Unraveling the mechanisms involved in zearalenone-mediated toxicity in permanent fish cell cultures

Toxicon 88 (2014) 44e61

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Unraveling the mechanisms involved in zearalenonemediated toxicity in permanent fish cell cultures Constanze Pietsch a, b, *, Jürg Noser c, Felix E. Wettstein d, Patricia Burkhardt-Holm b, e a

Zurich University of Applied Sciences (ZHAW), Institute of Natural Resource Sciences (IUNR), Gruental, P.O. Box, CH-8820 Waedenswil, Switzerland b Programm Man e Society e Environment, Department of Environmental Sciences, University of Basel, Vesalgasse 1, CH-4051 Basel, Switzerland c €ubernstrasse 12, CH-4410 Liestal, Switzerland Kantonales Laboratorium Basel, Gra d €nikon (ART), Research Station ART, Reckenholzstrasse 191, CH-8046 Zürich, Switzerland Agroscope Reckenholz-Ta e Department of Biological Sciences, University of Alberta, CW 405 Biological Sciences Building, T6G 2E9, Edmonton, AB, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 March 2014 Received in revised form 30 May 2014 Accepted 10 June 2014 Available online 17 June 2014

The world-wide occurrence of zearalenone (ZEN) as a contaminant in feed for farm animals and fish requires the evaluation of toxicity mechanisms of action of ZEN. The present study investigates possible metabolization of ZEN in fish cell lines suggesting that mainly glucuronidation takes place. It demonstrates that concentrations up to 20,000 ng ml
1 ZEN are capable of influencing cell viability in permanent fish cell cultures in a doseeresponse manner with different response patterns between the five tested cell lines, whereby lysosomes appeared to be the main target of ZEN. ZEN toxicity is often discussed in the context of oxidative stress. Our study shows a biphasic response of the cell lines when reactive oxygen species (ROS) production is monitored. Damage in cells was observed by measuring lipid peroxidation, DNA strand breaks, and alterations of intracellular glutathione levels. Metabolization of ZEN, especially at concentrations above 7500 ng ml
1 ZEN, does not prevent cytotoxicity. ZEN as an estrogenic compound may involve processes mediated by binding to estrogen receptors (ER). Since one cell line showed no detectable expression of ER, an ER-mediated pathway seems to be unlikely in these cells. This confirms a lysosomal pathway as a main target of ZEN in fish cells. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Mycotoxin Cytotoxicity Oxidative stress Genotoxicity Estrogenic action

1. Introduction Mycotoxins are widely distributed in our environment. Most mycotoxins are relatively stable in aquatic ecosystems, and thus, relevant concentrations can be found in  et al., 2001; Spengler et al., 2001; surface waters (Lagana

* Corresponding author. Zurich University of Applied Sciences (ZHAW), Institute of Natural Resource Sciences (IUNR), Gruental, P.O. Box, CH-8820 Waedenswil, Switzerland. Tel.: þ41 58 934 5613. E-mail address: [email protected] (C. Pietsch). http://dx.doi.org/10.1016/j.toxicon.2014.06.005 0041-0101/© 2014 Elsevier Ltd. All rights reserved.

Bucheli et al., 2008; Gromadzka et al., 2009; Kolpin et al., 2010). Mycotoxins in feed have been shown to possess multifold detrimental effects on aquatic organisms (Poston €ll et al., 2011; Hooft et al., 1982; Santacroce et al., 2008; Do et al., 2011; Sanden et al., 2012). Moreover, contamination of animal feed with mycotoxins has been found to occur (Roberts and Patterson, 1975; Bryden et al., 1980; Abdelhamid, 1990; Juszkiewicz and PiskorskaPliszczynska, 1992; Placinta et al., 1999; Ranjan and Sinha, 1991). Consequently, mycotoxins have become a worldwide interest.

C. Pietsch et al. / Toxicon 88 (2014) 44e61

Zearalenone (ZEN), a resorcylic acid lactone, is an important mycotoxin produced by fungi of the genus Fusarium. Fusarium-derived mycotoxins have been identified as aquatic micropollutants in surface waters (Bucheli et al., 2008; Hoerger et al., 2009). It has been reported that mycotoxins contribute to the estrogenic environmental exposure in aquatic systems (Hartmann et al., 2008). Especially ZEN has been shown to act as a typical estrogenic compound showing high estrogenic potencies via estrogen receptors of rainbow trout (Bucheli et al., 2005) and effects on reproduction of zebrafish (Danio rerio) and early life stages of fathead minnow (Pimephales promelas) have been described (Johns et al., 2009; Schwartz et al., 2010). Although metabolization of ZEN in fish has  et al., 2004), several merarely been described (Lagana tabolites of ZEN, namely a-ZEL and b-ZEL, have also estrogenic effects in fish (Matthews et al., 2000). Further metabolization of toxic compounds in phase II of the biotransformation pathway in vertebrates often includes glucuronidation via UDP-glucuronosyltransferases (UGTs) (Meech and Mackenzie, 1997; Grancharov et al., 2001). Glucuronidation, which was observed for ZEN as well (Warth et al., 2013), is the combining with glucuronic acid that leads to the formation of more hydrophilic derivatives. The toxic effects of ZEN in mammals and fish have been discovered. Many studies assessed toxic effects of ZEN and its metabolites in mammalian cell lines and ZEN has been found to be hepatotoxic (Maaroufi et al., 1996; Obremski  et al., 2001), immunotoxic (Marin et al., 1999; Conkova et al., 1996; Berek et al., 2001), and genotoxic (Ouanes et al., 2003; Abid-Essefi et al., 2004; Lioi et al., 2004; Gao et al., 2013). In fish, the mechanisms of toxicity of ZEN have not yet been demonstrated but effects on reproduction, development, and iron metabolism were observed (Johns et al., 2009; Schwartz et al., 2010; Wo zny et al., 2012; Bakos et al., 2013). However, a connection of the estrogenic potential and toxic effects of ZEN has not yet been investigated in fish. Generally, binding of estrogenic compounds to estrogen receptors that function as ligandactivated transcription factors leads to their conformational changes resulting in DNA binding and regulation of gene transcription (MacGregor and Jordan, 1998). Thus, treatment with estrogenic compounds regulates the expression of many genes including those which encode products involved in cell survival and apoptosis (Tanzer and Jones, 1997; Singer et al., 1998; Linford et al., 2001). From a study using mammalian cell lines it has been concluded that it is unlikely that the toxicity of this substance is solely due to its estrogenic potential because other mechanisms, such as oxidative stress, could be involved (Abid-Essefi et al., 2004). Generation of reactive oxygen species (ROS) is involved in important physiological functions of all aerobic organisms. However, an excess of ROS formation can lead to disturbances of metabolic pathways, depletions of cellular antioxidants, and damage to macromolecules, including DNA, proteins and lipids. This oxidative injury can result in a reduction of differentiation and proliferation of cells or can lead to apoptosis (Abid-Essefi et al., 2004; Hassen et al., 2007; Bouaziz et al., 2008). However, in fish, the generation of oxidative stress due to exposure to ZEN has not yet investigated. Thus, possible

