In Vitro Evaluation of the Individual and Combined Cytotoxic and Estrogenic Effects of Zearalenone, Its Reduced Metabolites, Alternariol, and Genistein.

Mycotoxins are toxic metabolites of filamentous fungi. Previous studies demonstrated the co-occurrence of Fusarium and Alternaria toxins, including zearalenone (ZEN), ZEN metabolites, and alternariol (AOH). These xenoestrogenic mycotoxins appear in soy-based meals and dietary supplements, resulting in the co-exposure to ZEN and AOH with the phytoestrogen genistein (GEN). In this study, the cytotoxic and estrogenic effects of ZEN, reduced ZEN metabolites, AOH, and GEN are examined to evaluate their individual and combined impacts. Our results demonstrate that reduced ZEN metabolites, AOH, and GEN can aggravate ZEN-induced toxicity; in addition, the compounds tested exerted mostly synergism or additive combined effects regarding cytotoxicity and/or estrogenicity. Therefore, these observations underline the importance and the considerable risk of mycotoxin co-exposure and the combined effects of mycoestrogens with phytoestrogens.

Protective effects of beta-cyclodextrins vs. zearalenone-induced toxicity in HeLa cells and Tg(vtg1:mCherry) zebrafish 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 × 104 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.

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

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.

Hormonal activity, cytotoxicity and developmental toxicity of UV filters.

Ultraviolet (UV) filters are commonly used compounds in personal care products and polymer based materials, as they can absorb solar energy in the UVA and UVB spectrum. However, they are able to bind to hormone receptors and have several and different types of hormonal activities determined by in vitro assays. One of the aims of this work was to measure the hormonal and cytotoxic activities of four frequently used UV filters using bioluminescence based yeast test organisms. Using Saccharomyces cerevisiae BLYES and BLYAS strains allowed the rapid and reliable detection of agonist and antagonist hormonal activities, whereas BLYR strain served to measure cytotoxicity. Results confirmed that all tested UV filters show multiple hormonal activities. Cytotoxicity is detected only in the case of benzophenone-3. Research data on the toxic effects of benzophenone-3, especially on aquatic organisms are scarce, so further investigations were carried out regarding its cytotoxic and teratogenic effects on bacteria and zebrafish (Danio rerio) embryos, respectively. Results revealed the cytotoxicity of benzophenone-3 not only to yeasts but to bacteria, as well as its ability to influence zebrafish embryo hatching and development.