Neurotoxic Potential and Cellular Uptake of T-2 ... - ACS Publications

Article pubs.acs.org/crt

Neurotoxic Potential and Cellular Uptake of T‑2 Toxin in Human Astrocytes in Primary Culture Maria Weidner,† Marlies Lenczyk,† Gerald Schwerdt,‡ Michael Gekle,‡ and Hans-Ulrich Humpf*,† †

Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstrasse 45, 48149 Münster, Germany Julius-Bernstein-Institute of Physiology, Martin-Luther-Universität Halle-Wittenberg, Magdeburger Strasse 6, 06097 Halle (Saale), Germany

‡

S Supporting Information *

ABSTRACT: The trichothecene mycotoxin T-2 toxin, which is produced by fungi of the Fusarium species, is a worldwide occurring contaminant of cereal based food and feed. The cytotoxic properties of T-2 toxin are already well described with apoptosis being a major mechanism of action in various cell lines as well as in primary cells of different origin. However, only few data on neurotoxic properties of T-2 toxin are reported so far, but in vivo studies showed different effects of T-2 toxin on behavior as well as on levels of brain amines in animals. To further investigate the cytotoxic properties of T-2 toxin on cells derived from brain tissue, normal human astrocytes in primary culture (NHA) were used in this study. Besides studies of cytotoxicity, apoptosis (caspase-3-activation, Annexin V) and necrosis (LDH-release), the cellular uptake and metabolism of T-2 toxin in NHA was analyzed and compared to the uptake in an established human cell line (HT-29). The results show that human astrocytes were highly sensitive to the cytotoxic properties of T-2 toxin, and apoptosis, induced at low concentrations, was identified for the first time as the mechanism of toxic action in NHA. Furthermore, a strong accumulation of T-2 toxin in NHA and HT-29 cells was detected, and T-2 toxin was subjected to metabolism leading to HT-2 toxin, a commonly found metabolite after T-2 toxin incubation in both cell types. This formation seems to occur within the cells since incubations of T-2 toxin with cell depleted culture medium did not lead to any degradation of the parent toxin. The results of this study emphasize the neurotoxic potential of T-2 toxin in human astrocytes at low concentrations after short incubation times.

1. INTRODUCTION

skeleton and the epoxide between position C12 and C13, which is responsible for the toxicological potential.1,2 With T-2 toxin being the most harmful toxin of the group of trichothecenes in animal studies3 its frequent occurrence in raw agricultural material as well as in processed grain based food and feed material (e.g., maize, wheat, barley, and oats) is a worldwide problem.1,2 Recently published studies of grain samples of different countries detected T-2 toxin as a common contaminant in wheat and barley with oats, however, being especially more susceptible to T-2 toxin contamination.4−7 The most common route of human exposure to T-2 toxin is the dietary intake. On the basis of a recent evaluation and collection of data concerning the occurrence and toxicity of T-2 and HT-2 toxin, the panel on contaminants in the food chain (CONTAM) of the European Food Safety Authority (EFSA) defined a group tolerable daily intake (TDI) of 100 ng/kg body weight for the sum of T-2 and HT-2 toxin.8 Nevertheless, only little information about the toxicokinetics of T-2 toxin in humans is available.8 However, after application in animals, T-2 toxin is rapidly absorbed and distributed in the organism without accumulation in specific organs.3,8 Furthermore, T-2

Trichothecenes are secondary fungal metabolites produced by different fungi of the Fusarium species (e.g. F. sporotrichioides and F. poae)1 and are generally referred to as mycotoxins. The most common mycotoxins from the type A subgroup of trichothecenes are T-2 and HT-2 toxin (Figure 1). The chemical structure of both is characterized by a sesquiterpenoid

Received: November 20, 2012 Published: January 30, 2013

Figure 1. Structure of T-2 and HT-2 toxin. © 2013 American Chemical Society

347

dx.doi.org/10.1021/tx3004664 | Chem. Res. Toxicol. 2013, 26, 347−355

Chemical Research in Toxicology

Article

Utilizing cells in primary culture, the situation in vivo can be mimicked closely since the cells are not modified (e.g., immortalized) but isolated directly from the corresponding tissue. As described in the literature, there can be a different response in primary cells and cell lines of the same origin after incubation with trichothecene mycotoxins.28 Therefore, the usage of primary cells in addition to human cell lines can give a more comprehensive depiction of certain reactions and behavior of human cells after toxin incubation. In this study, experiments with human astrocytes regarding the cytotoxicity and cell viability as well as studies investigating apoptotic (caspase-3-activation, Annexin V staining) or necrotic effects (LDH-release) were performed. Additionally, data on the uptake and accumulation of T-2 toxin in NHA were compared to uptake studies with an established human cell line (HT-29). This cell line was already used successfully in our group to investigate T-2 toxin metabolism in vitro: HT-29 cells metabolized T-2 toxin to a variety of compounds with HT-2 toxin being the main metabolite besides small amounts of neosolaniol and other more polar conjugates.15 Thus, data on uptake and more detailed information about metabolism kinetics in HT-29 cells were collected in this study and compared to human astrocytes.

toxin is metabolized by various reactions such as hydrolysis, hydroxylation, and conjugation.9 HT-2 toxin is not only a common cocontaminant in T-2 toxin contaminated grain commodities4,7 but also presents one of the main metabolites after T-2 toxin application in various in vitro and in vivo studies.10−13 In an established human colon carcinoma cell line (HT-29) as well as in human cells in primary culture (renal proximal tubule epithelial cells, RPTEC, and normal human lung fibroblasts, NHLF), HT-2 toxin was identified as the main (HT-29 and RPTEC) or even sole (NHLF) metabolite after T2 toxin incubation.14,15 Concerning the toxicity in general, T-2 toxin exerts systemic effects on many organ systems comprising the cardiovascular and central nervous system, bone marrow, liver, and the gastrointestinal tract.16 Various acute toxic effects in experimental animals such as diarrhea, vomiting, leukocytosis, skin irritation, and hemorrhage are reported.17,18 Additionally, chronic exposure can lead to anorexia, weight loss, diminished nutritional efficiency, neuro-endocrine changes, and immune modulation.3 T-2 toxin is also considered to be implicated in an outbreak of fatal alimentary toxic aleukia (ATA) affecting a large population in Russia between 1942 and 1948 where people had eaten overwintered grain infected with the T-2 toxin producing fungi Fusarium sporotrichioides and Fusarium poae.3,19,20 Moreover, the main toxic effects of T-2 toxin on the cellular level are the inhibition of eukaryotic protein synthesis, lowering of DNA and RNA synthesis, and apoptosis, which was proven in various cell lines.11,21,22 T-2 toxin and other trichothecene mycotoxins with structural similarities showed an induction of ribotoxic stress in Jurkat cells, which led to the activation of mitogen-activated protein kinases that are involved in the regulation of cell survival.23 Furthermore, studies of the cytotoxic potential of T-2 and HT-2 toxin on human cells in primary culture in vitro revealed apoptotic effects of T-2 and HT-2 toxin on RPTEC as well as on NHLF.14 There are only limited data on the effects of T-2 toxin on the central nervous system, and neurotoxicity was considered as not being one of the most critical effects of T-2 toxin by the CONTAM panel of the EFSA in 2011.8 However, rats given T2 toxin orally, showed reduced motor activity and performance in a passive avoidance test.24 Alterations in brain monoamine levels in certain areas of the rat brain after T-2 toxin administration have also been reported.25 Another in vivo study in pregnant rats exposed orally to T-2 toxin, revealed an induction of apoptosis in the fetal brain on day 13 of gestation.26 The ability of T-2 toxin to cross the blood−brain barrier (BBB) was additionally studied in vivo in adult mice after subcutaneous application of T-2 toxin, and an alteration of BBB permeability was reported.27 Since astrocytes are part of the neuronal unit located behind the cells of the BBB and alteration of the barrier function caused by T-2 toxin is described in literature in vivo,27 testing of the cytotoxicity on human astrocytes in vitro is relevant for the further understanding of possible neurotoxic effects of T-2 toxin. The objective of this study was to determine the neurotoxic potential of T-2 toxin by measuring the cellular uptake and apoptotic potential of T-2 toxin in normal human astrocytes (NHA) in primary culture. This cell type was used since very little data is available on the neurotoxic potential of T-2 toxin, but hints for effects of trichothecenes on distinct regions of the fetal brain or on levels of neurotransmitters in vivo are reported.8,26

