Using Tg(Vtg1:mcherry) Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

There are many endocrine disrupting compounds (EDC) in the environment, especially estrogenic substances. The detection of these substances is difficult ...

Szent István University

Zearalenone (ZEA, F2) is one of the most common mycotoxins and the only known mycoestrogen. It enters the food and feed chain from contaminated cereals and infiltrates into sewage or natural waters posing potential threat to exposed livestock, wildlife and humans. Therefore evaluation of its biological effects is of international importance. We performed toxicological tests on zebrafish (Danio rerio) larvae and adults. Developmental toxicity was assessed by an extended (5 days) fish embryo toxicity test (FET). Effects of early ZEA exposure were concentration-dependent with LC50 and LC10 values of 893 and 335 μg/L. In larvae exposed to 500 μg/L and above, ZEA induced similar phenotype to has (heart-and soul) showing defects in heart and eye development and upward curvature of the body axis. From 250 μg/L at 72 hpf the gap in the melanophore streak at the base of the tail fin was missing and the fin fold was abnormal, suggesting disturbance in the development of the adult tail fin primordium. Estrogenic potency was measured on the basis of Vitellogenin (Vtg) protein (adults) levels and relative abundance of vitellogenin-1 mRNA (vtg-1) (larvae and adults). qRT-PCR in larvae proved to be sufficient substitute to adult tests and sensitive enough to detect ZEA in 0.1 μg/L concentrations, that is close to levels observed in wastewaters. Developmental defects reveal that besides direct estrogenic effects, zearalenone might interact with other ontogenic pathways.

Developmental toxicity and estrogenic potency of zearalenone in zebrafish (Danio rerio).

Intraspecific morphological variability may reflect either genetic divergence among groups of individuals or response of individuals to environmental circumstances within the frame of phenotypic plasticity. Several studies were able to discriminate wild fish populations based on their scale shape. Here we examine whether the variations in the scale shape in fish populations could be related to genetic or environmental factors, or to both of them. In the first experiment, two inbred lines of zebrafish, Danio rerio (Hamilton 1822) reared under identical environmental conditions were compared. Secondly, to find out what effect environmental factors might have, offsprings were divided into two groups and reared on different diets for 12 weeks. Potential recovery of scales from an environmental effect was also assessed. Experimental groups could successfully be distinguished according to the shape of scales in both experiments, and the results showed that both genetic and environmental factors may notably influence scale shape. It was concluded that scale shape analysis might be used as an explanatory tool to detect potential variability of environmental influences impacting genetically homogeneous groups of fish. However, due to its sensitivity to environmental heterogeneity, the applicability of this technique in identifying intraspecific stock membership of fish could be limited. 

Impact of environmental and genetic factors on the scale shape of zebrafish, Danio rerio (Hamilton 1822): a geometric morphometric study.

Residues of anti-neoplastic drugs represent new and emerging pollutants in aquatic environments. Many of these drugs are genotoxic, and it has been postulated that they can cause adverse effects in aquatic ecosystems. 5-Fluorouracil (5-FU) is one of the most extensively used anti-neoplastic drugs in cancer therapy, and this article describes the results of the first investigation using a two-generation toxicity study design with zebrafish (Danio rerio). Exposure of zebrafish to 5-FU (0.01, 1.0 and 100 μg/L) was initiated with adult zebrafish (F0 generation) and continued through the hatchings and adults of the F1 generation, and the hatchings of the F2 generation, to day 33 post-fertilisation. The exposure did not affect survival, growth and reproduction of the zebrafish; however, histopathological changes were observed in the liver and kidney, along with genotoxic effects, at all 5-FU concentrations. Increases in DNA damage determined using the comet assay were significant in the liver and blood cells, but not in the gills and gonads. In erythrocytes, a significant, dose-dependent increase in frequency of micronuclei was observed at all 5-FU concentrations. Whole genome transcriptomic analysis of liver samples of F1 generation zebrafish exposed to 0.01 μg/L and 1 μg/L 5-FU revealed dose-dependent increases in the number of differentially expressed genes, including up-regulation of several DNA-damage-responsive genes and oncogenes (i.e., jun, myca). Although this chronic exposure to environmentally relevant concentrations of 5-FU did not affect the reproduction of the exposed zebrafish, it cannot be excluded that 5-FU can lead to degenerative changes, including cancers, which over long-term exposure of several generations might affect fish populations. The data from this study contribute to a better understanding of the potential consequences of chronic exposure of fish to low concentrations of anti-neoplastic drugs, and they demonstrate that further studies into multi-generation toxicity are needed.

Assessment of toxicity and genotoxicity of low doses of 5-fluorouracil in zebrafish (Danio rerio) two-generation study.

