Interactions of zearalenone with native and chemically modified cyclodextrins and their potential utilization

Journal of Photochemistry and Photobiology B: Biology 151 (2015) 63–68

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Interactions of zearalenone with native and chemically modified cyclodextrins and their potential utilization }szegi c,d, Lajos Szente e, Miklós Poór a,⇑, Sándor Kunsági-Máté b,c, Nikolett Sali c,d, Tamás Ko Beáta Peles-Lemli b,c a

Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Szigeti út 12, Pécs H-7624, Hungary Department of General and Physical Chemistry, University of Pécs, Ifjúság útja 6, Pécs H-7624, Hungary János Szentágothai Research Center, Ifjúság útja 20, Pécs H-7624, Hungary d Institute of Laboratory Medicine, University of Pécs, Ifjúság útja 13, Pécs H-7624, Hungary e CycloLab Cyclodextrin Research & Development Laboratory, Ltd., Illatos út 7, Budapest H-1097, Hungary b c

a r t i c l e

i n f o

Article history: Received 22 May 2015 Received in revised form 7 July 2015 Accepted 9 July 2015 Available online 11 July 2015 Keywords: Zearalenone Cyclodextrin Fluorescence spectroscopy Fluorescence enhancement Detoxification

a b s t r a c t Zearalenone (ZEA) is a widespread xenoestrogenic mycotoxin produced by several Fusarium species. ZEA can cause reproductive disorders in farm animals and hyperoestrogenic syndromes in humans; therefore, development of more sensitive analytical methods (to quantify the mycotoxin) as well as strategies for prevention of its toxic impacts is highly important. In this study, the interactions of ZEA with native and chemically modified cyclodextrins (CDs) were investigated using fluorescence spectroscopy. Furthermore, in vitro experiments on liver cells were also performed to test the potential effect of CDs on toxin uptake. Our results demonstrate that ZEA forms stable complexes with CDs (log K values are approximately 3.7–4.7) resulting in the considerable elevation of its fluorescence signal. In addition, some of the CDs show ability to inhibit the cellular uptake of ZEA, suggesting their potential suitability to develop new CD-based preventive/detoxification strategies against ZEA in the future. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Zearalenone (ZEA; previously called as F-2 toxin) is a mycotoxin produced by several Fusarium species (e.g. F. culmorum, F. graminearum, F. cerealis, etc.) [1]. ZEA occurs mainly in maize; however, other crops such as barley, rye, wheat and sorghum are also often contaminated with this mycotoxin [1,2]. Because of its wide occurrence and high thermal stability (ZEA is heat stable up to 150 °C) its complete eradication from the food chain does not seem feasible [3]. Chemically ZEA (3,4,5,6,9,10-hexahydro-14,16-dihydroxy-3methyl-1H-2-benzoxacyclotetradecin-1,7(8H)-dione) is a macrocyclic b-resorcylic acid lactone (see in Fig. 1). Despite that ZEA is a non-steroidal compound, it has xenoestrogenic effect on animals and humans [4]; therefore ZEA is belonging to the endocrine disruptor family. Because of its estrogenic activity, ZEA is able to cause reproductive disorders in farm animals and in some cases hyperoestrogenic syndromes in humans [1,5]. Furthermore, other

⇑ Corresponding author at: Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Medical School, Szigeti út 12, H-7624 Pécs, Hungary. E-mail address: [email protected] (M. Poór). http://dx.doi.org/10.1016/j.jphotobiol.2015.07.009 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.

toxic (e.g. haematotoxic, hepatotoxic, genotoxic, etc.) effects are also attributed to ZEA [1,6,7]. Cyclodextrins (CDs) are extensively studied molecules in the field of host–guest interactions. CDs have a conical structure with a hydrophobic interior and a hydrophilic exterior space [8]. The internal cavity can include a wide range of guest molecules; the stability and selectivity of these complexes are highly influenced by the chemical modification of native CDs [9,10]. The most abundant cyclodextrins are a-, b-, and c-CDs (are built up from six, seven and eight glucopyranose unites, respectively). Previous studies highlighted that b-CDs are able to form stable complexes with different mycotoxins such as citrinin, aflatoxin B1, ochratoxin A and zearalenone [11–15]. The complex formation commonly results in the fluorescence enhancement of the fluorescent mycotoxins therefore some of these interactions are suitable to develop more sensitive fluorescent analytical methods [16]. Furthermore, the development of different applications in order to remove mycotoxins from aqueous solutions (e.g. from different drinks) is also possible [17]. Recent studies showed that the interaction of ZEA with b-CDs results in the extensive increase of its fluorescence, and revealed the potential analytical utilization of the complex formation, e.g. in the cases of high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) with fluorescent