2. MATERIALS AND METHODS 2.1. Chemicals. T-2 toxin was biosynthetically prepared and isolated in our laboratory.29 Cell culture media (DMEM High Glucose; Quantum 333) and all used supplements were obtained from PAA Laboratories (Pasching, Austria). Fetal calf serum (FCS) was purchased from Biochrom AG (Berlin, Germany) and epidermal growth factor (EGF) from BD Bioscience (San Jose, USA). All other chemicals were obtained from Merck (Darmstadt, Germany), Roth (Karlsruhe, Germany), and Sigma-Aldrich (Steinheim, Germany). Purified water was generated by a Milli-Q Gradient A10 system (Millipore, Schwalbach, Germany). 2.2. Cell Culture. Normal human astrocytes (NHA) were obtained from Lonza Group AG (Basel, Switzerland). The human colon carcinoma cell line HT-29 was obtained from DSMZ (Braunschweig, Germany). HT-29 cells were cultivated in DMEM containing 1% penicillin/streptomycin and 10% FCS. The cells were subcultivated after reaching 90% confluence twice a week to a ratio of 1:10. NHA were cultivated using Quantum 333 medium with supplements according to a previous report.30 During cultivation, the medium was changed twice a week, and cells were subcultivated to a ratio of 1:3 every 6−8 days. For specific experiments, after reaching a microscopic confluence of 80−90% at about 2−3 days after seeding, the culture medium was changed to serum-free medium (DMEM/Ham’s F-12 medium with 15 mM HEPES and no other supplements) 24 h prior to toxin incubation (caspase-3-activation, protein content, LDH-release, and Annexin V assay). For the evaluation of toxicity and cell viability and for uptake studies, cells were maintained in regular culture medium before toxin incubation. T-2 toxin was diluted in serum-free medium from a 1 mM stock solution dissolved in acetonitrile/water (50:50; v/v) and incubated in concentrations from 1 nM to 200 μM for 6, 24, or 48 h, respectively. Cells serving as a negative control were incubated with an equal concentration of solvent without toxin in serum-free medium (control). All studies with NHA cells were performed using passages 3 to 7. During cultivation and incubation, all cells were kept at 37 °C and high humidity under 8.5% CO2 (HT-29) or 5% CO2 (NHA) atmosphere, respectively. 2.3. Cytotoxicity Assay. The determination of cytotoxic effects of T-2 toxin on NHA was carried out using the Cell Counting Kit-8 (CCK-8) from Dojindo Laboratories (Tokyo, Japan). The assay was completed as described previously.31 Briefly, after seeding 1 × 104 cells in 96-well plates with at least 100 μL of medium, T-2 toxin incubation was carried out over 24 and 48 h in concentration ranges from 1 nM to 348



Article

200 μM. After addition of the WST-8 solution [2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] and incubation according to the manufacturer’s manual, the absorbance of the formed formazan dye, which is directly proportional to the number of living cells per well, was measured with a FLUOstar Optima microplate reader (BMG Labtechnologies, Jena, Germany) at 450 nm. The absorbance of toxin-treated cells was compared to that of control cells. 2.4. Caspase-3-Activation. For studying caspase-3-activity, cell lysates from 1 × 105 (NHA) or 2 × 105 (HT-29) cells seeded in 24well plates and incubated with T-2 toxin (1 nM to 10 μM) for 6 and 24 h were used. The assay was performed according to the protocol described by Königs et al.31 For measuring caspase-3-activity, 33 μL of a reaction buffer mixture (50 mM PIPES, 10 mM EDTA, 0.5% CHAPS, 10 mM DTT, and 80 μM DEVD-AFC) was added to 30 μL of cell lysate in a black 96-well plate. After incubation at 37 °C for 6 h, the fluorescence of AFC (7-amino-4-trifluoromethylcoumarin), which is released in the presence of caspase-3, was measured at 400 nm excitation and 505 nm emission wavelength using a FLUOstar Optima microplate reader (BMG Labtechnologies, Jena, Germany). Caspase-3activity was quantitated with an AFC calibration curve (0.3−12.8 μM) and related to the cellular protein content. 2.5. Protein Content. For quantitation of cellular protein content, the bicinchoninic acid (BCA) assay kit from Sigma-Aldrich (Deisenhofen, Germany) was used as described in detail by Königs et al.31 Cells, 1 × 105 (NHA) or 2 × 105 (HT-29), were seeded in 24well plates with a minimum of 300 μL medium, and after incubation with T-2 toxin (1 nM to 10 μM) for 6 and 24 h, 15 μL of the cell lysate was incubated with 200 μL of a mixture of BCA and 4% copper sulfate (50:1) in a 96-well plate for 30 min at 37 °C following 1 h incubation at room temperature in the dark. The absorbance of the formed colored complex was measured at 560 nm with a FLUOstar Optima microplate reader (BMG Labtechnologies, Jena, Germany). The protein content of toxin-treated cells was quantitated via a calibration curve using bovine serum albumin (BSA) in concentrations from 50 to 500 μg/mL. 2.6. LDH Release. Cells, 1 ×105 (NHA) or 2 × 105 (HT-29), were seeded in 24-well plates with a minimum of 300 μL of medium and incubated with T-2 toxin (1 nM to 10 μM) for 6 and 24 h before cell media as well as cell lysates were used for quantitation of lactatedehydrogenase (LDH). The underlying mechanism of the assay is the LDH-catalyzed conversion of pyruvate and NADH to lactate and NAD+ according to a standard protocol.32 After incubation with T-2 toxin for 6 or 24 h, reaction buffer (100 mM HEPES, 0.14 g/L NADH, and 1.1 g/L sodium pyruvate, pH 7) was added to 40 μL of cell media or 15 μL of cell lysate to a total volume of 200 μL in a 96-well plate. The absorbance was measured at 355 nm with a FLUOstar Optima microplate reader (BMG Labtechnologies, Jena, Germany) every 2 min during incubation at 37 °C. LDH activity was evaluated as mU/ mL, and LDH release was determined as LDH activity in cell medium in % of total LDH release in comparison to that of control cells. 2.7. Flow Cytometric Determination of Apoptosis and Necrosis with Annexin V-FITC and 7-Amino Actinomycin (7AAD). For flow cytometric analysis of apoptosis and necrosis, 5 × 105 cells were seeded in 6-well plates with a minimum of 1.5 mL of medium and incubated with T-2 toxin (1 and 10 μM) for 8 and 10 h. After toxin incubation, cells were treated according to a report described in the literature33 with slight modifications. Briefly, cell medium with detached cells was removed and collected. The remaining attached cells were washed with phosphate-buffered saline (PBS; PAA Laboratories, Pasching, Austria) and detached using Accutase (PAA Laboratories). After combining cells and the collected culture medium, the mixture was centrifuged (200g, 5 min, 4 °C), and the supernatant was discarded. The cell pellet was washed with PBS supplemented with 5% FCS and centrifuged again as described above. Cells were resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4) and stained with Annexin Vfluorescein (Annexin V-FITC; Enzo Life Sciences, Lörrach, Germany) and 7-amino actinomycin D (7-AAD; Beckman Coulter, Krefeld, Germany). Samples were analyzed with a FC 500 flow cytometer

