Protective effects of beta-cyclodextrins vs. zearalenone-induced toxicity in HeLa cells and Tg(vtg1:mCherry) zebrafish embryos
Introduction
Zearalenone (ZEN; Fig. 1) is a xenoestrogenic mycotoxin produced by Fusarium species, which is a contaminant in cereals (e.g., maize and wheat), spices, and in different beverages (e.g., milk and beer) (Maragos, 2010; EFSA, 2017). Because of the high thermal stability and wide occurrence of ZEN, its removal from the food chain is difficult (Ryu et al., 1999). Based on cell and animal experiments, several adverse effects are attributed to ZEN, e.g., hepatotoxicity and genotoxicity (Zinedine et al., 2007; Cheraghi et al., 2015). Furthermore, ZEN can activate estrogen receptors in humans and animals, therefore, ZEN is an endocrine disruptor molecule which induces reproductive disorders (EFSA, 2017; Shier et al., 2001). ZEN is extensively metabolized in the body, during which reduced derivatives (zearalenols, zearalanone, and zearalanols) and glucuronic acid conjugates of ZEN and its reduced metabolites are produced (EFSA, 2017). Some of these metabolites (e.g., α-zearalenol and α-zearalanol) bind with significantly higher affinity to the estrogen receptors (and consequently exert higher toxicity) than ZEN (Shier et al., 2001; Filannino et al., 2011).
Cyclodextrins (CDs) are ring-shaped host molecules with a hydrophilic external part, which ensures excellent aqueous solubility, and an apolar internal cavity, which can accommodate lipophilic guest molecules (Szente and Szejtli, 1999; Szente et al., 2018). Therefore, they are frequently utilized by food, cosmetic, and pharmaceutical industries. The pharmaceutical application of beta-CDs is most common, due to their favorable cavity size for drugs (Challa et al., 2005). The native beta-CD (BCD) is often contained by orally administered drugs, however, its parenteral use is limited due to its nephrotoxicity and relatively low aqueous solubility of BCD (Jambhekar and Breen, 2016a). Methylated beta-CDs are absorbed from the gastrointestinal tract and cause nephrotoxic effects, therefore, they are not used neither orally nor parenterally (Jambhekar and Breen, 2016a). The sulfobutylated beta-CD is an excellent solubilizer without nephrotoxic adverse effect, thus, it is even suitable for parenteral application (Jambhekar and Breen, 2016b). Generally, the pharmaceutical industry applies drug-CD complexes with low binding constants to increase the aqueous solubility, gastrointestinal absorption, and/or cellular uptake of drugs (Jambhekar and Breen, 2016a). However, formation of highly stable CD complexes can strongly decrease the pharmacological effect and tissue uptake of drugs and other xenobiotics (Schaller and Lewald, 2016; Weiss-Errico et al., 2017).
Native and chemically modified beta-CDs can form stable complexes with mycotoxins, including aflatoxins (Dall’asta et al., 2003), citrinin (Poór et al., 2016), ochratoxin A (Poór et al., 2015a), and ZEN/zearalenols (Poór et al., 2017). The interaction of ZEN with beta-CDs has been reported in previous studies, demonstrating that native and chemically modified beta-CDs form highly stable complexes with ZEN (K is in the 104–105 L/mol range) (Dall’Asta et al., 2008; Dall’Asta et al., 2009; Poór et al., 2015b). Among beta-CDs tested, ZEN formed the most stable complexes with methyl and sulfobutyl derivatives (Poór et al., 2015b).
A beta-CD bead polymer has been shown recently to effectively remove ZEN and zearalenols added to aqueous solutions and corn beer samples (Poór et al., 2018). Furthermore, BCD strongly alleviated the toxic effect of ZEN in HepG2 cells, probably by limiting toxin uptake by the cells, as a result of the formation of highly stable mycotoxin-CD complexes (Poór et al., 2015b). Based on these observations, we hypothesize that CDs may also be effective as in vivo binders of ZEN.