45

oxidative stress due to exposure to ZEN was analyzed in the present study using fish cell lines to show the involvement of this pathway in the toxicity of this mycotoxin even outside mammalian cell systems. In aquatic toxicology, permanent fish cell cultures can be used as a tool to generate initial information on the metabolism and effects of pollutants (Babich et al., 1986; Segner, 2004). Well-targeted experiments may indicate whether metabolization of xenobiotics involves mechanisms of activation or deactivation and whether damage to biological targets such as lipids, DNA, and proteins can be expected. In the present study, five permanent fish cell lines were used for the first time to evaluate the comparative cytotoxic effects of ZEN in vitro and to delineate its doseeresponse relationship. This was necessary to show cell line-specific differences in toxicity of ZEN. The concentrations of ZEN have been chosen according to specifications in the literature on high ZEN (up to 1500 mg kg
1) in grains and feedstuff in Europe (Coppock et al., 1990; Veldman et al., 1992; Charmley et al., 1994; Whitlow and Hagler, 2005; Mankevi ciene_ et al., 2007; Driehuis et al., 2008). Mycotoxin contents in fish feed have rarely been investigated but recent data show that ZEN is highly prevalent in ingredients and complete feedstuffs in aquaculture (Måge et al., 2009; Santos et al., 2010; Pietsch et al., 2013). Thus, the aim of this study was to investigate the sensitivity of five fish cell lines, including cell lines derived from different tissues of rainbow trout, carp and salmon, towards this fungal metabolite with respect to organ- and species-specific differences. Therefore, the acute toxic effects and possible oxidative damage on these cell lines were assessed as a function of ZEN concentration by using colorimetric and fluorescent dyes. Possible cellular damage was further evaluated by assessing lipid peroxidation and DNA damages, and levels of reduced glutathione, as one of the most important antioxidants involved in phase II reactions. The involvement of direct estrogenic pathways in ZEN action was assessed by investigating the presence or absence of estrogen receptors alpha or beta in each of the cell lines by means of semi-quantitative RT-PCR. 2. Materials and methods 2.1. Chemicals All chemicals were obtained from SigmaeAldrich (Buchs, Switzerland) unless indicated otherwise. Zearalenone (ZEN; purity  99%; lot number 020M4056) was dissolved in ethanol. Solvent concentrations in the exposition media did not exceed 0.1% (v/v). Stock solutions were maintained in darkness at 4  C. 2.2. Cell culture and exposure All fish cell cultures and exposures were conducted at 19  C. The cells derived from rainbow trout liver (RTL-W1 cells) and gills (RTgill-W1 cells), and salmon head kidney (SHK-1) cells were maintained in L-15 medium at 19  C without addition of CO2. The cells from epithelial gonadal cell line of rainbow trout (RT EQ clone 8; strain No. 994) were maintained in Dulbecco's modified Eagle medium

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C. Pietsch et al. / Toxicon 88 (2014) 44e61

diluted 1:1 (v/v) with Ham F12 medium (LuBioScience, Lucerne, Switzerland). Carp brain (CCB) cells were cultured in Eagle minimum essential medium containing Earle's salts. The cell culture media contained 10% fetal bovine serum (FBS) for subculturing with the exception of the medium for RTL-W1 cells which contained 5% FBS. All cells were cultured, harvested, and seeded into 96-well microtitre plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) at a concentration of 30,000 cells per well as has been described by Pietsch et al. (2011). For exposure to chemicals cells were washed with Earle's medium and the reagent was added in serum-free Earle's medium for 24 h. All experiments were run in 3 independent replicates.

9.1 ml ml
1 hydrochloric acid (37%), 0.06% (w/v) butylated hydroxytoluene (BHT), and 866.9 ml destilled water. Subsequently, all samples were heated at 70  C for 90 min, cooled to room temperature, and centrifuged at 16,000 g for 10 min at room temperature. Standards containing malondialdehyde (MDA) concentrations ranging from 0 to 3200 nM MDA were prepared similarly. Optical densities of all samples were read at 532 nm (Infinite M200, Tecan €nnedorf, Switzerland). In parallel, aliquots of Group Ltd., Ma the cell homogenates were also used for protein determinations using the bicinchoninic acid (BCA) assay (Sigma) according to the manufacturer's protocol. 2.5. Comet assay

2.3. Measurement of cell viability and ROS production After exposure to different ZEN concentrations membrane integrity was assessed using the MTT assay according to Mosmann (1983), the neutral red (NR) test (Babich and Borenfreund, 1991) and the propidium iodide method. The concentration values resulting in 50% reduction of cell survival (EC50) were calculated for these viability assays by regression of the percentage of the response to ZEN against the natural logarithm of the compound concentration. The resulting regression equations were used to calculate the concentration of compound that would result in 50% reduction of cell viability. ROS production was measured using the fluorescent dye H2DCF-DA (LuBioScience, Lucerne, Switzerland). In addition, nitroblue tetrazolium chloride (NBT) tests were conducted. All assays have been described in detail by Pietsch et al. (2011). Each test was repeated three times and each microtitre plate had its own control treatment containing the solvent ethanol. Treatments on each plate were based on the mean of 8 replicate treatments per ZEN concentration or control incubation. 2.4. Lipid peroxidation assay Cell culture media containing 180,000 cells from each cell line were distributed in each well of separate 24-well plates (BD Falcon™ 24-well Multiwell Plate, Becton Dickinson AG, Allschwil, Switzerland) leading to comparable cell densities to those that have been used in the 96-well plate assays. Cells were allowed to adhere over night, washed with Earle's medium and treated with ZEN for 24 h at different concentrations in the same medium. A positive control was treated with 1.5 mM H2O2 in parallel. Thereafter, cells were washed with phosphate buffered saline (PBS), followed by a washing step with 200 ml versene (LuBioScience, Lucerne, Switzerland) and harvested by trypsinization. Trypsinization was terminated by addition of PBS containing 5% fetal calf serum (FCS). Cells were centrifuged for 3 min at 10,000 g at 4  C (Centrifuge 5415R, Eppendorf, Basel, Switzerland). The cell pellet was resuspended in PBS and homogenized using an UltraTurrax T8 (IKA Werke, Staufen, Germany) for 45 s at maximum speed. TBARs assay was conducted according to Rau et al. (2004) with the following modifications. A volume of 200 ml cell homogenate were mixed with 400 ml TBARs solution (Holt et al., 1986) containing 3.75 mg ml
1 thiobarbituric acid (TBA), 20% (w/v) trichloric acid (TCA),

Genotoxicity was analyzed as described by McKelveyMartin et al. (1993) with the following modifications. RTL-W1 cells were cultured in 24-well plates using 180,000 cells per well. After adherence of cells wells treated with the different ZEN concentrations in duplicates and after 24 h of incubation cells were harvested as described above. Two slides (Superfrost Plus, Gerhard Menzel GmbH, Braunschweig, Germany) coated with 90 ml of 1.0% normal melting agarose (w/v; SeaKem LE Agarose, Biozym Scientific GmbH, Hessisch Oldendorf, Germany; prepared in PBS) were mounted with 0.7% low melting agarose (w/v; SeaPlaque GTG Agarose, Biozym Scientific GmbH, Hessisch Oldendorf, Germany; prepared in PBS) containing the harvested cells from one well at 37  C. These slides were placed on cold metal plates for 5 min and dried at 37  C for further 5 min. Subsequently, additional 90 ml of 0.7% low melting agarose were spread on the slides as a protective layer, cooled for 5 min and dried at 37  C for further 5 min. Lysis of cells was accomplished by incubation of slides in a mixture of 100 mM ethylene-diamine-tetraacetic acid (EDTA) disodium salt hydrate, 2.5 M NaCl, 1% (v/v) Triton X100, 10% (v/v) dimethyl sulfoxide (DMSO), and 1% N-lauroylsarcosine sodium salt at pH 10.0 for 3 h at 4  C in the dark. Afterward, slides were electrophoresed at 25 V and 0.3 A for 25 min in ice-cold alkaline buffer containing 12 g L
1 NaOH, and 0.37 g L
1 EDTA using a horizontal electrophoresis tank (Maxigel ECO 2, Apelex, Evry Cedex, France) followed by neutralization of slides by using 400 mM TriseHCl (pH 7.4) for 2 min. Until staining with 1.56 mg ml
1 ethidium bromide slides were kept at 4  C in the dark. Individual comets were viewed using a Nikon epifluorescence microscope (Nikon Eclipse E400 equipped with a Nikon Digital Camera DXM1200F; Nikon AG, Egg, Switzerland) at a 200 magnification. Image analysis was done using the software TriTek CometScore™ v 1.5 (TriTek, Sumerduck, USA) and DNA damages were quantified as increases in tail moments (the product of the DNA percentage in the tail and the tail length) which are indicators of damage that is proportional to the number of strand breaks in cells. 2.6. Measurement of reduced and oxidized glutathione Cells from each cell line were cultured in 24-well plates, treated and harvested as described above. The resulting cell pellets were suspended in homogenization buffer