(Beckman Coulter), and emissions were measured at 525 nm for Annexin V-FITC and at 625 nm for 7-AAD. At least 10,000 cells were recorded for each sample, and every experiment was performed with two replicates. 2.8. Cellular Uptake. To study the cellular uptake of T-2 toxin, 1 × 105 (NHA) or 2 × 105 (HT-29) cells were seeded in 24-well plates with a minimum of 300 μL of medium and incubated with 10 μM T-2 toxin. After various time points (15 and 30 min, and 1, 2, 4, 6, 24, and 30 h), the culture medium was removed. Attached cells were washed with PBS followed by the addition of 200 μL of 1% triton-x solution. After centrifugation (10,000g, 10 min, 4 °C), the obtained cell lysate and the collected cell media were used for quantitation of T-2 and HT2 toxin (Section 2.11). In addition, cells were counted at each sampling time point with a CASY TT cell counter (Schärfe System, Reutlingen, Germany) to evaluate micromolar toxin concentrations in the cell lysate. The stability of T-2 toxin in aqueous medium at 37 °C for at least 48 h was previously shown in our group.14 2.9. Effects of Secreted Enzymes on the Metabolism of T-2 Toxin. For the analysis of the effects of secreted enzymes in cell culture medium on the metabolism of T-2 toxin, 1 × 106 cells were grown in 100 mm culture dishes and at least 10 mL of medium. After a growth period of 48 h, the supernatant medium was collected and incubated with 10 μM T-2 toxin for 30 h. After different time points (0, 15, and 30 min, and 1, 2, 4, 6, 24, and 30 h), samples were collected and analyzed by LC-MS/MS for their T-2 and HT-2 toxin content as described in Section 2.11. 2.10. Analyzing the Binding of T-2 Toxin to Cell Membranes. To analyze the binding of T-2 toxin to the cell membrane, cells were grown in 100 mm culture dishes and incubated with 10 μM T-2 toxin for 6 h. After removal of the culture medium, cells were washed with PBS and lysed as described in Section 2.8 with 2 mL of 1% triton-x solution. After centrifugation of the cell lysate, a mixture of acetonitrile/water (50:50; v/v) was added to the remaining cell pellet and extracted under shaking for 20 min. The extract was analyzed for T-2 and HT-2 toxin with LC-MS/MS after centrifugation (Section 2.11). 2.11. Quantitation of T-2 and HT-2 Toxin and Identification of HT-2 Toxin Glucuronide. 2.11.1. LC-MS/MS Measurements. For quantitation of T-2 and HT-2 toxin in cell lysate and cell medium, a matrix-matched calibration curve for the cell lysate and culture medium was used with different concentration levels (lysis buffer, T-2/ HT-2 toxin, 0.01−0.20 μg/mL; cell medium, T-2/HT-2 toxin, 0.2−5.0 μg/mL). At several time points after T-2 toxin incubation (15 and 30 min and 1, 2, 4, 6, 24, and 30 h), samples were collected, and d3-T-2 toxin was added to each sample as internal standard (lysis samples, 0.005 μg/mL; culture medium samples, 0.5 μg/mL) for quantitation. Analysis was performed on a HPLC-MS/MS system, and quantitative evaluation was completed by plotting the ratio of the peak area of the analytes to the peak area of the internal standard (d3-T-2 toxin) against the concentration of the analytes. 2.11.2. HPLC Parameters. Quantitation of T-2 and HT-2 toxin in cell media and cell lysate was carried out using LC-MS/MS with an Agilent 1100 series high performance liquid chromatography (HPLC) system (Agilent, Waldbronn, Germany) coupled to an API 4000 QTrap tandem mass spectrometer (Applied Biosystems, Darmstadt, Germany). Data acquisition was performed using Analyst 1.4.2 software (Applied Biosystems). Chromatographic separation was conducted on a 50 × 2.0 mm i.d., 2.4 μm, Pursuit UPS C18 column (Agilent, Waldbronn, Germany) with a 4 × 2.0 mm i.d. C18 guard cartridge (Phenomenex, Aschaffenburg, Germany). The analytes were separated using a binary gradient with acetonitrile supplemented with 5 mM ammonium acetate (solvent A) and water supplemented with 5 mM ammonium acetate (solvent B). Starting conditions were 20% A, increasing to 100% A in 8 min, keeping the conditions for 1 min before equilibrating at starting conditions for 6 min, resulting in a total run time of 15 min. Oven temperature was set to 40 °C, and flow rate was 300 μL/min and injection volume 30 μL. 2.11.3. MS/MS Parameters. The mass spectrometer was operated in the positive multiple reaction monitoring (MRM) mode with nitrogen serving as curtain gas (20 psi). For electrospray ionization, 349