The acute and sub-chronic effects of four cytostatic drugs-5-fluorouracil (5-FU), cisplatin (CisPt), etoposide (ET) and imatinib mesylate (IM)-on zebrafish (Danio rerio) were investigated. Acute tests were carried out in a static system in accordance with the OECD guideline 203 for adult fish and the draft guideline for fish embryos (FET test) in order to find the LC50 values of the four cytostatic drugs. Early-life stage toxicity test on zebrafish was conducted according the OECD guideline 210 using the cytostatic drugs 5-FU and IM in a semistatic system with the objective of investigating the sub-chronic effects of the cytostatic drugs on fish. In adult fish, the cytostatic drugs 5-FU and ET did not pass the limit test, thus, are considered non-toxic. In case of cisplatin, LC50 was calculated at 64.5 mg L(-1), whereas in case of IM, LC50 was at 70.8 mg L(-1). In the FET test, LC50 of 5-FU at 72-h post fertilization (hpf) was 2441.6 mg L(-1). In case of CisPt, LC50 was 349.9 mg L(-1) at 48 hpf and it progressively decreased to 81.3 mg L(-1) at 120 hpf. In addition, CisPt caused a significant delay in the hatch of larvae. In case of ET, LC50 values were not calculable as they were higher than 300 mg L(-1) at which concentration the substance crystallized in the solution. LC50 values of IM were 48 hpf; 158.3 mg L(-1) , 72 hpf; 141.6 mg L(-1), 96 hpf; 118.0 mg L(-1), and 120 hpf; 65.9 mg L(-1). In the Early-life Stage Test with 5-FU, embryonic deformities were not detected during the tests. Regarding mortalities, the 10 mg L(-1) concentration can be considered as LOEC, as statistically significant difference in mortalities was detected in this group alone. Concerning dry body weight and standard length, 1 mg L(-1) is the LOEC. In case of IM, the highest tested concentration (10 mg L(-1)) can be considered LOEC for mortalities, however, the treatment did not have an effect on the other investigated parameters (dry and wet weight, standard length). All four cytostatic drugs were characterized by low toxicity in zebrafish in acute and sub-chronic tests.

Acute and sub-chronic toxicity of four cytostatic drugs in zebrafish.

Since three bullhead catfish species were introduced to Europe in the late 19th century, they have spread to most European countries. In Hungary, the brown bullhead () was more widespread in the 1970s-1980s, but the black bullhead () has gradually supplanted since their second introduction in 1980. The introgressive hybridization of the two species has been presumed based on morphological examinations, but it has not previously been supported by genetic evidence. In this study, 11 different Hungarian habitats were screened with a new species-specific nuclear genetic, duplex PCR based, marker system to distinguish the introduced catfish species,, and, as well as the hybrids of the first two. More than 460 specimens were analyzed using the above markers and additional mitochondrial sequence analyses were also conducted on >25% of the individuals from each habitat sampled. The results showed that only 7.9% of the specimens from two habitats belonged to, and 92.1% were classified asof all habitats, whereas the presence ofwas not detected. Two specimens (>0.4%) showed the presence of both nuclear genomes and they were identified as hybrids of. An additional two individuals showed contradicting results from the nuclear and mitochondrial assays as a sign of a possible footprint of introgressive hybridization that might have happened two or more generations before. Surprisingly, the level of hybridization was much smaller than expected based on the analyses of the North American continent's indigenous stock from the hybrid zones. This phenomenon has been observed in several invasive fish species and it is regarded as an added level of complexity in the management of their rapid adaptation.

Species-specific markers provide molecular genetic evidence for natural introgression of bullhead catfishes in Hungary.

Environmental estrogens are a serious concern worldwide due to their ubiquity and adverse ecotoxicological and health effects. Chemical structure of these substances is highly diverse, therefore estrogenicity cannot be predicted on the basis of molecular structure. Furthermore, estimation of estrogenicity of environmental samples based on chemical analytics of suspects is difficult given the complex interaction of chemicals and the impact on estrogenicity. The full estrogenic impact of an environmental sample can thus only be revealed by a series of sensitive in vitro and in vivo ecotoxicological tests. Herein we describe a vitellogenin reporter transgenic zebrafish line (Tg(vtg1:mCherry)) that enables the detection of estrogenicity in the environmentally relevant, low concentration ranges in embryonic tests that are in accordance with 3Rs and relevant animal welfare regulations. The transgene construct used for the development of Tg(vtg1:mCherry) carried a long (3.4 kbp) natural vitellogenin-1 promoter sequence with a high number of ERE sites. A test protocol was developed based on our finding that the endogenous vitellogenin and the reporter show similar spatial expression pattern and both endogenous and vitellogenin reporter is only produced in the left hepatic lobe of 5 dpf zebrafish embryos. Seven generations of Tg(vtg1:mCherry) have been established, and the estrogen responsiveness was tested with different estrogenic substances and wastewater samples. Embryos were exposed from 3 to 5 days post fertilization (dpf). Fluorescence in embryos could be detected upon treatment with 17-ß-estradiol from a concentration of 100 ng/L, 17-α-ethynilestradiol from 1 ng/L, zearalenone from 100 ng/L and bisphenol-A from 1 mg/L. In the adult stage transgene activity appeared to be more sensitive to estrogen treatment, with detectable transgene activity from 5 ng/L 17-ß-estradiol concentration. The transgenic line Tg(vtg1:mCherry) was also suitable for the direct measurement of estrogenicity in wastewater samples without sample extraction. The detection of estrogenic activity using the reporter line was confirmed by the bioluminescent yeast estrogen screen.