C. Pietsch et al. / Toxicon 88 (2014) 44e61

containing 0.1 M potassium phosphate (pH 6.8), 154 mM KCl, and 5 mM diethylenetriaminepentaacetic acid (DTPA). These cell suspensions were homogenized for 15 s on ice (UltraTurrax T8, IKA Werke, Staufen, Germany) and cell debris was removed by centrifugation at 16,000 g for 3 min (Centrifuge 5415R, Eppendorf, Basel, Switzerland). Samples were then mixed with the same volume of ice-cold TCA buffer [40 mM HCl, 10 mM DTPA, 20 mM ascorbic acid, 10% (v/v) trichloroacetic acid (TCA)]. These mixtures were purified by centrifugation using Ultrafree® Centrifugal Filters (Millipore™, distributed by Carl Roth AG, Karlsruhe, Germany) and stored at
80  C until analyses. Reduced glutathione (GSH) was analyzed according to Senft et al. (2000) with the modifications for a microplate assay as described by Siraki et al. (2004). Therefore, 90 ml of the cell samples were mixed with 40 ml 1 M potassium phosphate buffer (pH 6.8). In parallel, additional wells were treated with 40 ml of 7.5 mM N-ethylmaleimide (NEM) in 1 M potassium phosphate buffer. After 15 min of incubation, 100 ml o-phthalaldehyde (OPA, 37.5 mM in 0.1 M potassium phosphate buffer, pH 6.8) were added. Fluorescence emission at 430 nm was read after 30 min of incubation in the dark using excitation at 345 nm. GSH standards were prepared from a 1 mM stock solution in redox-quenching buffer (RQB) containing 20 mM HCl, 5 mM DTPA and 10 mM ascorbic acid, pH 6.8. Traces of glutathione disulfide (GSSG) were removed by addition of 25 mg zinc powder followed by centrifugation at 16,000 g for 3 min (Centrifuge 5804 R, Eppendorf, Basel, Switzerland). Serial dilutions from this GSH stock solution were made using RQB containing 5% (v/v) TCA. In parallel, GSSG was analyzed from all samples by incubation of 90 ml of the cell extracts with 40 ml of 7.5 mM N-ethylmaleimide (NEM). Subsequently, 10 ml dithionite (DT) or RQB (as a blank) were added to the wells and the plates were incubated for 60 min at room temperature in the dark. Thereafter, 100 ml OPA solution were added and fluorescence values were measured as described above. GSSG dilutions in RQB containing 5% (v/v) TCA were used for the establishment of the standard curves. 2.7. Analyses of ZEN in cell culture media and cells Since ZEN is mainly metabolized in liver (Kiessling and  et al., 2004) initial investigations Pettersson, 1978; Lagana were conducted using the RTL-W1 cell line derived from hepatic tissue of rainbow trout. In order to analyze distribution of ZEN between media solutions and cells in an initial study, adherent RTL-W1 cells from cell culture flasks were washed with Earle's medium, exposed to 300 ng ml
1 ZEN for 24 h, and harvested by trypzinization. After centrifugation cell pellets were directly extracted by addition of 1 ml acetonitrile for 1 min, followed by centrifugation and transfer of 500 ml of the supernatants to HPLC vials. Volumes of 25 ml of the internal deuterated ZEN (D6-ZEN) standard were added to cell extracts. All samples were evaporated to complete dryness under a gentle air stream at 40  C. Subsequently, 1 ml of a water-acetonitrile-mixture (80/20; v/v) was added to the media samples. After that all samples were mixed for 10 s. For determination of zearalenone (ZEN) and its metabolites zearalanone (ZAN), alpha-zearalanol (a-ZAL), beta-zearalanol (b-ZAL), and

47

zearalenol (ZEL), which play a role during metabolization of ZEN in higher vertebrates (Schaut et al., 2008; Abid-Essefi et al., 2009), a HPLC-negative electrospray ionization (-ESI)-MS/MS analytical method was used as described by Hartmann et al. (2007). Quantification of ZEN was enabled by using the internal standard method with water/acetonitrile solutions (80/20; v/v), containing increasing amounts of ZEN and constant amounts of D6-ZEN. ZEN (99% purity), ZAN (98% purity), alpha-ZAL (97% purity), beta-ZAL (98% purity), alpha-ZEL (98%) and betaZEL (95% purity) were purchased from Sigma (Buchs, Switzerland). The deuterated internal standards D4-alphaZAL, D4-beta-ZAL, D4-alpha-ZEL (95% purity) and D4beta-ZEL (95% purity) were purchased from RIVM (Bilthoven, The Netherlands). D6-ZEN was prepared by basecatalyzed hydrogenedeuterium exchange on native ZEN as described in Miles et al. (1996). The purity of the internal standard for ZEN (D6-ZEN) was tested by scanning the masses of ZEN and Dn-ZEN (n ¼ 1e9). The relative amounts were 0.16%, 0.10%, 0.83%, 6.16%, 25.07%, 38.32%, 23.13%, 5.50%, 0.70% and 0.03% for ZEN, D1-ZEN, D2-ZEN, D3-ZEN, D4-ZEN, D5-ZEN, D6-ZEN, D7-ZEN, D8-ZEN, and D9-ZEN, respectively. Ratios of the different grades of deuterated ZEN's did not change over time. Therefore, D6-ZEN was found to be a suitable internal standard and the other deuterated ZEN products did not influence quantification. Instrument linearity was determined with ZEN calibration solutions prepared in Earle's medium. The procedure to determine method/instrument precision and method detection/quantification limit is described in Bucheli et al. (2008). The ion suppression was determined by the method of standard addition (Freitas et al., 2004). An ion suppression for ZEN of 17% was calculated (which was compensated for by normalization to D6-ZEN). The limit of detection (LOD) obtained from the signal to noise ratio was between 1.5 and 5 ng ml
1 ZEN, whereas a limit of quantitation (LOQ) was between 4.5 and 8 ng ml
1 ZEN, depending on the analyzed matrix. Quality control parameters were determined in Earle's medium. Involvement of phase II reactions in metabolization of ZEN in fish cells was investigated in single exposures exemplarily after incubation of only RTL-W1 cells with 300 ng ml
1 ZEN in Earle's medium for 24 h. De-glucuronidation was achieved using b-glucuronidase from Helix pomatia according to Frandsen (2007) with the following modifications. Sodium acetate solution was added to the remaining cell media samples from all cell cultures to a final concentration of 100 mM, the pH was adjusted to 5.5, and 8.8 U ml
1 bglucuronidase in 0.2% NaCl were supplemented. Subsequently, samples were mixed and incubated for 24 h at 37  C. The de-glucuronidation was stopped by addition of ethanol, followed by centrifugation and storage of the supernatant at
20  C. Since the b-glucuronidase from mollusks also shows sulfatase activity the enzymatic deconjugation was also conducted at pH 6.2 to favor this enzymatic reaction. In parallel, acid hydrolysis was conducted as described by Frandsen (2007). In a separate experiment high performance liquid chromatography with diode array detection (HPLC-DAD) was used to quantify ZEN metabolization in all cell lines. To assess the amount of free ZEN and its conjugates with

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glucuronic acid produced by each cell line, cells were treated as follows. A volume of 5 ml cell suspension was cultured at initial concentrations of 225,000 cells per ml in cell culture flasks (25 cm2) over night. Subsequently, cells were washed with serum-free Earle's medium and ZEN solutions with concentrations ranging from 0 to 20,000 ng ml
1 ZEN in Earle's medium were applied in duplicate for 24 h. To confirm nominal exposure concentrations and ZEN stability analytically mycotoxin concentrations in exposure media were verified by sampling freshly prepared solutions before exposure and media directly from the cell culture flasks after exposure of cells. Non-adherent cells were removed from medium samples by centrifugation at 300 g for 3 min and added to the samples from adherent cells harvested directly after sampling of the cell media to assess ZEN concentrations in cells. One part of media samples was mixed with the same volume acetonitril and stored at
20  C before analyses. From each cell culture flask cells were also harvested by trypsinization which was stopped by addition of PBS containing 5% FCS. Cells were then centrifuged and resuspended in PBS. One part of this suspension was supplemented with acetonitril, incubated for 15 min with the same volume of acetonitril, centrifuged at 300 g for 3 min and stored at
20  C for later analyses. The other part of the cell suspension was incubated with b-glucuronidase for 24 h as described above before addition of acetonitrile as has been described for the preparation of cell media samples. Analyses were performed on a HPLC-DAD (high performance liquid chromatography with diode array detection) by fluorescence detection on a HP 1046A using a LiChrospher 100 RP8 column (250 mm) with a cutoff filter at 407 nm. The mobile phase consisted of acetonitrile and nanopure water (40:60, v:v), reaching a ratio of 60:40, (v:v) after 20 min, followed by a ratio of 95:5 (v:v) for further 5 min at a flow rate of 1 ml min
1. The excitation wavelength of the fluorescence detector was set at 274 nm, while the emission wavelength was 466 nm. The limit of detection was 25 ng ml
1 and the mean recovery was approximately 88%. 2.8. RNA isolation, PCR and sequencing of ERa and ERb from cell cultures and liver tissue of fish

DNA Polymerase dNTPack (Roche Applied Science, Basel, Switzerland). Oligonucleotid primers for PCR measurements were designed based on known sequences for fish ERa and ERb (Supplementary Table) and were obtained from Microsynth (Balgach, Switzerland). All primers were validated for PCR by sequencing after gel extraction of PCR products according to the manufacturer's protocol (QIAquick gel extraction kit, Qiagene, USA) followed by direct sequencing with the BigDye sequencing chemistry (Applied Biosystems), analysis on an ABI 3130xl genetic analyzer (Applied Biosystems) and subsequent comparison by Basic Local Alignment Search Tool (BLAST) using the databases at the National Center for Biotechnical Information (NCBI) at the National Library of Medicine. Polymerase chain reaction were performed in Taq polymerase buffer using 1 U Taq polymerase, MgCl2 (1.5 mM), dNTPs (200 mM), and primers (200 nM) in a total volume of 20 ml. PCR conditions included 4 min at 94  C, followed by 40 s at 94  C, 40 s at annealing temperature and 40 s at 72  C, and a final extension for 10 min at 72  C using a PCR system (Eppendorf Mastercycler EP Gradient S 96, Eppendorf, Basel, Switzerland). PCR reactions were optimized with respect to the selection of the annealing temperature. The PCR products were transferred to 3% agarose gel containing 0.005% ethidium bromide (stock solution 10 mg ml
1, v/v; Carl Roth AG, Karlsruhe, Germany) and after horizontal gel electrophoresis in TAE buffer (242 g TRIS-HCl, 37.2 g sodium EDTA, 57.1 ml glacial acetic acid in 1 L distilled water, pH 7.5) for 35 min at 100 mV PCR products were visualized using AlphaImager®EP (Alpha Innotech distributed by Biozym Scientific GmbH, Hessisch Oldendorf, Germany). 2.9. Statistial analyses Data of ROS measurements, cell viability, lipid peroxidation, glutathione levels and comet assay determinations are presented as the mean ± standard error of the mean (SEM) from 3 independent cell cultures. Effects of treatments were determined by comparison of treatment groups to controls using Friedman test followed by Wilcoxon test (SPSS 16.0 for Windows). A p value of <0.05 was accepted as being statistically significant. 3. Results

Cells were grown to confluence in cell culture flasks (TPP Techno Plastic Products AG, Trasadingen, Switzerland), and harvested by trypsinization. Thereafter, cells were washed with 1 ml PBS and frozen at
80  C in 300 ml TRIzol (Invitrogen AG, Basel, Switzerland). RNA was isolated from cell culture samples and snap-frozen liver € gi et al. samples of carp and rainbow trout according to Bo (2002) using a phenol-chloroform method including a DNase treatment according to the manufacturer's protocol (DNA-free™ kit, Ambion®, distributed by Life Technologies Europe B.V., Zug, Switzerland). Total RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems distributed by Life Technologies Europe B.V., Zug, Switzerland) according to the manufacturer's directions. Two micrograms of the resulting cDNA were diluted in nuclease-free water and used for semiquantitative PCR using the FastStart Taq

3.1. ZEN concentrations in exposure media and cell extracts The experiments with RTL-W1 cells showed that ZEN is largely not detectable as free ZEN by LC-MS/MS in exposure media after exposure to 300 ng ml
1 ZEN for 24 h (Table 1). Metabolites of ZEN, namely zearalanone (ZAN), alphazearalanol (a-ZAL), beta-zearalanol (b-ZAL), and zearalenol (ZEL) could not be identified in media after cell exposure. Enzymatic de-conjugation increased the detectable amount of free ZEN in exposure media to 76e82% of the amount that was initially applied to the cells, depending on the pH that was used for the de-conjugation. This suggests that ZEN had been taken up by these cells and was released to the surrounding medium as ZEN-glucuronide. Acid hydrolysis as described by Frandsen (2007) was not valuable to assess glucuronidation of ZEN since it only

C. Pietsch et al. / Toxicon 88 (2014) 44e61 Table 1 ZEN in cell media from RTL-W1 cells for 24 h with 300 ng ml
1 ZEN. Samples were treated with b-glucuronidase prior to analyses by LC-MS/ MS. ZEN ng ml
1 ZEN in Earle's medium without cell exposure ZEN after 24 h cell exposure ZEN after 24 h cell exposure treated with b-glucuronidase pH 5.5 ZEN after 24 h cell exposure treated with b-glucuronidase pH 6.2 ZEN after 24 h cell exposure treated by acid hydrolysis

272 20 223 208 29

marginally increased the amount of free ZEN in the samples. Analyses of ZEN in cell culture media by HPLC-DAD from all cell lines after 24 h of incubation with this mycotoxin (Fig. 1) showed that in all cell media exposure to ZEN for 24 h reduced the detectable amount of free ZEN compared to the initial media concentrations. Especially, initial concentrations of less than 625 ng ml
1 ZEN were no longer detectable after 24 h of exposure to cells. The fate of ZEN at higher concentrations, however, differed between the cell lines. When applied to the gill cell line an average of 24% of the initial applied ZEN concentration could be recovered in the medium after 24 h, whereas the other cell lines derived from rainbow trout (RTL-W1 and RT EQ clone 8 cells) showed decreasing ZEN concentrations in the medium averaging between 35 and 38% of the initial exposure medium concentration. In the medium of SHK-1 cells and in CCB cells averages of approximately 50% of the initially applied ZEN concentrations could be detected after 24 h and especially at ZEN concentrations above 10,000 ng ml
1 the capacity to remove ZEN from the exposure medium decreased considerably in these cell lines.

49

ZEN analyses from cell extracts showed different results (Fig. 1). At exposure medium concentrations of 313 ng ml
1 ZEN could rarely be detected in any of the cell lines. In addition, at higher exposure medium concentrations, only low ZEN amounts were accumulated in RT EQ clone 8 cells and CCB cells (showing average values of 20% and 27%, respectively). SHK-1 cells showed an average ZEN value of 48% at exposure concentrations higher than 313 ng ml
1 ZEN. The RTL-W1 and RTgill-W1 cells showed a linear trend between the internal cell concentrations and the initial mycotoxin concentrations in the exposure media that were applied. Especially at exposure medium concentrations above 7,500 ng ml
1 ZEN the mycotoxin concentrations in these cell extracts amounted between 82 and 85% of the initial concentration of the surrounding medium indicating that ZEN is taken up and accumulated to a certain extent in these cell lines. De-glucuronidation for 24 h improved the ZEN detection by HPLC-DAD in the exposure media with initial concentrations of less than 625 ng ml
1 ZEN (Fig. 2) which suggests that at low ZEN concentrations the mycotoxin is conjugated in the cells and released to the surrounding medium rapidly. Increased ZEN concentrations were also observed in media from RTL-W1 and RTgill-W1 cells after incubation with the highest ZEN concentration followed by de-glucuronidation (þ53% and þ184%, respectively) compared to samples without enzymatical de-conjugation. In cell extracts less ZEN was detectable in samples treated with b-glucuronidase compared to untreated cell extracts when low ZEN concentrations were used. Less ZEN was especially found after de-glucuronidation of cell extracts from RTL-W1 and RTgill-W1 cells treated with ZEN. However, concentrations of 5,000 ng ml
1 or higher often resulted in higher detection of free ZEN in extracts from the other cell lines when glucuronidase treatment was applied compared to extracts without de-glucuronidation. This

150

cells medium

ZEN [ng ml-1]

125 100 75 50 25

EtOH 313 625 1025 2500 5000 7500 10000 15000 20000

EtOH 313 625 1025 2500 5000 7500 10000 15000 20000

EtOH 313 625 1025 2500 5000 7500 10000 15000 20000

EtOH 313 625 1025 2500 5000 7500 10000 15000 20000

EtOH 313 625 1025 2500 5000 7500 10000 15000 20000

0

RTL-W1

RTgill-W1

RT EQ clone 8

SHK-1

CCB

nominal ZEN concentrations [ng

ml-1]

Fig. 1. ZEN concentrations in medium and cell extracts after 24 h of exposure in the permanent fish cells cultures (RTL-W1, RTgill-W1, RT EQ clone 8, SHK-1, and CCB cells) expressed as percentages of the exposure media before incubation with cells.

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C. Pietsch et al. / Toxicon 88 (2014) 44e61 cells

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nominal ZEN concentrations [ng

ml-1]

Fig. 2. ZEN concentrations in medium (A) and cell extracts (B) after 24 h of exposure in the permanent fish cells cultures (RTL-W1, RTgill-W1, RT EQ clone 8, SHK1, and CCB cells) followed by b-glucuronidase treatment for 24 h expressed as percentages of the exposure media before ZEN incubation of cells.

de-glucuronidation led to 30e33% higher ZEN levels detected in cell extracts when RT EQ clone 8, SHK-1 or CCB cells were treated with 20,000 ng ml
1 ZEN for 24 h. 3.2. Influence on cell viability Cell viability studies demonstrated that after 24 h of incubation with the mycotoxin ZEN a biphasic response was observed in RTL-W1, SHK-1 and in CCB cells in NR assays, whereas the same assay in RTgill-W1 and RT EQ clone 8 cells showed only a decreased NR uptake at concentrations above 625 ng ml
1 ZEN and 1250 ng ml
1 ZEN, respectively (Fig. 3). Cell viability was also measured using the MTT assay determining membrane integrity and mitochondrial activity (Fig. 4). This assay revealed a biphasic response in all cell lines which was especially pronounced in CCB cells (Fig. 4E). Membrane integrity of cells was also investigated using the PI assay showing strongly increased membrane damages at concentrations higher than 7,500 ng ml
1 ZEN in all cell lines (Fig. 5). This assay also revealed that the loss of membrane integrity was very pronounced in RT EQ clone 8 cells which showed increased damages to membranes at all ZEN concentrations used. Looking at the EC50 values (the concentration values resulting in 50% reduction of cell response in control incubations) for these tests, the assessment of membrane integrity using these assays yielded different results (Table 2). The NR assays yielded lower EC50 values than the MTT assays. EC50 values for the PI assays were found to be quite similar for all fish cell lines and showed higher values than for the NR assay.

(Fig. 6). Similarly, a biphasic effect of ZEN on ROS production in all cell cultures was also measurend by the fluorescent dye H2DCF-DA (Fig. 7). The ROS production after treatment of cells with low concentration of ZEN ranging from 313 to 625 ng ml
1 ZEN was found to be significantly reduced with the exception of SHK-1 cells. All cell lines showed increased ROS production at moderate ZEN concentrations and again decreased oxidative stress at high ZEN concentrations. 3.4. Lipid peroxidation The measurement of MDA as an indicator of lipid peroxidation in cell extracts showed that in RTL-W1 cells treatment with ZEN led to reduced values of lipid peroxidation. In RTgill-W1 cells and SHK-1 cells a similar reduction of lipid damages was observed at ZEN concentrations between 1,250 and 15,000 ng ml
1 ZEN but not at 20,000 ng ml
1 ZEN. The lack of significance at exposure concentrations of 20,000 ng ml
1 ZEN in these two cell lines is probably influenced by low protein concentrations in the samples which were used for MDA calculations caused by high cytotoxic effects of the mycotoxin. In contrast, RT EQ clone 8 cells showed reduced MDA values at ZEN concentrations between 1,250 and 5,000 ng ml
1 ZEN, but increased lipid peroxidation at 10,000 and 20,000 ng ml
1 ZEN (Fig. 8c). Similarly, ZEN-treated CCB cells showed lower MDA values at 313 ng ml
1 ZEN and increased lipid peroxidation at mycotoxin concentrations above 5,000 ng ml
1 ZEN (Fig. 8E). 3.5. DNA damage

3.3. Influence on ROS production The results from the NBT assay showed a biphasic response to ZEN were obtained using for most cell lines

ZEN-treated RTL-W1 cells were heavily damaged at all ZEN concentrations compared to untreated control cells when analyzed using the alkaline comet assay (Fig. 9). At

C. Pietsch et al. / Toxicon 88 (2014) 44e61

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120 Vitality (% control)

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*

* *

* *

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40

* * * * * *

20 0 EtOH

313

625

1250 2500 5000 7500 10000 15000 20000

ZEN (ng/ml) Fig. 3. Membrane integrity and lysosomal activity determined by uptake of neutral red uptake by permanent fish cells cultures (A ¼ RTL-W1, B ¼ RTgill-W1, C ¼ RT EQ clone 8, D ¼ SHK-1, E ¼ CCB) after incubation with ZEN for 24 h, mean ± SE, difference to solvent controls: * ¼ P < 0.05; ** ¼ P < 0.01, *** ¼ P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

the highest ZEN concentrations the tail moments are increased 23-folde26-fold compared to cells treated with the solvent only. The 50% effect concentration of ZEN for DNA damages in RTL-W1 cells was found to be 2009 ng ml
1 ZEN. 3.6. Glutathione contents The values for reduced glutathione (GSH) were decreased by treatment of RTL-W1 cells with ZEN concentrations lower than 1250 ng ml
1 ZEN (Fig. 10A). At high ZEN concentrations both, GSH and GSSG, increased in these cells. A decrease of GSH concentrations in cells treated with high ZEN concentrations was observed in RTgill-W1 and CCB cells, although GSSG levels were not

significantly influenced. The effects of ZEN on glutathione levels were not as pronounced in RT EQ clone 8 and SHK-1 cells. 3.7. PCR and sequencing of ERa and ERb from cell cultures Both, estrogen receptor alpha (ERa) and the estrogen receptor beta isoforms (ERb1 and ERb2), have been identified in normal liver tissue of carp but not in the sample from the CCB cell culture (see pictures in the Supplement). In contrast, ERb has been found in trout liver tissue and in RT EQ clone 8 cells and in SHK cells while it was absent in RTL-W1 cells and RTgill-W1 cells. ERa1 and ERa2 were identified in all samples of rainbow trout origin but not in the SHK-1 cell line.

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ZEN (ng/ml) Fig. 4. MTT conversion by different cells lines (A ¼ RTL-W1, B ¼ RTgill-W1, C ¼ RT EQ clone 8, D ¼ SHK-1, E ¼ CCB) after incubation with ZEN for 24 h, mean ± SE; difference to solvent controls: * ¼ P < 0.05, ** ¼ P < 0.01, *** ¼ P < 0.001.

4. Discussion Small amounts of ZEN can rapidly be metabolized by permanent fish cell lines. ZEN was found to be metabolized to zearalanone (ZAN), alpha-zearalanol (a-ZAL), betazearalanol (b-ZAL), and zearalenol (ZEL) in a mammalian cell line (Schaut et al., 2008; Videmann et al., 2008). However, these metabolites of ZEN could not be detected in RTLW1 cell cultures after exposure to ZEN for 24 h. This indicates that metabolization of ZEN to these derivatives has not taken place in these cells after this exposure period. The opposite was found in vivo whereby ZEN was metabolized to a-ZEL and b-ZEL in trout liver (Lagana et al., 2004) and to aZEL in carp (Pietsch, unpublished data) which implies that the ability to hydroxylate ZEN via 3a- and 3b-hydroxysteroid dehydrogenases (Olsen et al., 1981) has been lost in the RTLW1 cell line that has been used in the present study.

We were able to show that glucuronidation mediated by UDP-glucuronosyltransferases (UGTs) plays a role in metabolization of ZEN in fish cells which has also been shown for mammalian systems (Pfeiffer et al., 2010). Multiple UGTs have been identified in fishes which emphasizes the importance of this biotransformation pathway (Pesonen and Andersson, 1987; George and Taylor, 2002). However, our investigations indicate that glucuronidation is probably not the only biotransformation process that takes place in ZEN-treated fish cells since deglucuronidation often led to less detectable free ZEN than expected. This is especially evident in the three cell lines derived from rainbow trout where the sum of detectable ZEN in media and cell extracts results in less ZEN than initially applied. The decrease in detection of ZEN is more pronounced in cell extracts which suggests that biotransformation enzymes further metabolized the ZEN that was

C. Pietsch et al. / Toxicon 88 (2014) 44e61

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ZEN (ng/ml)

2'000 PI fluorescence (% control)

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PI fluorescence (% control)

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EtOH

** *

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**

**

313

625

** *

** *

** *

** *

** *

** *

** *

1250 2500 5000 7500 10000 15000 20000

ZEN (ng/ml) Fig. 5. Influence of ZEN on membrane integrity measured as PI fluorescence in permanent fish cell cultures (A ¼ RTL-W1, B ¼ RTgill-W1, C ¼ RT EQ clone 8, D ¼ SHK-1, E ¼ CCB) for 24 h, mean ± SE; difference to solvent controls: * ¼ P < 0.05, ** ¼ P < 0.01, *** ¼ P < 0.001.

de-glucuronidated to other metabolites within 24 h. The involvement of undetected ZEN metabolites is also indicated by the fact that the analysis of media and cell extracts does not lead to 100% recovery of the initial applied concentrations. This is supported by the observation that absorption of ZEN to culture flasks can be excluded because

Table 2 EC50 values of cell viability assays (NR, MTT and PI) in fish cell cultures after exposure to ZEN for 24 h.

RTL-W1 RTgill-W1 RT EQ clone 8 SHK-1 CCB

NR50 ng ml
1

MTT50 ng ml
1

PI50 ng ml-

8,973 2,837 4,819 4,197 3,828

17,903 >20,000 >20,000 >20,000 5,046

12,104 11,693 11,828 11,296 12,386

the incubation of ZEN in culture flasks without cells for 24 h did not markedly influence ZEN recovery in the exposure media (Table 1). Thus, it is assumed that other metabolites of ZEN may have been formed that could not be detected by our HPLC-DAD method. Further well-targeted experiments should indicate whether these possible metabolites may include catechol derivatives of ZEN that are formed by aromatic hydroxylation and which are known for their oxidative DNA damage (Fleck et al., 2012). This may be a major explanation for the cytotoxic effects that have been found for ZEN in the present study. Cytotoxic effects of ZEN which were comparable to those observed in mammalian cell lines (Abid-Essefi et al., 2004; Kouadio et al., 2005) could be confirmed in the present study using fish cell lines by means of three different cell viability assays in our study. The results obtained in this study using in vitro models show that fish cells are sensitive to fungal metabolites such

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ZEN (ng/ml) Fig. 6. Influence on the ROS production as determined by NBT assay in several permanent fish cells (A ¼ RTL-W1, B ¼ RTgill-W1, C ¼ RT EQ clone 8, D ¼ SHK-1, E ¼ CCB) due to exposure to ZEN for 24 h, mean ± SE; difference to solvent controls: * ¼ P < 0.05, ** ¼ P < 0.01, *** ¼ P < 0.001.

as ZEN in a concentration-dependent manner which is the first report of cytotoxic effects of this fungal metabolite on permanent fish cell cultures. The cytotoxicity assessment using neutral red (NR) correlated well with results obtained with the tetrazolium salt reduction (MTT) technique although the latter method showed higher EC50 values. Still, the EC50 values calculated for the MTT assay for the RTL-W1 (17,903 ng ml
1 ZEN) and the CCB cells (5,046 ng ml
1 ZEN) are lower than most values reported for mammalian cell lines which certainly is also due to the fact that 10% FBS is used for ZEN exposure in most mammalian systems while the exposure media in the present study did not contain FBS. For example, the cytotoxicity of ZEN assessed by this assay after 24 h exposure of human HepG2 cells to ZEN in medium containing 10% FBS

ranged from 12,735 to 57,306 ng ml
1 (Hassen et al., 2007; Ayed-Boussema et al., 2008). Cetin and Bullerman (2005) also used cell culture media containing 10% FBS and reported IC50 values (the concentration values resulting in 50% inhibition of cell response) of more than 90,000 ng ml
1 ZEN for different mammalian cell lines (CHOeK1, Caco-2, V79 and HepG2) after 48 and 72 h of exposure to this mycotoxin, whereas C5eO cells showed a value of 24,000 ng ml
1 ZEN at 72 h of exposure to ZEN. In contrast, Kouadio et al. (2005) showed lower IC50 values after 72 h of exposure to concentrations ranging from 4,775 to 7,959 ng ml
1 ZEN for similar treated Caco-2 cells grown in media containing 10% FBS. Moreover, an insect cell line incubated with ZEN showed an EC50 of 5,571 ng ml
1 for the cytotoxicity as measured by the MTT assay

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ZEN (ng/ml) Fig. 7. ROS production by different cells lines (A ¼ RTL-W1, B ¼ RTgill-W1, C ¼ RT EQ clone 8, D ¼ SHK-1, E ¼ CCB) after exposure to ZEN for 24 h measured by fluorescence of oxidized DCF followed by emission at 535 nm after excitation at 485 nm, mean ± SE, difference to solvent controls: * ¼ P < 0.05, ** ¼ P < 0.01, *** ¼ P < 0.001.

(Fornelli et al., 2004). However, since the absence of FBS does not negatively influence cell viability of fish cells such as the RTgill-W1 cells (Michel et al., 2014) and resulted in even better differentiation of rainbow trout cells and sustained growth in vitro for over 15 days (Mothersill et al., 1995), this exposure system is appropriate for cell viability testing. Thus, from the MTT values for our cell lines, which indicate influences on mitochondrial functionality, it can be concluded that fish cell lines are more sensitive or at least similar in sensitivity to ZEN compared to mammalian or insect cell lines. The EC50 values obtained for the NR assay (ranging from 2,837e8,973 ng ml
1 ZEN) showed lower values than for the MTT assays. These values are considerably lower than the IC50 of 34,625 ng ml
1 ZEN

reported for CHOeK1 cells treated for 24 h with ZEN in the presence of 10% FBS (Ferrer et al., 2009). Decreased uptake of NR due to ZEN exposure has also been shown in Caco2 cells in a concentration-dependent manner revealing an IC50 of 4,775 ng ml
1 (Kouadio et al., 2005). Thus, it can be assumed that despite the fact that serum absence influenced the sensitivity of the fish cells, these are very sensitive to ZEN. Although ZEN does not seem to specifically target the protein synthesis machinery such as has been reported for the mycotoxin deoxynivalenol (Kouadio et al., 2005), apoptotic effects of ZEN involving a mitochondrial signaling pathway have been confirmed in human HepG2 cells (Ayed-Boussema et al., 2008). A loss of membrane integrity

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C. Pietsch et al. / Toxicon 88 (2014) 44e61

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ZEN (ng/ml) Fig. 8. Lipid peroxidation expressed as ng malondialdehyde (MDA) per mg protein in different cells lines (A ¼ RTL-W1, B ¼ RTgill-W1, C ¼ RT EQ clone 8, D ¼ SHK-1, E ¼ CCB) after exposure to ZEN for 24 h, mean ± SE, difference to solvent controls: * ¼ P < 0.05, ** ¼ P < 0.01, *** ¼ P < 0.001.

120

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* * *

* * *

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30 0 EtOH

313

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ZEN (ng/ml) Fig. 9. Genotoxic effects of ZEN measured as tail moments in the alkaline comet assay in RTL-W1 cells after exposure to different concentrations of ZEN for 24 h, mean ± SE, difference to solvent controls: *** ¼ P < 0.001.

indicating apoptotic events in cells was also observed in all cell lines by means of the propidium iodide (PI) assays. The observations of destabilization of lysosomes determined by the NR assay together with damages to membranes and mitochondria revealed by the PI assay and the MTT test strongly indicate that apoptosis takes place in these cells. The PI assay showing rather similar EC50 values for all cell lines was more sensitive than the MTT test. Thus, it can be concluded that at exposure concentrations higher than approximately 11,000 ng ml
1 ZEN membrane integrity of all cell types is impaired. Induction of apoptosis has been shown in several mammalian cell lines by treatment with ZEN (Ouanes et al., 2003; Abid-Essefi et al., 2004). Since the EC50 values of the NR assay are much lower than for the other assays it may be assumed that ZEN exerts specific effects on lysosomes and/or endosomes before more general toxic effects on cell membranes occur. A central role of

C. Pietsch et al. / Toxicon 88 (2014) 44e61

57

Fig. 10. Reduced (GSH) and oxidized glutathione (GSSG) in different cells lines (A ¼ RTL-W1, B ¼ RTgill-W1, C ¼ RT EQ clone 8, D ¼ SHK-1, E ¼ CCB) after exposure to ZEN for 24 h, mean ± SD, difference to solvent controls: * ¼ P < 0.05.

a lysosomal pathway in the cytotoxicity of ZEN was also found in a human embryonic kidney cell line (Gao et al., 2013). However, the involvement of specific reactions causing toxicity of ZEN should be the subject of further research. At least a major role of estrogenic modes of action in toxicity of ZEN which has occasionally been assumed (Wasowicz et al., 2005) can be excluded in the fish cell lines since cell lines such as the CCB cells without identifiable expression of nuclear estrogen receptors still show high sensitivity to this mycotoxin. Similar to our results, mammalian cell lines show similar cytotoxicity to ZEN although they differentially express estrogen receptors (Abid-Essefi et al., 2004). This suggests that an estrogen receptor-mediated action in cells is rather unlikely to be the prime target of ZEN in these cell lines. However, since estrogens are also capable of initiating second messenger signaling events, including mobilization of intracellular calcium (Morley et al., 1992), release of cyclic AMP (Razandi

et al., 1999), and activation of the mitogen-activated protein kinases (Watters et al., 1997; Singh et al., 1999), further research is needed to investigate these possible mechanisms in fish cells. Furthermore, it is possible that ZEN toxicity involves oxidative stress as a potential mode of action. Cytotoxic effects in combination with ROS produced due to ZEN treatment of mammalian cells (Abid-Essefi et al., 2004; Hassen et al., 2007) have also been observed in our fish cell lines. Loss of mitochondrial membrane potential due to rapid ROS production and damage at the mitochondrial pathway by ZEN has also been observed in CHOeK1 cells (Ayed-Boussema et al., 2008; Ferrer et al., 2009). Since ROS production may influence membrane function fluorescence dyes such as H2DCF-DA may yield more reliable results than absorbance measurements using NBT. However, cells under way in apoptosis may still maintain their metabolic activity to a certain extent and are counted as living cells in

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the MTT method (Bouaziz et al., 2008). Therefore, also other end points of cell function have been included in our experiments. Overproduction of ROS is known to cause depletion of cellular antioxidants and oxidative injury in cells including DNA damage, oxidation of proteins and lipid peroxidation (Mates, 2000; Hassen et al., 2007). Lipidperoxidation due to ZEN treatment in vitro and in vivo has been reported (Kouadio et al., 2005; Hassen et al., 2007; Abbes et al., 2007; Ferrer et al., 2009). However, the occurence of lipid peroxidations in the fish cell lines was often not as pronounced as the effects observed in other assays. Therefore, it can be concluded that oxidative stress in these cells may not progress through membrane damages. Moreover, damage to DNA by ROS can lead to genotoxicity (Epe, 1991). ZEN-induced DNA damages were very pronounced in RTL-W1 cells. This confirms the genotoxic potential of ZEN which has already been shown in mammalian cell lines (Ouanes et al., 2003; Abid-Essefi et al., 2004; Lioi et al., 2004; Venkataramana et al., 2014). Moreover, increased DNA strand breaks in RTL-W1 cells were found at even lower concentrations than reported previously in a human cell line (Gao et al., 2013) and the fish cell line proved to be more sensitive to this mycotoxin than Vero or Caco-2 cells by showing a lower EC50 value in the alkaline comet assay than for the determination of DNA synthesis inhibition in the mammalian cells (2009 ng ml
1 ZEN versus 2865 and 4139 ng ml
1 ZEN, respectively) (Abid-Essefi et al., 2004). Furthermore the exposure to ZEN generated a considerable amount of reactive oxygen species in mammalian cells leading to apoptosis which could be counteracted by the supplementation with antioxidants such as vitamin E (El Golli Bennour et al., 2008) and N-acetyl cysteine (Venkataramana et al., 2014). In our study, we showed that glutathione as an antioxidant is influenced by ZEN exposure, although less reduced GSH levels not necessary led to increased GSSG levels. This is probably due to the fact that whole-cell extracts do not allow investigating the exact regulation of GSSG levels in different cellular compartments (Morgan et al., 2013). However, increased ROS production was accompanied by decreased GSH levels at 1250 ng ml
1 in RTL-W1 cells and at 5,000 ng ml
1 in RTgill-W1 cells. No coherence between ROS production and GSH levels was found in RT EQ clone 8 cells and CCB cells, and reduced ROS production showed also reduced GSH levels in SHK-1 cells at the same time. Thus, cell-specific differences in ROS metabolization and GSH homeostasis must be assumed. These findings confirm that exposure to ZEN leads to oxidative stress in cells. The increased GSH to GSSG ratio at concentrations above 2,500 ng ml
1 ZEN that occurred in some of the cell lines was probably due to rebound effects that already have been observed in liver tissue after treatment with several compounds which have been reported to deplete GSH (Plummer et al., 1981). This effect commonly comes into action 12e48 h after initial stress probably due to activation of GSH-providing enzymes (Deneke and Fanburg, 1989). The antioxidant N-acetyl cysteine counteracted the ZEN-mediated ROS generation and effects on mitochondrial membrane potential in human neuroblastoma cells

(Venkataramana et al., 2014). However, the use of the antioxidant hydroxytyrosol revealed that ROS production in human embryonic kidney cells due to ZEN exposure could be reduced whereas the genotoxic potential was not influenced (Gao et al., 2013). This proved that there is no direct causal connection between oxidative stress and DNA strand breaks in these cells which might also apply to the present study on fish cell lines. Moderate concentrations of ZEN induced an increase of absorption values in the NR and the MTT tests, and we were able to show that ZEN has concentration-dependent biphasic effects on cell viability. Biphasic effects in biological systems caused by xenobiotics are thought to be adaptive responses by which an initial disruption of homeostasis induces compensatory biological processes (Calabrese and Baldwin, 2002). Accordingly, biphasic effects have been described for cell lines treated with the mycotoxin deoxynivalenol or plant-derived toxins such as isoflavones (de Lemos, 2001; Chen and Donovan, 2004; Guo et al., 2004; Diesing et al., 2011; Pietsch et al., 2011) but not for exposure to ZEN. Increased values in the MTT or NR tests at low ZEN concentrations are certainly not due to cell proliferations since the absence of FBS in the exposure media prevents cell proliferation as has been shown for the RTgill-W1 cells (Michel et al., 2014). Thus, the biphasic effects that have been observed in the cytotoxicity assays and the ROS measurements in the present study may have other reasons. On the one hand it has been proposed that carcinogens may induce repair mechanisms that not only reverse damages provoked by the exogenous chemical but also background damages (Conolly and Lutz, 2004). Thus, at low concentrations of a toxicant the induction of repair mechanisms may over-compensate the incremental lesions. These findings have been demonstrated for DNA damages (Conolly and Lutz, 2004). Thus, this hypothesis may also be true for the effects of ZEN on fish cells since for this substance the production of DNA adducts and genotoxic effects in mammals have already been shown (PfohlLeszkowicz et al., 1995; Ouanes et al., 2003; Abid-Essefi et al., 2004; Lioi et al., 2004) although the carcinogenicity is still a matter of controversy (Becci et al., 1982; NTP, 1982). On the other hand minor DNA damages e.g. at low toxicant concentrations are thought to affect the cell cycle to allow a sufficient DNA repair before replication (Conolly and Lutz, 2004) whereas high concentrations of a cytotoxic substance may accelerate cell divisions resulting in a regenerative hyperplasia. Effects of ZEN on cell cycle progression have been described in two mammalian cell lines (AbidEssefi et al., 2004). At the moment there is no evidence if this hypothesis may be true for the present study using fish cell lines, but further research will be employed to describe possible effects on cell cycle progression in fish cells. In conclusion, we were able to show that (I) ZEN is principally metabolized by glucuronidation in the tested fish cell lines, and (II) metabolization of ZEN does not prevent cytotoxicity whereby lysosomal functions are affected first. In addition, the results show that (III) the estrogenic potential of ZEN is not determining the cytotoxic effects in the cell lines via estrogen receptor a and b. Further, (IV) the cytotoxic and genotoxic potential of ZEN is accompanied by a substantial degree of intracellular

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oxidative stress generated by this substance, but recent research shows that lysosomes are the main targets of ZEN toxicity and oxidative stress is considered to be a byproduct of disruption of cell homeostasis (Gao et al., 2013). Thus, the present study allows new insights to the mechanisms of action of ZEN although whereby all these effects are induced remains still not completely understood. Especially, the impairment of lysosomes and further progress of cell toxicity as well as the involvement of metabolization of ZEN to catechols and possible quinone and semiquinone intermediates which probably promote ROS production in fish cells should be the subject of further research. Acknowledgments The authors like to thank Prof. Dr. Kristin Schirmer (EAWAG, Dübendorf, Switzerland), Dr. Dannevig (National Veterinary Institute, Oslo, Norway), Dr. Edwige Quillet (Jouy-en-Josas Cedex, France), and the Fish Disease Research Unit at the School of Veterinary Medicine (Hannover, Germany) for providing the cell lines. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2014.06. 005. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.toxicon.2014.06.005. References Abbes, S., Zouhour, O., Salah-Abbes, J.B., Abdel-Wahhab, M.A., Oueslati, R., Bacha, H., 2007. Preventive role of aluminosilicate clay against induction of micronuclei and chromosome aberrations in bone-marrow cells of Balb/c mice treated with zearalenone. Mutat. Res. 631, 85e92. Abdelhamid, A.M., 1990. Occurrence of some mycotoxins (aflatoxins, ochratoxin A, citrinin, zearalenone and vomitoxin) in various Egyptian feeds. Arch. Tierernahr. 40 (7), 647e664. Abid-Essefi, S., Ouanes, Z., Hassen, W., Baudrimont, I., Creppy, E.E., Bacha, H., 2004. Cytotoxicity, inhibition of DNA and protein syntheses and oxidative damage in cultured cells exposed to zearalenone. Toxicol. In Vitro 18, 467e474. Abid-Essefi, S., Bouaziz, C., El Golli-Bennour, E., Ouanes, Z., Bacha, H., 2009. Comparative study of toxic effects of zearalenone and its two major metabolites alpha-zearalenol and beta-zearalenol on cultured human Caco-2 cells. J. Biochem. Mol. Toxicol. 23, 233e243. Ayed-Boussema, I., Bouaziz, C., Rjiba, K., Valenti, K., Laporte, F., Bacha, H., Hassen, W., 2008. The mycotoxin zearalenone induces apoptosis in human hepatocytes (HepG2) via p53-dependent mitochondrial signaling pathway. Toxicol. In Vitro 22, 1671e1680. Babich, H., Borenfreund, E., 1991. Cytotoxicity and genotoxicity assays with cultured fish cells: a review. Toxicol. In Vitro 5, 91e100. Babich, H., Shopsis, C., Borenfreund, E., 1986. In vitro cytotoxicity testing of aquatic pollutants (cadmium, copper, zinc, nickel) using established fish cell lines. Ecotoxicol. Environ. Saf. 11, 91e99.

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