Article

cells (RPTEC), with 230 nM (±18 nM),15 and normal human lung fibroblasts (NHLF) with 500 nM (±100 nM).14 These data clearly show that human astrocytes (NHA) used in this study are very sensitive to the exposure of T-2 toxin resulting in a low IC50 value. To compare the results of NHA with an established cell line derived from human colon carcinoma cells (HT-29), the same concentrations of T-2 toxin were incubated for 24 and 48 h (Figure S1, Supporting Information). The IC50 value for HT-29 cells was calculated with 741 nM (±302 nM), and therefore, HT-29 cells were by a factor of 30 less sensitive against T-2 toxin exposure than NHA. In the literature, several different IC50 values are described for various cell types of human and animal origin ranging from 0.2 to 150 nM T-2 toxin.35 Differences in the response of primary cells and cell lines after mycotoxin exposure have already been reported in the literature for an immortalized human kidney cell line (IHKE) being more sensitive (IC50 value: 19 nM) to T-2 toxin exposure than primary cells from the same origin (RPTEC; IC50 value: 200 nM).14 For another trichothecene mycotoxin, deoxynivalenol, differences in the reaction of primary cells and tumor-derived cells of the same human tissue were investigated, and an approximately 7-fold lower IC50 value was observed for human primary hepatocytes in comparison to the IC50 value of 41.4 μM for the HepG2 cell line (human hepatocellular liver carcinoma cell line).28 Despite the high difference in the IC50 value of NHA and HT-29 cells, which is most likely due to the different origin of the used cells (cf. cancer-derived cell line in contrast to unmodified primary astrocytes), NHA are very sensitive to the exposure with T-2 toxin resulting in a strong depletion of viable cells in a concentration-dependent manner starting at concentrations of 10 nM (Figure 2). 3.2. Caspase-3-Activation. To further investigate the cytotoxic effects of T-2 toxin on human astrocytes (NHA), caspase-3-activation was used to differentiate between apoptotic and necrotic cell death after toxin exposure. Therefore, the activation of the enzyme caspase-3 was measured as a marker for apoptosis in comparison to that of solvent-treated control cells (set to 100%). The concentrations of T-2 toxin ranged from 1 nM to 10 μM, and the results of caspase-3-activation after incubation for 6 and 24 h are displayed in Figure 3. The early time points of 6 and 24 h were chosen since the results of the viability assay indicated a strong cytotoxic effect starting at 10 nM after 24 h of incubation (Section 3.1). After incubation of 5 μM T-2 toxin for 6 h on human astrocytes, caspase-3-activity increased to 200 ± 36% compared to that of control cells. After incubation of 10 μM T-2 toxin for 6 h, caspase-3-activity increased to 233 ± 29% (Figure 3A). Although the enhancement of caspase-3-activity is only minor between incubations of 5 μM (200%) and 10 μM (233%) T-2 toxin for 6 h, the observed caspase-3-activity is still rather high compared to that of literature data. Considering the effects of alkaloid mycotoxins on NHA, the incubated alkaloids ergotamine and α-ergocryptine showed a caspase-3-activation of 250−350% compared to that of solvent-treated control cells, at concentrations of 10 and 20 μM after an incubation period of 24 h.30 The results presented in our study show a caspase-3activation induced by T-2 toxin in NHA already 6 h after toxin incubation (Figure 3A). Compared to the literature, other mycotoxins exhibit effects in the same cells in similar ranges but after longer incubation periods,30 indicating that T-2 toxin causes an intense and fast response in human astrocytes leading

ion voltage was set to 5500 V. Zero-grade air was used as nebulizer gas (35 psi) and as drying gas (45 psi) heated to 350 °C. Each transition reaction was monitored for 50 ms. The ammonium adducts of the analytes and internal standard [M + NH4]+ were analyzed with the following transitions and listed declustering potential (DP), collision energy (CE), and cell exit potential (CXP): T-2 toxin, 484−215 (DP 65 V, CE 24 V, CXP 14 V), 484−305 (DP 65 V, CE 19 V, CXP 19 V); d3-T-2 toxin, 487−215 (DP 65 V, CE 24 V, CXP 14 V), 487−308 (DP 65 V, CE 19 V, CXP 19 V); HT-2 toxin 442−215 (DP 54 V, CE 18 V, CXP 16 V), 442−263 (DP 54 V, CE 22 V, CXP 18 V), HT-2 toxin glucuronide 618−215 (DP 61 V, CE 28 V, CXP 17 V), 618−245 (DP 61 V, CE 24 V, CXP 19 V). Both quadrupoles were set at unit resolution. The first given transition served as the quantifier, the second as qualifier. 2.12. Statistical Analysis. All measurements are displayed as the mean value ± SEM. Experiments were performed in at least three different cell passages with a minimum of six wells per group for the determination of cytotoxicity, a minimum of three different experiments per group for LDH-release, caspase-3-activation, and protein content, and two separate wells for each group and time point for uptake studies. Therefore, a minimum of 6−18 samples was analyzed for each characterized parameter. For the determination of significant differences, the unpaired Student’s t-test was used, and results with p ≤ 0.05 were regarded as statistically significant. On the basis of cytotoxicity data after 48 h of toxin incubation, the medium effective concentrations (IC50 values) were calculated using Sigma Plot version 12.0 according to the literature.34

3. RESULTS AND DISCUSSION 3.1. Cytotoxic Effects of T-2 Toxin. To evaluate the cytotoxicity of T-2 toxin on human astrocytes in primary culture, the CCK-8 assay, measuring the metabolic activity of mitochondrial and cytoplasmic dehydrogenases in viable cells, was performed (Figure 2). T-2 toxin was applied to NHA cells

Figure 2. Viability of normal human astrocytes (NHA) depending on the T-2 toxin concentrations (1 nM−200 μM) after 24 and 48 h of incubation time, determined by the CCK-8 assay. Number of analyzed samples n = 18 over three individual passages; mean ± SEM; * indicates significant differences from the control (p ≤ 0.05).

in concentrations ranging from 1 nM to 200 μM for 24 and 48 h. The cell viability was already significantly different compared to that of the solvent-treated control cells (p ≤ 0.05) after the incubation of 10 nM T-2 toxin for 24 h resulting in a decrease of cell viability to 70%. The IC50 value for NHA was calculated with 24 nM (±4.5 nM) based on the effects after 48 h of incubation time. This IC50 value for NHA is lower than IC50 values for T-2 toxin described in the literature for other human cell types in primary culture, renal proximal tubule epithelial 350



Article

Consequently, the effects observed after T-2 toxin incubation in this study show a fast and intense apoptotic reaction of NHA measured by an increased caspase-3-activity after 6 and 24 h of incubation. 3.3. LDH Release. An apoptotic effect of T-2 toxin on human astrocytes could be identified by caspase-3-activation (Section 3.2), but a differentiation to a necrotic effect should further be investigated in these cells. Therefore, concentrations of 1 nM to 10 μM T-2 toxin were applied to NHA for 6 and 24 h, and lactate dehydrogenase (LDH) release into the culture medium was measured. During necrosis, cells swell, and the cell membrane starts to disintegrate releasing intracellular enzymes such as LDH into the cell culture medium. Therefore, LDH can be utilized as a marker for necrosis.14 The results for LDH release after T-2 toxin incubation are summarized in Figure 4.

Figure 3. Concentration- and time-dependent caspase-3-activation in NHA after 6 h (A) and 24 h (B) incubation with T-2 toxin (10 nM− 10 μM (A); 10 nM−1 μM (B)). Caspase-3-activation is expressed as percent in comparison to solvent-treated control cells (set to 100%). Number of analyzed samples n = 9 over three individual passages; mean ± SEM; * indicates significant differences from the control (p ≤ 0.05).

Figure 4. Release of LDH in NHA after T-2 toxin incubation (10 nM−10 μM) for 6 and 24 h. Quantitation of LDH-release is shown as percent in comparison to solvent-treated control cells (set to 0%). Number of analyzed samples n = 9 over three individual passages; mean ± SEM; * indicates significant differences from the control (p ≤ 0.05).

to an early activation of caspase-3 as a key enzyme in the apoptotic pathway. After 24 h of incubation, caspase-3-activity was further elevated even with lower concentrations of T-2 toxin (Figure 3B). At higher T-2 toxin concentrations (5 and 10 μM), caspase-3-activity could not be evaluated due to the strong cytotoxic effects of T-2 toxin resulting in a high number of dead cells as observed visually. Thus, only very low or nondetectable protein contents per well were determined by the BCA assay, which could not be used for further calculations of the caspase3-activity at these concentrations. Nevertheless, after 24 h of incubation with T-2 toxin, a dose-dependent elevation of caspase-3-activity was observed starting at a concentration of 0.1 μM and reaching statistical significance at a concentration of 1 μM (Figure 3B). Compared to control cells, the application of 500 nM T-2 toxin to human astrocytes for 24 h led to a caspase-3-activation of 252 ± 48% and was additionally elevated after the incubation of 1 μM T-2 toxin with 555 ± 84% caspase-3-activity being significantly enhanced (p ≤ 0.05) in comparison to that of control cells. These findings are consistent with literature data investigating the apoptotic effect of T-2 toxin in human primary kidney cells and lung fibroblasts where T-2 toxin led to caspase-3-activation (approximately 650% compared to control cells) after the incubation of 1 μM for 24 h.14 Caspase-3-activation determined by flow cytometry was also reported after T-2 toxin application in a human MOLT-4 cell line 5 h after toxin application.36 Results for ergot alkaloids tested on the same cell type as used in this study (NHA) show a caspase-3-activity of 450−550% for the substance ergocristine after 24 h of incubation but using higher concentrations (10 or 20 μM, respectively).30

No LDH release is detectable after the incubation of various concentrations of T-2 toxin for 6 h. With a prolonged incubation time of 24 h, LDH release was identified in a dose−response manner starting from 0.5 μM to 10 μM (3− 29% LDH release compared to control cells) with significance (p ≤ 0.05) compared to control cells at 1 μM and above. These results suggest the occurrence of a secondary necrosis after the induction of apoptosis. This effect was already reported for apoptotic cells in in vitro systems as no scavengers for apoptotic cells (e.g., macrophages) are present.37 In comparison to the literature data of T-2 toxin applied to primary kidney cells and lung fibroblasts for 24 h, no significant effects on LDH release were reported14 in contrast to our findings. The maximum LDH release at concentrations of 5 and 10 μM T-2 toxin with up to 30% compared to control cells is explainable since the cells were visibly impaired after incubation with these T-2 concentrations. This effect corresponds with the lowered protein values after the incubation of 5 and 10 μM T-2 toxin for 24 h as reported above (Section 3.2). Therefore, the induction of apoptosis remains the main effect after T-2 toxin application in NHA, followed by necrotic effects after enhanced incubation periods presumably as a secondary effect. 3.4. Flow Cytometric Determination of Apoptosis and Necrosis with Annexin V-FITC and 7-Amino Actinomycin (7-AAD). In order to verify the relation between apoptosis and cell membrane integrity, induced by T-2 toxin in human astrocytes, Annexin V-FITC (Annexin V) combined with 7-amino actinomycin (7-AAD) staining was performed, 351



Article

and cells were analyzed by flow cytometry. Besides the quantitative assays, Annexin V/7-AAD staining was conducted to confirm the results of the early caspase-3-activation (6 h after toxin application; Section 3.2). Therefore, results of one representative experiment with two replicates and counting of at least 10,000 cells per replicate were evaluated qualitatively. T2 toxin was applied to NHA in concentrations of 1 and 10 μM for 8 and 10 h. The principle of the performed assay is based on the translocation of phosphatidylserine (PS) from the inner to the outer layer of the cell membrane during the early phase of apoptosis.33 Annexin V coupled with FITC binds specifically to surface PS, and simultaneous staining with 7-AAD as a viability dye allows the differentiation between apoptotic and necrotic cell death. The results after T-2 toxin incubation of NHA are displayed in Figure 5 showing an increase in the Annexin V positive and 7-AAD negative population in a time- and concentrationdependent manner in comparison to that of control cells. The population of necrotic cells (Annexin V and 7-AAD positive: upper right sector) remains consistent and does not increase after T-2 toxin incubation in contrast to the apoptotic cells, which can be differentiated and identified by this assay

(Annexin V positive and 7-AAD negative: lower right sector). The distinct increase in apoptotic cells is visible at concentrations of 10 μM T-2 toxin after 8 h of incubation with further enhancement after 10 h of incubation (Figure 5C and F). Further, Annexin V binding as proof of apoptosis after 6 h of T-2 toxin incubation of a T lymphoblastic human cell line (MOLT-4) was already reported.36 In our study, human astrocytes in primary culture were analyzed by flow cytometry with Annexin V-FITC and 7-AAD staining after T-2 toxin incubation, and an apoptotic effect was detectable at 10 μM T-2 toxin after 8 and 10 h of incubation, which proves the results from caspase-3-activation in NHA (Section 3.2). 3.5. Cellular Uptake. Experiments on cellular uptake were performed in primary human astrocytes (NHA) and in a human colon carcinoma cell line (HT-29). The results after exposure to 10 μM T-2 toxin for various time points are shown in Figure 6 presenting a strong accumulation as well as the formation of HT-2 toxin in both cell types. The initially applied T-2 toxin concentration is exceeded in HT-29 as well as in NHA cells leading to an intracellular concentration of 200 μM (NHA) and 150 μM (HT-29), respectively, after 15 min of exposure to T-2 toxin (Figure 6A and C). In NHA, the cellular accumulation increases in a timedependent manner exhibiting a peak level 1 h after incubation with an about 30-fold increased intracellular concentration of T-2 toxin compared to the surrounding cell medium. The early detection of T-2 toxin in the cell lysate 15 min after incubation indicates a fast uptake of T-2 toxin. After reaching the peak concentration of up to 320 μM (1 h), T-2 toxin is metabolized to HT-2 toxin rapidly since the cellular level of T-2 toxin decreases from 2 to 30 h and the HT-2 toxin concentration increases in cell lysate and cell medium. Interestingly, the cells are unable to completely eliminate the absorbed toxin since a plateau of about 100 μM T-2 toxin is present in the cell lysate from 6 to 30 h. In HT-29 cells, the cellular accumulation of T-2 toxin is 15fold compared to the initially applied toxin and therefore about 2 times lower than the peak concentration in NHA. Still, T-2 toxin is already present in the analyzed cell lysate 15 min after toxin incubation revealing a fast uptake of T-2 toxin. However, focusing on the ability to metabolize the accumulated toxin, the cellular concentration in HT-29 cells was already significantly decreased 2 h past incubation (30% of initial cellular concentration) yielding a nondetectable level of T-2 toxin after 24 h. This faster elimination of T-2 toxin from the cytoplasmic compartment in comparison to NHA might be linked to the concurrent formation of HT-2 toxin, the deacetylated major metabolite of T-2 toxin, already described in HT-29 cells.15 Regarding the formation of HT-2 toxin, both cell types are able to metabolize T-2 toxin to HT-2 toxin, yet with different kinetics. After 15 min of T-2 toxin incubation, the intracellular HT-2 toxin concentration is similar in both cell types (50 μM) despite the different concentrations of accumulated T-2 toxin in the cell lysate (NHA, 200 μM; HT-29, 150 μM). HT-2 toxin levels increase between 2 to 4 h of incubation time in HT-29 cells, reaching the maximum concentration of 150 μM per cell after 6 h and then decreasing to 100 μM per cell at 24 h and to similar levels thereafter. The concentration of HT-2 toxin in NHA remains at the same level (50 μM per cell) for the first 4 h after T-2 toxin application and increases only slightly after 6 h before reaching the maximum concentration of 200 μM after 30

Figure 5. Induction of apoptosis in NHA after the application of T-2 toxin evaluated by flow cytometric analysis after Annexin V-FITC and 7-AAD staining. Solvent-treated control cells (A) and incubation of 1 μM T-2 toxin (B) and 10 μM T-2 toxin (C) for 8 h; control cells (D), 1 μM T-2 toxin (E), and 10 μM T-2 toxin (F) incubated for 10 h. Apoptotic cells are located in the lower right quadrant of each panel. 352



Article

Figure 6. Time-dependent uptake of T-2 toxin (10 μM) and formation of HT-2 toxin in NHA (A) and HT-29 cells (C) (concentrations measured in the cell lysate were normalized to cell number and cell volume [μmol/cell]); analysis of T-2 and HT-2 toxin concentrations in cell media after incubation of T-2 toxin (10 μM) for various time points in NHA (B) and HT-29 cells (D). Number of analyzed samples n = 6 over three individual passages; mean ± SEM.

determine if T-2 toxin can penetrate the blood−brain barrier and disrupt astrocyte viability. Regarding the corresponding cell medium, the amount of applied T-2 toxin decreases in both cell types (Figure 6B and D) with a simultaneous increase of the HT-2 concentration. The shift in concentration levels of T-2 and HT-2 toxin occurs in HT-29 cells already between 2 and 4 h after toxin incubation, whereas this change appears in NHA between 6 and 24 h. Again, the amount of T-2 toxin is not thoroughly reduced in NHA cells compared to that in the human cell line where T-2 toxin is not detectable anymore after 24 h, while the concentration in NHA is still about 25% of the originally applied toxin (1 μg/mL) after 24 h. Another metabolite of T-2 toxin in HT-29 cells was described as the glucuronic acid conjugate of HT-2 toxin.15 HT-2 toxin glucuronide was likewise detected as a phase II metabolite in vitro after incubations of T-2 toxin with liver microsomes of different animals as well as in vivo in pig urine after oral administration of T-2 toxin.39 This metabolite also occurs in the presented study in traces in cell media of HT-29 cells incubated with T-2 toxin but was not detectable in NHA (data not shown). Therefore, the ability to form phase II conjugates is possible in the human cell line but not in human astrocytes. This might be another explanation for the difference in cytotoxicity in both cell types. 3.6. Effects of Secreted Enzymes on the Metabolism of T-2 Toxin. In order to investigate the metabolism of T-2 toxin by secreted enzymes, experiments with used cell culture medium were performed. Since T-2 toxin enters both studied cell types and the formed metabolite HT-2 toxin is detectable

h. The formation of HT-2 toxin is relevant since the cytotoxic effect of HT-2 toxin was already studied in various in vitro test systems showing an effect similar to that of T-2 toxin causing apoptosis in human Jurkat cells or primary human kidney cells.14,15,38 Therefore, the observed cytotoxic effects in NHA could be induced by both T-2 and HT-2 toxins. Albeit there is a difference in the extent of the accumulation of applied T-2 toxin per cell, the used primary cells as well as the cell line are metabolizing T-2 toxin to HT-2 toxin rapidly, indicating a short half-life of T-2 toxin after application in vitro. However, T-2 toxin appears to be able to enter the cells already after short incubation periods (15 min), and human astrocytes are accumulating the toxin to a higher level per cell than the cancer cell line (HT-29) and keep additionally a certain level of intracellular T-2 toxin for up to 30 h. The strong cytotoxic effects of T-2 toxin on NHA (Section 3.1) are most likely facilitated by the high cellular accumulation and fast uptake of T-2 toxin triggering apoptotic reactions in NHA. Cytotoxic effects in HT-29 cells were not as severe as in NHA (cf. IC50 values; Section 3.1). The uptake in HT-29 cells was also not as distinct as in NHA, and since HT-29 cells were able to eliminate or metabolize the entire accumulated T-2 toxin from the cell interior, cytotoxic reactions might not be as easily triggered as in NHA, where a plateau of T-2 toxin remains within the cell interior up to 30 h after exposure. The observation of the high accumulation and sensitivity of NHA against T-2 toxin treatment in vitro leads to the question of whether T-2 toxin is able to reach astrocytes in vivo by crossing the blood−brain barrier. Further studies are needed to 353



Article

activation, LDH release, and Annexin V staining. Distinct elevations in caspase-3-activity were already reported 6 h after exposure to T-2 toxin increasing up to 24 h. Uptake studies with HT-29 and NHA cells revealed a fast cellular uptake and high accumulation in both cell types leading to a 15- to 30-fold increased concentration in the intracellular compartment. However, the human colon carcinoma cell line did not accumulate T-2 toxin to the same extent as the primary human astrocytes. The fast uptake in both cell types (T-2 toxin is already present in the cytoplasmic cell compartment 15 min after toxin application) followed by a high accumulation emphasizes the strong impact of T-2 toxin. Human astrocytes are very sensitive to T-2 toxin treatment leading to strong cytotoxic responses already after short incubation periods. Further studies with T-2 toxin and its penetration across the blood−brain barrier are needed to evaluate the ability of T-2 toxin to reach astrocytes to unfold cytotoxic effects as described in this study. Simultaneously, the formation of HT-2 toxin, a major T-2 toxin metabolite, was detected in both cell types and seems to take place in the cell interior since no effect of secreted enzymes in the cell culture medium nor binding to the tritoninsoluble cell pellet was observed. Thus, the observed apoptotic effects in NHA are likely caused by a combination of T-2 and HT-2 toxin. Therefore, both compounds, T-2 and HT-2 toxin, should be considered for risk assessment. Additionally, human astrocytes were shown to be very sensitive against T-2 toxin treatment, indicating the neurotoxic properties that were until now not taken into account but should certainly be implemented when discussing toxic effects of T-2 toxin.

in the cell lysate as well, the metabolism is likely to take place in the inner cell compartments. However, metabolism reactions performed by excreted enzymes are also possible. Therefore, media from cells cultivated for 24 h were collected and incubated with 10 μM T-2 toxin for 6 h without cells, and samples were analyzed after various time points (data not shown). In the media of HT-29 cells, the initially incubated amount of T-2 toxin decreased only slightly after 24 h, and only small amounts of HT-2 toxin were formed after 24 to 30 h (about 3.5% of initially applied toxin). Similar effects were seen in the media of NHA where HT-2 toxin was detectable in small amounts between 24 and 30 h after toxin incubation (about 2.3% of initially applied toxin). These findings support the hypothesis that the metabolism of T-2 toxin takes place after uptake into the cell interior and is not conducted by secreted enzymes outside the cells. 3.7. Binding of T-2 Toxin to Cell Membranes. Another aspect when analyzing the uptake and metabolism of xenobiotics in cells is the binding to the cell membrane. Since T-2 toxin is rather lipophilic, binding to the cell membrane might be possible; however, the detection of high amounts of T-2 toxin in the cell lysate indicates a thorough uptake of T-2 toxin into the cell interior. After T-2 toxin incubation for 6 h followed by cell lysis, the remaining cell pellet containing, e.g., cell debris, cell membranes, and cell nuclei, was extracted with acetonitrile/water (50:50; v/v). The amount of T-2 and HT-2 toxin in the extract of the cell pellet was compared to the amount detected in the corresponding cell lysate (sum set to 100%). In HT-29 cells, 12% T-2 toxin was distributed in the cell lysate, and only 4% was found in the cell membrane extract. HT-2 toxin was also detected with 57% in the cell lysate and 27% in cell membrane extract, resulting in a total distribution of 69% of the applied toxin in the cell lysate and only 31% in the cell pellet (calculated as the sum of T-2 and HT-2 toxin). A similar tendency was found for NHA cells with 54% T-2 toxin in the cell lysate, 11% in the cell membrane, 24% HT-2 toxin in the cell lysate, and 10% in the cell pellet, leading to 78% toxin in the cell lysate and 21% in the cell pellet extract (calculated as the sum of T-2 and HT-2 toxin) (data not shown). The amounts of T-2 and HT-2 toxin in the cell lysate and cell pellet extract 6 h after toxin incubation reflect the observed metabolism pattern as described above (Section 3.5) with T-2 toxin being rapidly metabolized in HT-29 cells (12% in cell lysate; 4% in cell pellet) under simultaneous formation of HT-2 toxin (57% in lysate; 27% in cell pellet) in contrast to the slower metabolism of T-2 toxin in NHA (54% T-2, 11% HT-2 toxin in cell lysate; 24% T-2, 10% HT-2 toxin in cell pellet). Our findings can be compared to the literature data where a time-dependent distribution of radiolabeled [3H]T-2 toxin in different fractions of perfused rat liver was analyzed. Plasma membrane fractions contained 38% of the radiolabel 5 min after exposure, but levels decreased to less than 1% after 2 h of perfusion. However, the radiolabel in fractions of smooth endoplasmic reticulum and mitochondrial fraction increased up to 2 h of perfusion, whereas the label in the nuclear fraction remained persistent at 7% from 30 min to 2 h.40

■

ASSOCIATED CONTENT

S Supporting Information *

Cell viability of HT-29 cells after the incubation of various T-2 toxin concentrations (1 nM−200 μM) after 24 and 48 h of incubation time. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Westfälische Wilhelms-Universität Münster, Institute of Food Chemistry, Corrensstrasse 45, 48149 Münster, Germany. Phone: +49 251 8333391. Fax: +49 251 8333396. E-mail: [email protected]. Funding

We thank the Deutsche Forschungsgemeinschaft (DFG), Bonn, for funding (HU-730/7-2 and GE 905/12-2). Notes

The authors declare no competing financial interest.

■

ABBREVIATIONS 7-AAD, 7-amino actinomycin D; BBB, blood−brain barrier; BCA, bicinchoninic acid; CE, collision energy; CXP, collision cell exit potential; DMEM, Dulbecco’s modified Eagle’s medium; DP, declustering potential; EGF, epidermal growth factor; FCS, fetal calf serum; HPLC, high-performance liquid chromatography; LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry; LDH, lactate dehydrogenase; MRM, multiple reaction monitoring; NHA, normal human astrocytes; NHLF, normal human lung fibroblasts; PBS, phosphate buffered saline; RPTEC, renal proximal tubule epithelial cells

4. CONCLUSIONS Apoptotic effects of T-2 toxin on human astrocytes in primary culture are reported in this study investigated by caspase-3354



■

Article

damage and triggers apoptosis via caspase pathway in human cervical cancer cells. Toxicology 262, 153−161. (23) Shifrin, V. I., and Anderson, P. (1999) Trichothecene mycotoxins trigger a ribotoxic stress response that activates c-Jun Nterminal kinase and p38 mitogen-activated protein kinase and induces apoptosis. J. Biol. Chem. 274, 13985−13992. (24) Sirkka, U., Nieminen, S. A., and Ylitalo, P. (1992) Acute neurobehavioural toxicity of trichothecene T-2 toxin in the rat. Pharmacol. Toxicol. 70, 111−114. (25) Wang, J., Fitzpatrick, D. W., and Wilson, J. R. (1998) Effects of the trichothecene mycotoxin T-2 toxin on neurotransmitters and metabolites in discrete areas of the rat brain. Food Chem. Toxicol. 36, 947−953. (26) Doi, K., and Uetsuka, K. (2011) Mechanisms of mycotoxininduced neurotoxicity through oxidative stress-associated pathways. Int. J. Mol. Sci. 12, 5213−5237. (27) Ravindran, J., Agrawal, M., Gupta, N., and Lakshmana Rao, P. V. (2011) Alteration of blood brain barrier permeability by T-2 toxin: Role of MMP-9 and inflammatory cytokines. Toxicology 280, 44−52. (28) Königs, M., Schwerdt, G., Gekle, M., and Humpf, H.-U. (2008) Effects of the mycotoxin deoxynivalenol on human primary hepatocytes. Mol. Nutr. Food Res. 52, 830−839. (29) Beyer, M., Ferse, I., and Humpf, H.-U. (2009) Large-scale production of selected type A trichothecenes: the use of HT-2 toxin and T-2 triol as precursors for the synthesis of d3-T-2 and d3-HT-2 toxin. Mycotoxin Res. 25, 41−52. (30) Mulac, D., and Humpf, H.-U. (2011) Cytotoxicity and accumulation of ergot alkaloids in human primary cells. Toxicology 282, 112−121. (31) Königs, M., Lenczyk, M., Schwerdt, G., Holzinger, H., Gekle, M., and Humpf, H.-U. (2007) Cytotoxicity, metabolism and cellular uptake of the mycotoxin deoxynivalenol in human proximal tubule cells and lung fibroblasts in primary culture. Toxicology 240, 48−59. (32) Bergmeyer, H. U. (1970) Methoden der enzymatischen Analyse, Verlag Chemie, Weinheim, Germany. (33) Vermes, I., Haanen, C., Steffens-Nakken, H., and Reutelingsperger, C. (1995) A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods 184, 39−51. (34) DeLean, A., Munson, P. J., and Rodbard, D. (1978) Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose-response curves. Am. J. Physiol. 235, E97−102. (35) Gutleb, A. C., Morrison, E., and Murk, A. J. (2002) Cytotoxicity assays for mycotoxins produced by Fusarium strains: a review. Environ. Toxicol. Pharmacol. 11, 309−320. (36) Minervini, F., Fornelli, F., Lucivero, G., Romano, C., and Visconti, A. (2005) T-2 toxin immunotoxicity on human B and T lymphoid cell lines. Toxicology 210, 81−91. (37) Mikami, O., Yamamoto, S., Yamanaka, N., and Nakajima, Y. (2004) Porcine hepatocyte apoptosis and reduction of albumin secretion induced by deoxynivalenol. Toxicology 204, 241−249. (38) EU (2001) Opinion of the Scientific Committee on Food on Fusarium Toxins Part 5: T-2 Toxin and HT-2 Toxin, Scientific Committee on Food (SCF), Brussels, Belgium. (39) Welsch, T., and Humpf, H.-U. (2012) HT-2 toxin 4-glucuronide as new T-2 toxin metabolite: enzymatic synthesis, analysis, and species specific formation of T-2 and HT-2 toxin glucuronides by rat, mouse, pig, and human liver microsomes. J. Agric. Food Chem. 60, 10170− 10178. (40) Pace, J. G., and Watts, M. R. (1989) Hepatic subcellular distribution of [3H]T-2 toxin. Toxicon 27, 1307−1311.

REFERENCES

(1) Desjardins, A. E., Hohn, T. M., and McCormick, S. P. (1993) Trichothecene biosynthesis in Fusarium species: chemistry, genetics, and significance. Microbiol. Rev. 57, 595−604. (2) Sudakin, D. (2003) Trichothecenes in the environment: relevance to human health. Toxicol. Lett. 143, 97−107. (3) Bouaziz, C., Abidessefi, S., Bouslimi, A., Elgolli, E., and Bacha, H. (2006) Cytotoxicity and related effects of T-2 toxin on cultured Vero cells. Toxicon 48, 343−352. (4) Edwards, S. G. (2009) Fusarium mycotoxin content of UK organic and conventional oats. Food Addit. Contam., Part A 26, 1063− 1069. (5) Schothorst, R. C., and van Egmond, H. P. (2004) Report from SCOOP task 3.2.10 “collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states”. Toxicol. Lett. 153, 133−143. (6) Schwake-Anduschus, C., Langenkämper, G., Unbehend, G., Dietrich, R., Märtlbauer, E., and Münzing, K. (2010) Occurrence of Fusarium T-2 and HT-2 toxins in oats from cultivar studies in Germany and degradation of the toxins during grain cleaning treatment and food processing. Food Addit. Contam., Part A 27, 1253−1260. (7) van der Fels-Klerx, H., J., and Stratakou, I. (2010) T-2 toxin and HT-2 toxin in grain and grain-based commodities in Europe occurrence, factors affecting occurrence, co-occurrence and toxicological effects. World Mycotoxin J. 3, 349−367. (8) EFSA Panel on Contaminants in the Food Chain (CONTAM) (2011) Scientific opinion on the risks for animal and public health related to the presence of T-2 and HT-2 toxin in food and feed. EFSA J. 12, 2481. (9) Wu, Q., Dohnal, V., Huang, L., Kuca, K., and Yuan, Z. (2010) Metabolic pathways of trichothecenes. Drug. Metab. Rev. 42, 250−267. (10) Johnsen, H., Odden, E., Johnsen, B. A., and Fonnum, F. (1988) Metabolism of T-2 toxin by blood cell carboxylesterases. Biochem. Pharmacol. 37, 3193−3197. (11) Li, Y., Wang, Z., Beier, R. C., Shen, J., Smet, D. D., De Saeger, S., and Zhang, S. (2011) T-2 Toxin, a trichothecene mycotoxin: Review of toxicity, metabolism, and analytical methods. J. Agric. Food Chem. 59, 3441−3453. (12) Ohta, M., Ishii, K., and Ueno, Y. (1977) Metabolism of trichothecene mycotoxins. I. Microsomal deacetylation of T-2 toxin in animal tissues. J. Biochem. 82, 1591−1598. (13) Trusal, L. R. (1986) Metabolism of T-2 mycotoxin by cultured cells. Toxicon 24, 597−603. (14) Königs, M., Mulac, D., Schwerdt, G., Gekle, M., and Humpf, H.U. (2009) Metabolism and cytotoxic effects of T-2 toxin and its metabolites on human cells in primary culture. Toxicology 258, 106− 115. (15) Weidner, M., Welsch, T., Hübner, F., Schwerdt, G., Gekle, M., and Humpf, H.-U. (2012) Identification and apoptotic potential of T-2 toxin metabolites in human cells. J. Agric. Food Chem. 60, 5676−5684. (16) Dugyala, R. R., Kim, Y. W., and Sharma, R. P. (1994) Effects of aflatoxin B1 and T-2 toxin on the granulocyte-macrophage progenitor cells in mouse bone marrow cultures. Immunopharmacology 27, 57−65. (17) Fairhurst, S., Marrs, T. C., Parker, H. C., Scawin, J. W., and Swanston, D. W. (1987) Acute toxicity of T-2 toxin in rats, mice, guinea-pigs, and pigeons. Toxicology 43, 31−49. (18) Ueno, Y. (1985) The toxicology of mycotoxins. Crit. Rev. Toxicol. 14, 99−132. (19) Bennett, J. W., and Klich, M. (2003) Mycotoxins. Clin. Microbiol. Rev. 16, 497−516. (20) Foroud, N. A., and Eudes, F. (2009) Trichothecenes in cereal grains. Int. J. Mol. Sci. 10, 147−173. (21) Bouaziz, C., Sharaf El Dein, O., El Golli, E., Abid-Essefi, S., Brenner, C., Lemaire, C., and Bacha, H. (2008) Different apoptotic pathways induced by zearalenone, T-2 toxin and ochratoxin A in human hepatoma cells. Toxicology 254, 19−28. (22) Chaudhari, M., Jayaraj, R., Bhaskar, A. S. B., and Lakshmana Rao, P. V. (2009) Oxidative stress induction by T-2 toxin causes DNA 355


Neurotoxic Potential and Cellular Uptake of T-2 ... - ACS Publications

Recommend Documents