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Fig. 1. Fluorescence emission spectra of 2 lM ZEA in 0.05 M ammonium acetate buffer at pH 5.0 (A) and 10.0 (B), in the presence of increasing DIMEB concentrations (0– 1000 lM) [kexc = 315 nm].

detector (FLD) [15,18–21]. Complex formation of ZEA with b-cyclodextrin (BCD), hydroxypropyl-b-cyclodextrin (HPBCD) and heptakis-2,6-di-O-methyl-b-cyclodextrin (DIMEB) was proved and characterized by Dall’Asta et al. [15,18]. In our study, the interactions of zearalenone with native and chemically modified cyclodextrins were investigated using fluorescence spectroscopy. In the first step, the fluorescence behavior of ZEA was examined at different pH values. Thereafter, CD-mediated fluorescence enhancement of ZEA was tested as well as the stability constants of ZEA-CD complexes were determined. Finally, the toxic impact of ZEA alone and in presence of CDs was investigated on HepG2 liver cell line, in order to test our hypothesis that some of the CDs may be suitable to inhibit the cellular uptake of ZEA. 2. Materials and methods 2.1. Reagents All of the applied reagents and solvents were of spectroscopic or analytical grade. Zearalenone (ZEA), bovine serum albumin (BSA), DMEM (Dulbecco’s Modified Eagle’s Medium) – high glucose (4500 mg/L), fetal bovine serum (FBS), DAPI (40 ,6-diamidino-2-phe nylindole), penicillin/streptomycin solution (all from Sigma– Aldrich), Coomassie Brilliant Blue G-250, spectroscopic grade methanol and acetonitrile (all from Reanal), b-cyclodextrin (BCD), hydroxypropyl-b-cyclodextrin (HPBCD), randomly methylated-b-cyclodextrin (RAMEB), heptakis-2,6-di-O-methyl-b-cyclodextrin (DIMEB), sulfobutyla ted-b-cyclodextrin (SBCD), (2-hydroxy-3-N,N,N-trimethylamino)p ropyl-b-cyclodextrin (QABCD), c-cyclodextrin (GCD) and hydroxy propyl-c-cyclodextrin (HPGCD) (all from Cyclolab Ltd.) were used as received. 5000 lM stock solution of ZEA was prepared in ethanol (Reanal, spectroscopic grade) and stored at 4 °C, protected from light. Spectroscopic measurements of ZEA in absence and presence of cyclodextrins were performed in 0.05 M ammonium acetate buffer (pH 5.0 and 10.0), in order to avoid the interferences of potential interaction of ZEA with alkali and/or alkaline earth metal ions. 2.2. Fluorescence spectroscopic measurements Fluorolog s3 spectrofluorometric system (Jobin–Yvon/SPEX) was applied for steady state fluorescence spectroscopic measurements. All analyses were performed in the presence of air at +25 °C. Binding constants (K) of ZEA-cyclodextrin complexes were determined by the Benesi–Hildebrand equation, assuming 1:1 stoichiometry:

I0 1 1 ¼ þ ðI  I0 Þ A A  K  ½Hn

ð1Þ

where K is the binding constant, I0 is the initial fluorescence intensity of ZEA (in the absence of cyclodextrins), I is the fluorescence intensity of ZEA in the presence of the host molecule with concentration [H], while A is a constant and n is the number of binding sites. 2.3. Tissue cultures HepG2 cells (human liver, ATCC: HB-8065) were cultured in DMEM with 10% FBS, penicillin (100 U/mL) and streptomycin (100 lg/mL) in 25 cm2 sterile plastic flasks (VWR). Cells were grown at 37 °C in humidified atmosphere, in the presence of 5% CO2. Cells were plated into 24-well sterile plastic plates (VWR); and after attachment of cells (24 h), the medium was replaced with fresh one and HepG2 cells were treated with 0–100 lM ZEA in the absence and in the presence of CDs. Measurements were performed after 24-h incubation. All sterile work was carried out in an Aireguard-126300 (Nuaire) vertical laminar box. 2.4. Quantification of the number of living cells In order to investigate the number of living cells, total DNA content/well was determined. The wells were washed twice with ice-cold PBS and the emptied wells were filled with 500 lL of 5% perchloric acid (PCA). Estimation of the number of the attached cells was performed by DAPI staining of PCA fixed cells. The wells were gently washed with McIlvaine’s buffer (citric acid/Na2HPO4, pH 7.0) then 200 lL of a 10 lg/mL DAPI solution in the above buffer was pipetted into each well (24-well plates). Staining was performed at room temperature (30 min). After washing, 200 lL McIlvaine’s buffer was pipetted into each well and the fluorescence of the samples was determined with a plate reader (Perkin Elmer EnSpire Multimode reader) applying 355 nm excitation and 460 nm emission wavelengths using area scan mode (cylindrical scan with 80 measuring points/well). Fluorescence intensity was expressed as the sum of the 80 measuring points. Unstained and untreated cells were used as blanks. To confirm the results derived from DNA staining, quantification of total protein levels/well was also performed. Intracellular proteins from the cells in the wells were solubilized by 500 lL of 1 M NaOH (15 min incubation at room temperature). Total intracellular protein levels were quantified by the Bradford reaction with the measurement of absorbance at 595 nm, applying purified BSA as standard (concentration range: 20–100 mg/L). Then 20 lL

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of the solubilized sample was pipetted into 96-well plates, and 200 lL Bradford reagent (10 mg Coomassie Brilliant Blue G-250, 5 mL 96% ethanol, 10 mL 85% phosphoric acid in 100 mL aqueous solution) was added into each well. Intracellular protein levels were determined using the plate reader. 2.5. Statistics Data shown (mean ± SEM) are derived from at least three independent experiments. Statistical analyses were performed by One-Way ANOVA test (IBM SPSS Statistics, Version 21). The level of significance was set at p < 0.05.

ZEA was observed without changes of its fluorescence emission wavelength maxima (Figs. 1A and 2). The examined c-cyclodextrins (GCD and HPGCD) caused the weakest fluorescence enhancement of ZEA (6- to 8-fold); furthermore, QABCD induced lower as well as HPBCD and SBCD resulted in similar (approximately 17-fold) increase of fluorescence intensities of ZEA than the native BCD (Fig. 2 and Table 1). BCD was followed by RAMEB, while the highest fluorescence emission intensity at 455 nm was noticed in the presence of DIMEB, resulting in more than 19-fold higher fluorescence compared to the signal of ZEA itself (Fig. 2 and Table 1). 3.3. Stability of zearalenon–cyclodextrin complexes

3. Results 3.1. Fluorescence properties of zearalenone in ammonium acetate buffer ZEA has two excitation maxima located at 275 nm and at 315 nm. To investigate the spectral properties of its nonionic and deprotonated forms fluorescence emission spectra of ZEA were recorded in 0.05 M ammonium acetate buffer at pH 5.0 and pH 10.0. Using 275 and 315 nm as excitation wavelengths the same results were noticed: at pH 5.0 ZEA showed emission maximum at 455 nm, while at pH 10.0 the emission maximum of the mycotoxin at 413 nm was observed. 3.2. Effects of b-cyclodextrins on the fluorescence of zearalenone In order to examine the similarities or potential differences between the interaction of CDs with nonionic and deprotonated forms of ZEA, increasing CD concentrations (0–1000 lM) were added to 2 lM ZEA in 0.05 M ammonium acetate buffer at low (pH = 5.0) and at high (pH = 10.0) pH levels, where nonionic and deprotonated (or anionic) form of ZEA predominates, respectively. Interestingly, at pH 10.0 (compared to data obtained at pH 5.0) considerably higher CD concentrations were needed to induce remarkable fluorescence enhancement of ZEA (Fig. 1). Furthermore, a marked red shift of the fluorescence emission maximum was observed (413 ? 455 nm). Then increasing quantities (0–2000 lM) of different cyclodextrins were added to standard amount of ZEA (2 lM) in 0.05 M ammonium acetate buffer (pH 5.0). Fluorescence emission spectra were recorded applying 315 nm as excitation wavelength. In presence of CDs considerable increase of the fluorescence intensity of

