels of 25 ng/L EE2. The ovaries of these nonspawning females were regressed and mean ovary somatic index (OSI) was significantly below the reference OSI determined in nonexposed females prior to spawning. Our results suggest an adverse impact of EE2 on male fertilization capacity and demonstrate a significant reduction in testis somatical index after exposure to 10 and 25 ng/L EE2. For both males and females, a dose dependent VTG induction was measured. Levels of VTG in fish plasma were significantly correlated with measured gonadosomatic indices. Minor effects were observed for OP. No significant effects on spawning or fertilization success were observed in this study, though OSI of nonspawning females was reduced at levels of 25 μg/L OP and higher. No changes in plasma protein levels were measured in male and female fish exposed to OP. The results from this study demonstrate that OP and especially EE2 can adversely affect the normal reproduction success of male and especially female zebrafish, with relevance for population effects. Received: 20 February 2001/Accepted: 13 June 2001     

Effects of Sevin (carbaryl insecticide) on early life stages of zebrafish (Danio rerio)

Sevin brand carbaryl insecticide is one of the most commonly used insecticides in the United States, with great potential for leaching into ground- and surface water reserves. Its possible teratogenic effects were tested on zebrafish eggs in four dilutions of decreasing concentration. The average mortality rate was low, indicating that Sevin does not directly kill embryos at these concentrations. Eggs and embryos were consistently smaller than the control starting at 24 h after spawning until hatching. Embryos in the highest concentration took up to twice as long to hatch as the control. This delayed hatching time increases vulnerability to predation. In addition, as minnows are lower on the food chain, bioaccumulation of Sevin in tissues may increase in larger predators, affecting their metabolism and reproduction.     

Comparative embryotoxicity and proteotoxicity of three carrier solvents to zebrafish (Danio rerio) embryos

The present study examines the effects of ethanol (ETOH), dimethyl sulfoxide (DMSO), and acetone on zebrafish embryos and the implications of the observed results on the use of these solvents to zebrafish early life stage tests. The embryos were exposed to different concentrations (0.0, 0.0001, 0.001, 0.01, 0.1, 0.05, 1, 1.5, and 2.0% v/v) of the respective solvents by diluting reagent-grade solvent with reconstituted water [DIN 38415-6-Suborganismische Testverfahren (Gruppe T) Teil 6: Giftigkeit gegenüber Fischen. Deutsches Institute für Normung e.V]. The following endpoints were investigated (mortality, hatching rate, abnormalities, heart rate, and hsp 70 induction). No effect on survival was recorded for both acetone and DMSO even up to the highest concentration. On the other hand, embryos exposed to 1.5% and 2.0% ethanol showed a significant reduction in survival rate. No developmental defects occurred with any of the solvents at the 0.1% concentration. However, starting with 1.0%, weak to very pronounced abnormalities (weak pigmentation, edema, crooked bodies, eye defect, tail defect, reduced heartbeat, and abnormal hatching) were observed depending on the solvent type and the concentration used. Ethanol has been shown to be the most embryotoxic solvent while DMSO and acetone have comparably lesser effects. Heat shock protein 70 was induced by all solvents but at different concentration ranges. DMSO has been shown to be the most potent inducer of stress proteins. Based on the study, the chemicals tested here may be used as carrier solvents in the zebrafish embryo assay at levels below 1.5, 1.5, and 1% v/v for acetone, DMSO, and ethanol, respectively. For stress protein analysis of the exposed embryos, however, the solvent levels should be below 0.1%, 0.01%, and 1.5%, respectively. Additional and separate investigations utilizing other biomarkers should be carried out to further validate the suitability of using these solvents in a typical zebrafish embryo assay.     

Histopathology as a tool for the evaluation of endocrine disruption in zebrafish (Danio rerio)

The importance of histology as a tool in the evaluation of endocrine disruption in fish depends on the choice and interpretation of appropriate endpoints, as is illustrated by the analysis of the effects of exposure to the estrogen 17beta-estradiol (E2) and the nonaromatizable androgen 17-methyldihydrotestosterone (MDHT). The E2 led to the disappearance of vitellogenic oocytes in the ovary and an increased area of relatively large, eosinophilic cells in the testis, which were identified as spermatogonia under high-power magnification; this was a relative increase, as was shown by histomorphometry, because of a decreased size of spermatogenic cysts and a relative decrease of spermatocyte cysts. The E2 also induced an accumulation of acidophilic fluid in vessels and interstitial spaces, confirmed by immunohistochemistry as vitellogenin, and basophilia in the liver also associated with the production of vitellogenin. The MDHT induced activation of Sertoli cells in the testis and a decreased presence of vitellogenic oocytes and a reduced growth of previtellogenic oocytes in the ovary. These observations indicate the advantages of examining multiple organ systems on whole-body sections and the application of adequate magnifications. Inclusion of additional techniques such as morphometry and immunohistochemistry is valuable to further uncover insidious effects of endocrine disruptors.     

Elevated temperature applied during gonadal transformation leads to male bias in zebrafish (Danio rerio)

Temperature effects on sex determination or differentiation exist in many fish species, with high temperatures predominantly producing more males. The present study aimed at elucidating the genetic background of temperature effects on sex differentiation in zebrafish. Experimental fish were generated by matings between 4 or 6 golden females and a normal or a mitotic gynogenetic male, respectively. All the larvae were reared at 28.5°C until they were divided into 3 groups per full-sib family, a control group raised at 28.5°C and 2 treatment groups reared at 35°C from 20 to 30 dpf or 25 to 35 dpf, respectively. Backcross progenies, reared at 28.5°C, were derived from F1 temperature-treated sons (35°C, 25-35 dpf) that were sired by a mitotic gynogenetic male and their corresponding mothers. No significant differences were observed regarding the survival rate between the control and treatment groups. Significant differences in the phenotypic male proportions from the controls were observed in groups treated at 35°C. The sex ratio in zebrafish was influenced by the male spawner, the female spawner, and a significant interaction of genotype by temperature. Backcross experiments point to a continuum of major genetic, minor genetic, and environmental factors in the expression of the phenotypic sex in zebrafish.     

Early life-stage toxicity in zebrafish (Danio rerio) following embryonal exposure to selected polychlorinated biphenyls

Polychlorinated biphenyls are a widespread aquatic contaminant. In this article, specific polychlorinated biphenyl congeners were examined for embryo and early life-stage toxicity in zebrafish (Danio rerio). A set of three polychlorinated biphenyl congeners (non-ortho polychlorinated biphenyl 126, mono-ortho polychlorinated biphenyl 28 and di-ortho polychlorinated biphenyl 153) were tested. The typical lesions observed were yolk sac edema, vertebra defect, craniofacial malformations (double head, triple retina), anaxial body and inhibition of swim bladder inflation. Moreover, embryo and larval mortality increased and hatching success decreased. The severity of abnormalities and mortalities were concentration- and congener-dependent. Of the compounds tested, polychlorinated biphenyl congener 126 was found to be highly toxic to the fish embryos following exposure. The Lethal Concentration 50 values for polychlorinated biphenyl 28, polychlorinated biphenyl 126, polychlorinated biphenyl 153 calculated by probit analysis were 3.270, 1.298 and 5.375 ppm, respectively. The inhibition of swim bladder inflation was the most sensitive endpoint measured, and it is suggested that the inhibition of swim bladder inflation may be mediated by mechanism with an aryl hydrocarbon receptor activation.     

Integrated condition indices as a measure of whole effluent toxicity in zebrafish (Danio rerio)

Toxic exposure of organisms interferes with organismal integrity at the biochemical level and ultimately gives rise to effects at the individual level. These effects may result in reductions in ecologically relevant characteristics such as growth, reproduction, and survival. A chronic toxicity test with zebrafish (Danio rerio) was conducted where fish were exposed to 50, 75, and 100% effluent for 28 d under flow-through conditions. Effects of effluent exposure were determined using endpoints of physiological (respiration during swimming), growth (condition, length, and weight), and reproductive (spawning and hatching) processes within the same population. Results clearly indicate that the condition and growth of zebrafish is depressed by exposure to the effluent. Also, increased oxygen consumption was found after 14, 21, and 28 d of exposure. Reproduction proved to correlate well with the condition of the motherfish in the control, and spawning and hatching were significantly depressed by effluent exposure. These results indicate that the evaluation of endpoints describing different ecologically relevant processes provides a rational assessment of the cause-effect relationships of effluent toxicity. This approach can quantify effects on different biological processes and can determine the interactions that occur between these different processes.     