Estrogen sensitive liver transgenic zebrafish (Danio rerio) line (Tg(vtg1:mCherry)) suitable for the direct detection of estrogenicity in environmental samples.

The use of microinjection of newly fertilized zebrafish eggs as an appropriate tool for qualifying the biodetoxification properties of toxin-degrading microbes was investigated. Ochratoxin A (OTA), bacterial degradation products of OTA and bacterial metabolites of the Cupriavidus basilensis ŐR16 strain were microinjected. Results showed that variations in the injected droplet size, and thus treatment concentrations, stayed within ±20%, moreover embryo mortality did not exceed 10% in controls, that is in accordance with the recommendations of the OECD 236 guideline. The highest lethality was caused by OTA with a significantly higher toxicity than that of bacterial metabolites or OTA degradation products. However, toxicity of the latter two did not differ statistically from each other showing that the observed mortality was due to the intrinsic toxicity of bacterial metabolites (and not OTA degradation products), thus, the strain effectively degrades OTA to nontoxic products. Sublethal symptoms also confirmed this finding. RESULTS: confirmed that microinjection of zebrafish embryos could be a reliable tool for testing the toxin-degrading properties of microbes. The method also allows comparisons among microbial strains able to degrade the same toxin, helping the selection of effective and environmentally safe microbial strains for the biodetoxification of mycotoxins in large scale.

Biological evaluation of microbial toxin degradation by microinjected zebrafish (Danio rerio) embryos.

The African catfish or sharp tooth catfish (Clarias gariepinus) is one of the important species (due to its high environmental tolerance and easily controllable breeding habits) that can significantly contribute to reducing hunger in many countries. It is farmed in numerous African, Asian, and European countries. Moreover, during the last decades its production has grown significantly worldwide. Currently, following the carp, this species is produced in the second largest volume in Hungary. Despite its economic importance, the stocks have been maintained without genetic control or guided breeding. Molecular genetic data on bred populations or strains are very limited. In order to investigate the genetic structure of the stocks, 49 new microsatellite markers were characterized and tested on 32 individuals from a Hungarian farmed stock. All these markers were polymorph. The number of alleles per locus ranged from 2 to 11. The observed and expected overall heterozygosities were between 0.519 and 0.544 respectively and the overall inbreeding coefficient (Fis: 0.063) does not reveal the presence of inbreeding. However, 63% of the markers showed significant deviations from HWE. The results suggest that the maintenance of genetic variation within the stock require high attention in closed bred populations. These new markers provide a useful tool for population and conservation genetics of natural and bred African catfish populations.

Development and characterization of 49 novel microsatellite markers in the African catfish, Clarias gariepinus (Burchell, 1822).

Zearalenone is a xenoestrogenic mycotoxin produced by Fusarium species. High exposure with zearalenone induces reproductive disorders worldwide. Cyclodextrins are ring-shaped host molecules built up from glucose units. The apolar cavity of cyclodextrins can entrap so-called guest molecules. The formation of highly stable host-guest type complexes with cyclodextrins can decrease the biological effect of the guest molecule. Therefore, cyclodextrins may be suitable to decrease the toxicity of some xenobiotics even after the exposure. In this study, the protective effect of beta-cyclodextrins against zearalenone-induced toxicity was investigated in HeLa cells and zebrafish embryos. Fluorescence spectroscopic studies demonstrated the formation of stable complexes of zearalenone with sulfobutyl-, methyl-, and succinyl-methyl-substituted beta-cyclodextrins at pH 7.4 (K = 1.4-4.7 × 10 L/mol). These chemically modified cyclodextrins considerably decreased or even abolished the zearalenone-induced loss of cell viability in HeLa cells and mortality in zebrafish embryos. Furthermore, the sublethal effects of zearalenone were also significantly alleviated by the co-treatment with beta-cyclodextrins. To test the estrogenic effect of the mycotoxin, a transgenic bioindicator zebrafish model (Tg(vtg1:mCherry)) was also applied. Our results suggest that the zearalenone-induced vitellogenin production is partly suppressed by the hepatotoxicity of zearalenone in zebrafish. This study demonstrates that the formation of stable zearalenone-cyclodextrin complexes can strongly decrease or even abolish the zearalenone-induced toxicity, both in vitro and in vivo. Therefore, cyclodextrins appear as promising new mycotoxin binders.

Katalin Bakos

Department of Aquaculture,
Institute of Aquaculture and Environmental Safety,
Faculty of Agricultural and Environmental Sciences,
Department of Aquaculture, Institute of Aquaculture and Environmental Safety, Faculty of Agricultural and Environmental Sciences

Szent István University

Using Tg(Vtg1:mcherry) Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds