using-tg-vtg1mcherry-zebrafish-embryos-to-test-estrogenic-effects

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

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.

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.

Protective effects of beta-cyclodextrins vs. zearalenone-induced toxicity in HeLa cells and Tg(vtg1:mCherry) zebrafish embryos.

The biodegradation and biodetoxification ability of five prominent mycotoxins, namely aflatoxin B1 (AFB1), ochratoxin-A (OTA), zearalenone (ZON), T-2 toxin (T-2) and deoxynivalenol (DON) of Cupriavidus genus were investigated. Biological methods are the most appropriate approach to detoxify mycotoxins. The Cupriavidus genus has resistance to heavy metals and can be found in several niches such as root nodules and aquatic environments. The genus has 17 type strains, 16 of which have been investigated in the present study. According to the results, seven type strains can degrade OTA, four strains can degrade AFB1, four strains can degrade ZON and three strains can degrade T-2. None of the strains can degrade DON. The biodetoxification was measured using different biotests. SOS-chromotest was used for detecting the genotoxicity of AFB1, the BLYES test was used to evaluate the oestrogenicity of ZON, and the zebrafish embryo microinjection test was conducted to observe the teratogenicity of OTA, T-2 and their by-products. Two type strains, namely C. laharis CCUG 53908 and C. oxalaticus JCM 11285 reduced the genotoxicity of AFB1, whilst C. basilensis DSM 11853 decreased the oestrogenic of ZON. There were strains which were able to biodegrade more than two mycotoxins. Two strains degraded two mycotoxins, namely C. metalliduriens CCUG 13724 (AFB1, T-2) and C. oxalaticus (AFB1, ZON) whilst two strains C. pinatubonensis DSM 19553 and C. basilensis degraded three toxins (ZON, OTA, T-2) and C. numazuensis DSM 15562 degraded four mycotoxins (AFB1, ZON, OTA, T-2), which is unique a phenomenon amongst bacteria.

Mycotoxin Biodegradation Ability of the Cupriavidus Genus.

T-2 mycotoxin degradation and detoxification efficiency of seven bacterial strains were investigated with zebrafish microinjection method in three steps ((1) determination of mycotoxin toxicity baseline, (2) examination of bacterial metabolites toxicity, (3) identification of degradation products toxicity). Toxicity of T-2 was used as a baseline of toxic effects, bacterial metabolites of strains as control of bacterial toxicity and degradation products of toxin as control of biodegradation were injected into one-cell stage embryos in the same experiment. The results of in vivo tests were checked and supplemented with UHPLC-MS/MS measurement of T-2 concentration of samples. Results showed that the  NI1 strain was the only one of the seven tested ( AK38,  N361,  N774,  NI2,  N58,  NZS14), which was appropriated to criteria all aspects (bacterial and degradation metabolites of strains caused lower toxicity effects than T-2, and strains were able to degrade T-2 mycotoxin). Bacterial and degradation metabolites of the NI1 strain caused slight lethal and sublethal effects on zebrafish embryos at 72- and 120-h postinjection. Results demonstrated that the three-step zebrafish microinjection method is well-suited to the determination and classification of different bacterial strains by their mycotoxin degradation and detoxification efficiency.

Edina Garai

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