Neuropharmaceuticals in the environment: mianserin-induced neuroendocrine disruption in zebrafish (Danio rerio) using cDNA microarrays

Because of their environmental occurrence and high biological activity, human pharmaceuticals have received increasing attention from environmental and health agencies. A major bottleneck in their risk assessment is the lack of relevant and specific effect data. We developed an approach using gene expression analysis in quantifying adverse effects of neuroendocrine pharmaceuticals in the environment. We studied effects of mianserin on zebrafish (Danio rerio) gene expression using a brain-specific, custom microarray, with real-time polymerase chain reaction as confirmation. After exposure (0, 25, and 250 microg/L) for 2, 4, and 14 d, RNA was extracted from brain tissue and used for microarray hybridization. In parallel, we investigated the impact of exposure on egg production, fertilization, and hatching. After 2 d of exposure, microarray analysis showed a clear effect of mianserin on important neuroendocrine-related genes (e.g., aromatase and estrogen receptor), indicating that antidepressants can modulate neuroendocrine processes. This initial neuroendocrine effect was followed by a "late gene expression effect" on neuronal plasticity, supporting the current concept regarding the mode of action for antidepressants in mammals. Clear adverse effects on egg viability were seen after 14 d of exposure at the highest concentration tested. Based on the specific molecular impact and the effects on reproduction, we conclude that further investigation of the adverse effects on the brain-liver-gonad axis is needed for a correct ecological risk assessment of antidepressants.     

Gene expression changes in female zebrafish (Danio rerio) brain in response to acute exposure to methylmercury

Methylmercury (MeHg) is a potent neurotoxicant and endocrine disruptor that accumulates in aquatic systems. Previous studies have shown suppression of hormone levels in both male and female fish, suggesting effects on gonadotropin regulation in the brain. The gene expression profile in adult female zebrafish whole brain induced by acute (96 h) MeHg exposure was investigated. Fish were exposed by injection to 0 or 0.5 μg MeHg/g. Gene expression changes in the brain were examined using a 22,000-feature zebrafish microarray. At a significance level of p < 0.01, 79 genes were up-regulated and 76 genes were down-regulated in response to MeHg exposure. Individual genes exhibiting altered expression in response to MeHg exposure implicate effects on glutathione metabolism in the mechanism of MeHg neurotoxicity. Gene ontology (GO) terms significantly enriched among altered genes included protein folding, cell redox homeostasis, and steroid biosynthetic process. The most affected biological functions were related to nervous system development and function, as well as lipid metabolism and molecular transport. These results support the involvement of oxidative stress and effects on protein structure in the mechanism of action of MeHg in the female brain. Future studies will compare the gene expression profile induced in response to MeHg with that induced by other toxicants and will investigate responsive genes as potential biomarkers of MeHg exposure.     

Changes in cellular energy budget as a measure of whole effluent toxicity in zebrafish (Danio rerio)

Organisms exposed to suboptimal conditions face an a priori cost of combating stress in terms of metabolic resources. The energy available for maintenance, growth, and reproduction, based on the biochemical analysis of the energy budget rather than on the direct measurement of those endpoints, may therefore provide a sensitive measure of stress in an organism. The aim of this study was to validate changes in energy budget as an ecologically relevant sublethal parameter by comparing these responses with physiological, growth, and reproductive endpoints. A toxicity test with zebrafish (Danio rerio) was conducted where fish were exposed to a control and 50, 75, and 100% effluent for 28 d under flow-through conditions. Effects of effluent exposure were measured as changes in glycogen, lipid, and protein budgets. Furthermore, the observed effects were linked to different processes like growth, condition, respiration, and reproduction within the same populations. Our results indicate that changes in lipid budgets in exposed fish were the most sensitive endpoint. Excellent relations were found between cellular energy budgets, relative condition factor, and respiration (r2 = 0.795, p < 0.001; and r2 = 0.735, p < 0.001, respectively), but relations were poorer for reproductive output (r2 = 0.410, p < 0.034). As a whole, especially changes in lipid energy budgets provide a sensitive and fast indicator of altered condition in zebrafish under the given exposure regime and allow linking cellular effects to other endpoints within the same exposed populations.     

Development and validation of an enzyme-linked immunosorbent assay to measure vitellogenin in the zebrafish (Danio rerio)

In this study, an enzyme-linked immunosorbent assay (ELISA) was developed to quantify vitellogenin (Vtg) in zebrafish (Danio rerio). Zebrafish Vtg (zf-Vtg) was purified from whole-body homogenates of estradiol-exposed zebrafish, and polyclonal antibodies against zf-Vtg were raised. Using purified zf-Vtg as a standard and anti-zf-Vtg antibodies (DR-264), a competitive ELISA method was set up and validated. The working range of the assay is from 1 to 30 ng/ml (20-80% binding), and the detection limit is 0.4 ng/ml for purified zf-Vtg. In whole-body homogenates samples, the practical detection limit is higher than that for purified Vtg (40 ng/ml) due to matrix effect. The intra- and interassay variations were 4.7% and 14%, respectively, at 50% binding (n = 36). Its usefulness to detect changes in Vtg concentration in other cyprinid fish was also tested. In addition, the assay was used to assess Vtg induction in male zebrafish exposed to 17beta-estradiol (E2). Exposure of male zebrafish to 0.1, 1, 10, and 100 microg/L of E2 for 7 d led to a Vtg induction from the lowest concentration. The results show the suitability of the developed ELISA to quantify Vtg inductions in zebrafish, the cross-reactivity of DR264 antibodies with commonly used cyprinids, and the potential of zf-Vtg induction as a sensitive biochemical endpoint that could be used to detect estrogenic properties of chemical substances.     

Effects of ethynylestradiol on the reproductive physiology in zebrafish (Danio rerio): time dependency and reversibility

Environmental pollution with natural or synthetic estrogens may pose a serious threat to reproduction of wildlife species. This study describes the effects of 17-alpha-ethynylestradiol (EE2) on fish reproductive organs in a laboratory model. Adult zebrafish were semistatically exposed to nominal concentrations of 0, 10, and 25 ng/L EE2 for 24 d and then transferred to EE2-free medium. Gonadosomatic index (GSI), plasma vitellogenin concentration (VTG), and histology of the gonads (control and 10 ng/L only) were examined as a function of time. It was found that EE2 has an adverse impact on both male and female reproductive organs. Notably in females, gonadal changes were observed through histological evaluation after 3 d of exposure to 10 ng/L EE2. and this was followed by a reduction of GSI at day 6 of exposure. In males, a reduction of GSI and altered testis histology was found after 24 d of exposure to 10 ng/L. The observed effects on the ovary after EE2 exposure, combined with complete recovery after 24 d, is considered to be triggered by feedback at the level of the pituitary. In both males and females, VTG was induced in response to EE2 and normalized during the recovery period. The observed correlation between VTG and ovarian somatic index (OSI) demonstrates that excessive VTG induction may be predictive for adverse effects of EE2 on ovarian function in female zebrafish. These results indicate that long-term stimulation by synthetic estrogens such as EE2 might impair reproductive function in zebrafish in a reversible manner.     

Concentration-response concept in ecotoxicoproteomics: effects of different phenanthrene concentrations to the zebrafish (Danio rerio) embryo proteome

Concentration-response experiments, based on the testing of less replicates in favour of more exposure concentrations, represent the typical design of choice applied in toxicological and ecotoxicological effect assessment studies using traditional endpoints such as lethality. However, to our knowledge this concept has not found implementation in the increasingly applied OMICS techniques studying thousands of molecular endpoints at the same time. The present study is among the first applying the concentration-response concept for an ecotoxicoproteomics study. The effects of six different concentrations in the low effect range (相似文献   

Long-term exposure to environmentally relevant doses of methyl-tert-butyl ether causes significant reproductive dysfunction in the zebrafish (Danio rerio)

Methyl-tert-butyl ether (MTBE), an anthropogenic chemical used as a gasoline additive, is being detected at an increasing frequency in the environment. The acute lethal concentration that kills 50% of the fish test population and the chronic effects of exposure to MTBE were investigated in the zebrafish (Danio rerio). Chronic exposure over three weeks to effective MTBE conceritrations as low as 0.11 mg/L induced a significant increase in the vitellogenin concentration of male fish. The impact of a chronic, eight-week exposure at effective concentrations ranging from 0.44 to 220 mg/L had no significant effect on fecundity, fertilization, or hatch rate but highly significant impacts on sperm motility. Spermatozoa of all MTBE-exposure groups showed a significantly lower straight-line velocity and lower average path velocity compared to those of the nonexposed group. These results suggest that chronic exposure to MTBE negatively affects fish sperm motility at concentrations that are environmentally relevant and several orders of magnitude lower than concentrations inducing acute effects.     

Developmental toxicity in zebrafish (Danio rerio) embryos after exposure to manufactured nanomaterials: buckminsterfullerene aggregates (nC60) and fullerol

The present paper summarizes, to our knowledge, the first study regarding the developmental toxicity of stable buckminsterfullerene aggregates suspended in water (nC60) using zebrafish (Danio rerio) as a vertebrate model. Zebrafish embryo survival, hatching rate, heartbeat, and pericardial edema were noted and described within 96 h of exposure. Fullerol (a hydroxylated C60 derivative, C60(OH)16-18) at 50 mg/L did not exert toxicity to zebrafish embryos. In contrast, nC60 at 1.5 mg/L delayed zebrafish embryo and larval development, decreased survival and hatching rates, and caused pericardial edema. Toxicity was mitigated by adding an antioxidant (glutathione), which suggests that a free radical-induced mechanism or another form of oxidative stress played a role in developmental toxicity.