Applying the fluorescence emission intensities determined at 455 nm (kexc = 315 nm) in pH 5.0 ammonium acetate buffer (Fig. 2), we calculated the stability constants (K) of ZEA-CD complexes using the Benesi–Hildebrand equation (Fig. 3; see details in Section 2.2). GCD, HPGCD and QABCD form less stable complexes with ZEA compared to BCD. Furthermore, ZEA-HPBCD complex shows similar stability than ZEA-BCD, while SBCD and methyl-substituted b-cyclodextrins (RAMEB and DIMEB) have higher affinities toward ZEA compared to the native BCD (Table 1). The highest K value was quantified for ZEA-DIMEB complex (with approximately 5-fold higher stability than ZEA-BCD). 3.4. Fluorescence of zearalenone dissolved in methanol and acetonitrile compared to in aqueous buffer without and with BCD or DIMEB Since previous studies highlighted that ZEA shows much higher fluorescence in organic solvents than in water [22], we tested the effects of methanol and acetonitrile on the fluorescence properties of ZEA using 275 nm as excitation wavelength. Therefore, 2 lM ZEA solutions were prepared in methanol and acetonitrile then fluorescence spectra were recorded, in order to compare them with the spectra previously measured in ammonium acetate buffer (pH 5.0) in absence and presence of 2 mM BCD or DIMEB. Acetonitrile and methanol resulted in 8-fold and 10-fold elevation of the fluorescence signal of ZEA, respectively (Fig. 4). Emission wavelength maximum of the toxin did not show changes. 3.5. Impacts of BCD and DIMEB on the toxic effect of zearalenone in vitro First, the dose-dependent effect of ZEA on HepG2 cells was investigated. Concentration dependent decrease of living HepG2 cells was observed after 24-h treatment with 20–100 lM ZEA (Fig. S1). Total DNA and protein values were in a very good correlation, indicating that the applied methods truly describe the number of living cells in the wells of the plates. Since 40 lM ZEA led to approximately 40–50% decrease of living cells in our preliminary

Table 1 Stabilities of ZEA-CD complexes (the unit of K is dm3/mol) and fluorescence enhancement of ZEA (2 lM) by CDs (2000 lM) compared to the fluorescence of ZEA alone in 0.05 M ammonium acetate buffer (pH 5.0) [kexc = 315 nm, kem = 455 nm].

Fig. 2. Fluorescence emission intensities of ZEA in the presence of increasing CD concentrations (0–2000 lM) in 0.05 M ammonium acetate buffer (pH 5.0) [kexc = 315 nm, kem = 455 nm].

BCD HPBCD RAMEB DIMEB SBCD QABCD GCD HPGCD

log K (R2)

I/I0

3.99 3.94 4.36 4.76 4.20 3.75 3.79 3.76

17.5 17.1 18.5 19.2 16.8 14.9 6.3 7.5

(0.999) (0.999) (0.999) (0.998) (0.999) (0.999) (0.998) (0.998)

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Fig. 3. Benesi–Hildebrand plots of ZEA-CD complexes (n = 1) based on fluorescence emission intensities of ZEA in absence and presence of increasing CD concentrations (0– 2000 lM) in 0.05 M ammonium acetate buffer (pH 5.0) [kexc = 315 nm, kem = 455 nm].

Fig. 4. Benesi–Hildebrand plots of ZEA-BCD and ZEA-DIMEB complexes (n = 1) in 0.05 M ammonium acetate buffer (pH 5.0) using 275 nm as excitation wavelength (left), and fluorescence intensities of 2 lM ZEA in methanol (MeOH), in acetonitrile (ACN), and in 0.05 M ammonium acetate buffer (pH 5.0) in absence and presence of 2 mM BCD or DIMEB (right) [kexc = 275 nm, kem = 455 nm].