There are numerous of endocrine disruptors in the environment, especially estrogenic xenobiotics. Sensitive biomonitor/bioindicator organisms are commonly applied to test xenoestrogenic effects. Among these biomonitoring organisms, several fish models, including zebrafish, exist (Chen et al., 2010; Fetter et al., 2014; Bakos et al., 2019). The main advantage of zebrafish as a biosensor is the transparent body of embryos and larvae; therefore, the fluorescence signal of a reporter protein can be easily studied in vivo in the living animal (Strähle et al., 2012). Zebrafish embryo is widely used as a model in developmental toxicology tests (Braunbeck et al., 2005; Scholz et al., 2008) because the developing and transparent zebrafish can be assessed conveniently for lethality and developmental abnormalities from fertilization through larval stages. Furthermore, the development of zebrafish embryos is very similar to the embryogenesis in higher vertebrates (including humans); therefore, this species is highly suitable for the investigation of the fundamental processes underlying embryonic development (Nagel, 2002; Weigt et al., 2011). In addition to animal protection, it is also favorable that the same individual fish can be studied throughout the treatment (Segner, 2009). In our experiments, we used a vitellogenin reporter transgenic zebrafish line, the Tg(vtg1:mCherry) (Bakos et al., 2019).
In this study, we examined the hypothesis that beta-CDs can limit the toxic effects of ZEN, employing BCD and its chemically modified derivatives, namely sulfobutylated beta-cyclodextrin (SbBCD), randomly methylated beta-cyclodextrin (RAMEB), succinyl-beta-cyclodextrin (SucBCD), and succinyl-methyl-beta-cyclodextrin (SuRAMEB) (Fig. 1). The stability of ZEN-CD complexes was tested in a physiological buffer by fluorescence spectroscopy. In our previous study, the cytotoxic effects of ZEN in the absence and presence of CDs were examined on HepG2 cell line (Poór et al., 2015b). Because HepG2 liver cells may significantly biotransform ZEN, the toxic actions of the mycotoxin were examined in HeLa (cervical cancer) cell line, in the absence and presence of CDs. The cytotoxicity of ZEN and CDs were evaluated based on ATP levels/well. Furthermore, the acute toxicity of ZEN was also examined on zebrafish embryos, in the absence and presence of CDs. Our results demonstrate that CDs can strongly alleviate the ZEN-induced toxicity both in vitro and in vivo.
Section snippets
Reagents
Zearalenone (ZEN), Dulbecco’s Modified Eagle Medium (DMEM), and fluorescamine (Fluram) were purchased from Sigma-Aldrich (St. Louis, MO, US). Cyclodextrins, including beta-cyclodextrin (BCD), sulfobutylated beta-cyclodextrin (SbBCD), randomly methylated beta-cyclodextrin (RAMEB), succinyl-beta-cyclodextrin (SucBCD), and succinyl-methyl-beta-cyclodextrin (SuRAMEB) were provided by CycloLab Cyclodextrin Research and Development Laboratory, Ltd (Budapest, Hungary). Bioluminescent ATP Assay Kit
Interaction of ZEN with beta-CDs in physiological buffer
Whereas the complex formation of ZEN with some beta-CDs has been reported, the interaction of SucBCD and SuRAMEB with ZEN has not been tested. Furthermore, previous experiments did not try to approximate extracellular physiological conditions; therefore, our spectroscopic experiments were performed in PBS buffer (pH 7.4). Each tested CD induced a strong increase in the fluorescence of ZEN (which is the sign of complex formation), showing the following order in the fluorescence enhancement:
Conclusions
In summary, the protective effects of native and chemically modified beta-CDs on ZEN-induced toxicity were investigated in HeLa cells and in zebrafish embryos. The chemically modified beta-CDs that formed more stable complexes with ZEN had considerably stronger protective effect on HeLa cells and zebrafish embryos against the toxic consequences of ZEN-exposure. Since beta-CDs strongly decreased or even abolished the ZEN-induced toxicity both in our in vitro and in vivo models, it is reasonable
Declarations of interest
The authors declare no conflict of interest. We have full control of all primary data and we agree to allow the journal to review our data if requested.
Acknowledgements
Supported by the ÚNKP-18-3 New National Excellence Program of the Ministry of Human Capacities (Zelma Faisal). Miklós Poór and Zelma Faisal are thankful for support of the Hungarian National Research, Development and Innovation Office (FK125166). This project was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (Miklós Poór and Mátyás Cserháti). This work was supported by the National Research, Development and Innovation Office (NKFIH) from the National
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These authors contributed equally to this work.