studies, this concentration was selected in order to test the potential protective effects of CDs against ZEA-induced toxicity during

the following experiments. Cells were treated with ZEA (40 lM) and BCD (1.0 or 2.5 mM) alone as well as in combination. As Fig. 5 demonstrates, the applied BCD concentrations alone did not have neither positive nor negative effect on HepG2 cells. The treatment with ZEA resulted in remarkable reduction of cell number, similarly to our previous observations. However, the co-treatment of ZEA-exposed cells with BCD significantly alleviated the toxic impact of ZEA, in a concentration dependent fashion. Approximately 18% and 25% increase of the number of living cells was observed in the presence of 1.0 and 2.5 mM BCD, respectively (compared to that of control). Similar experiments were also performed with DIMEB, which forms more stable complexes with ZEA compared to the native BCD. Interestingly, even 1.0 mM DIMEB caused significant decrease of living cells (approximately 20–30%) therefore it did not have positive effect neither in its combination with ZEA (data not shown). Furthermore, 0.5 mM DIMEB did not have harmful impact on HepG2 cells under the applied circumstances; however, no significant positive or negative changes on ZEA-exposed cells were observed in the presence of 0.5 mM DIMEB (data not shown).

Fig. 5. Number of living HepG2 cells in the absence and in the presence of ZEA (40 lM) and/or BCD (1.0 and 2.5 mM) after 24-h treatment based on total DNA content/well (see details in Section 2.4). Total protein measurements were in a very good agreement with the results suggested by DAPI staining (data not shown). The applied concentrations of BCD significantly alleviated the viability loss induced by ZEA (⁄p < 0.05).

4. Discussion In agreement with previous studies we also determined two fluorescence excitation wavelength maxima of ZEA [22]. Using 275 nm as excitation wavelength higher fluorescence emission

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signals were observed compared to the excitation at 315 nm, thus this is better to apply for quantitative analytical measurements (e.g. HPLC-FLD), due to the higher sensitivity. Therefore, the fluorescence enhancement of ZEA by CDs and organic solvents was investigated using both excitation wavelengths. On the other hand, the application of 315 nm is associated with much less spectroscopic difficulties and interferences. For this reason, during the investigation of the complex formations of ZEA with CDs 315 nm was used as excitation wavelength. However, confirmatory measurements were also performed applying 275 nm, these results showed very good correlation (data not shown). Deprotonation of ZEA led to the blue shift of its fluorescence emission maxima (455 nm ? 413 nm). The pKa1 value of ZEA (7.6) suggests that under acidic and physiological conditions the nonionic form of ZEA predominates in aqueous solution [23]. At higher pH (10.0) where the deprotonated form of ZEA predominates, much higher CD concentrations were needed to induce spectral changes. The shift of the emission maximum of ZEA from 413 nm (the fluorescence emission maximum of deprotonated ZEA) to 455 nm (the fluorescence emission maximum of nonionic ZEA) strongly suggests that CDs form much more stable complexes with nonionic ZEA compared to its deprotonated form. Because the deprotonation of the toxin causes neither more relevant fluorescence enhancement by CDs nor more efficient complex formation with CDs, we did not continue the investigations at higher pH levels. Then ZEA-CD interactions were investigated in pH 5.0 ammonium acetate buffer because at this pH the nonionic form of ZEA highly predominates. In each case, the complex formation of ZEA with CDs resulted in the strong fluorescence enhancement of the mycotoxin. Since water molecules are able to effectively quench the fluorescence of ZEA, the presence of organic solvents or detergents as well as the apolar cavity of CDs can act as a protection toward the quenching effect [15,18,22]. b-cyclodextrins (except of QABCD) induced much higher fluorescence enhancement of ZEA compared to c-cyclodextrins (GCD and HPGCD). Among the tested chemically modified b-cyclodextrins only the methylated derivatives (RAMEB and DIMEB) caused higher fluorescence enhancement of ZEA than BCD. This is in a good agreement with the study of Dall’Asta et al. [15], where the complex formation of ZEA with BCD, HPBCD and DIMEB were examined. It is also very interesting that the randomly methylated-CD (RAMEB) exerted weaker impact to ZEA than the 2,6-di-O-methylated derivative (DIMEB). Based on previous studies [15,18] and our data (Table 1), b-CDs are able to form stable complexes with ZEA (mainly with its nonionic form). The stability of ZEA-CD complexes is approximately 100-fold higher compared to previously reported aflatoxin B1-CD and citrinin-CD complexes [11,12]. In agreement with previous studies [15,18], our results also suggest 1:1 stoichiometry during the complex formation (Fig. 3). Complex stability of ZEA with the native BCD is approximately 1.6-fold higher than with GCD, HPGCD or QABCD, and similar with HPBCD (Table 1). The stability constants of ZEA complexes with SBCD, RAMEB and DIMEB were 1.6-fold, 2.3-fold and 4.9-fold higher than with the native BCD, respectively. Similar difference between BCD and DIMEB was described by Dall’Asta et al. [15]. Since the presence of different organic solvents results in the fluorescence enhancement of ZEA as well [24] and because of we did not find data regarding the direct comparison of fluorescence enhancement of ZEA by organic solvents vs. CDs, we tested the effectiveness of methanol and acetonitrile (the most commonly applied organic components of reversed-phase HPLC eluents) vs. 2 mM BCD and DIMEB in ammonium acetate buffer (pH 5.0). Our results indicate that the application of methanol and/or acetonitrile can strongly enhance the fluorescence signal of ZEA and in this way increase the sensitivity of its fluorescence detection. However,

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the comparison with CDs in aqueous solution highlights that the utilization of BCD or DIMEB leads to much higher fluorescence enhancement than that was observed in methanol or acetonitrile (Fig. 4). Previous studies [18,20,24] and our observations strongly suggest the very good suitability of CDs to improve the sensitivity of fluorescent analytical methods, even in absence of organic solvents. Fig. 4 also demonstrates that very similar fluorescence enhancement of ZEA by BCD and DIMEB was determined using 275 nm as excitation wavelength compared to data shown in Table 1 (where kexc = 315 nm). Furthermore, the calculated stability constants of ZEA-BCD and ZEA-DIMEB complexes (3.94 and 4.71, respectively) also showed a good correlation (see Benesi–Hildebrand plots in Fig. 4). The magnitude of complex stabilities (log K  3.7–4.7) was determined for ZEA-CD complexes allowed us to test the potential protective effects of b-cyclodextrins against ZEA on in vitro cell culture. Since b-CDs form stable complexes with ZEA, we hypothesized that the entrapment of ZEA by CDs may decrease the cellular uptake of the mycotoxin, and in this way alleviate the ZEA-induced toxicity on HepG2 cells. BCD was nontoxic at the applied 1.0 and 2.5 mM concentrations. Furthermore, co-treatment of ZEA-exposed cells with BCD effectively alleviated the ZEA-induced toxicity on HepG2 cells (Fig. 5). On the other hand, even 1.0 mM DIMEB alone caused significant decrease of living cells. This phenomenon can be explained by two facts: (1) DIMEB forms very stable complex with cholesterol (log K = 5.74) [25], which is an important constituent of cell membranes, therefore millimolar concentration of DIMEB can disrupt cell physiology [26,27]. (2) DIMEB can be taken up by fluid-phase endocytosis into cells and its cellular accumulation is probably also able to disrupt cell physiology [28]. When cells were treated only with 0.5 mM DIMEB, no negative effects on HepG2 cells were observed. However, co-treatment of ZEA-exposed cell with 0.5 mM DIMEB failed to alleviate ZEA-induced toxicity, despite of its much stronger affinity toward ZEA compared to BCD. This observation can be explained again by the complex formation of CDs with cholesterol. BCD forms much more stable complex with ZEA (log K = 3.99) than with cholesterol (log K = 3.28) [25], therefore BCD can permanently entrap the mycotoxin molecule, resulting in the poor uptake of ZEA by HepG2 cells. In contrast, DIMEB shows substantially higher preference toward cholesterol (log K = 5.74) than toward ZEA (log K = 4.76), thus the guest molecule (ZEA) can be easily exchanged by cholesterol, causing the loss of the protective effect of DIMEB. These observations suggest that we need to find chemically modified CDs which form much more stable complex with ZEA than with cholesterol, if we would like to apply CDs even in vivo detoxification. Acknowledgements The authors thank Zsófia Gerner for her assistance in the experimental work. This work was supported by PTE ÁOK-KA-2013/15 grant. Financial support of the Environmental industry related innovative trans- and interdisciplinary research team development in the University of Pécs knowledge base (SROP-4.2.2.D-15/1/Kon v-2015-0015) is highly appreciated. The present scientific contribution is dedicated to the 650th anniversary of the foundation of the University of Pécs, Hungary. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jphotobiol.2015. 07.009.

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