Cellular responses to T-2 toxin and/or deoxynivalenol that induce cartilage damage are not specific to chondrocytes

The relationship between T-2 toxin and deoxynivalenol (DON) and the risk of Kashin-Beck disease is still controversial since it is poorly known about their selectivity in cartilage damage. We aimed to compare the cytotoxicity of T-2 toxin and DON on cell lines representative of cell types encountered in vivo, including human chondrocytes (C28/I2), human hepatic epithelial cells (L-02) and human tubular epithelial cells (HK-2). In addition, we determined the distribution of T-2 toxin and DON in Sprague-Dawley (SD) rats after a single dose exposure. T-2 toxin or DON decreased proliferation in a time- and concentration-dependent manner and their combination showed a similar antagonistic effect in C28/I2, L-02 and HK-2 cells. Moreover, we observed cell cycle arrest and apoptosis, associated with increased oxidative stress and decline in mitochondrial membrane potential induced by T-2 toxin and/or DON. In vivo study showed that T-2 toxin and DON did not accumulate preferentially in the knee joint compared to liver and kidney after an acute exposure in SD rats. These results suggest that T-2 toxin and/or DON inhibit proliferation and induce apoptosis through a possible mechanism involving reactive oxygen species-mediated mitochondrial pathway that is not specific for chondrocytes in vitro or joint tissues in vivo.

Determination of cytotoxicity. The MTT assay was used to determine the cytotoxicity after exposure to T-2 toxin and/or DON. Briefly, cells in 96-well plates were treated with five dilutions of T-2 toxin (from 2.5 to 40 ng/ml) or DON (from 100 to 1600 ng/ml), or a fixed constant ratio of the two mycotoxins (T-2 + DON, ratio = 1:40) 11 . Following 24, 48 and 72 h of exposure, the medium was replaced by fresh medium containing 20 μl (5 mg/ml PBS) MTT. After 4 h of incubation, the medium was removed and 150 μl DMSO was added to dissolve the formazan. Measurement of the absorbance was performed with an automatic ELISA reader (Infinite M200, Tecan, Switzerland) at 490 nm.
The combined effects of T-2 toxin and DON mixtures were analyzed using an isobologram method 34,35 . According to the isobologram analysis, a combination index (CI) value was calculated for quantification of synergism or antagonism for two drugs, as below: where D is the dose (or concentration) of a drug, D m is the median-effect dose (e.g., IC50, ED50, or LD50) that inhibits the system under study by 50%, f a is the fraction affected by D (e.g., percentage inhibition/100), and m is the coefficient signifying the shape of the dose-effect relationship. Dm and m values are used for calculating the CI values, where CI < 1, CI = 1, and CI > 1 indicate synergistic, additive, and antagonistic effects, respectively 35 . Cells were treated with T-2 toxin and/or DON in 6-well plates for 24 h, then were collected by digestion with trypsin, and fixed with 2.5% glutaraldehyde, post-fixed in 0.1% osmium tetroxide and embedded in Epon epoxy resin. Ultrathin sections were cut, stained with 0.1% lead citrate and 10% uranyl acetate. The ultrastructure of cells was observed with a transmission electron microscope (HITACHI H-7650, Tokyo, Japan).
Hoechst 33324 staining. The morphological alterations of nuclei associated with apoptosis were observed with a Hoechst 33224 staining kit from Sigma Chemical Co. (St. Louis, MO, USA). Briefly, the cells were cultured in 6-well plates and exposed to T-2 toxin (10 ng/ml) or DON (800 ng/ml), alone or in combination for 24 h, the medium was removed, and cells were washed twice with PBS. Following incubation of the cells with 10 μg/ml Hoechst 33342 dye for 20 min at 37 °C, fragmented and intact nuclei were observed with an inverted fluorescence microscope (Nikon, Tokyo, Japan).
Flow cytometry analysis of cell cycle and apoptosis. The cell cycle was determined using a cell cycle assay kit (CWBIO, Beijing, China) according to the manufacturer's instructions. Following treatment with T-2 toxin (10 ng/ml) or DON (800 ng/ml), alone or in combination, in 6-well plates for 24 h, the cells were collected and washed once with cold PBS and fixed in 95% cold ethanol for 2 h. Then the fixed cells were washed once with PBS, treated with 400 μl propidium iodide (PI) dye solution, and incubated at 37 °C in the dark for 30 min before measurement.
An Annexin V-FITC/PI detection kit (4 A Biotech Co., Ltd, Beijing, China) was used to determine the cell apoptosis rates following the treatment of mycotoxins, as described above. Briefly, the cells were harvested, washed with cold PBS twice, and resuspended in 100 μl of binding buffer. After addition of 5 μl of Annexin V-FITC and incubation for 5 min in the dark, 10 μl of 20 mg/ml PI dye solution was added followed by addition of 400 μl of PBS for measurement.
Flow cytometric analysis (FACS) of cell cycle progression and apoptosis rates were performed using a Facscalibur Flow Cytometer (Becton Dickinson, Mountain View, CA, USA). Cell cycle and apoptosis data were acquired with the CellQuest software (BD Biosciences).

Measurement of ROS.
The oxidative stress level was assessed by measurement of ROS with a fluorescent dye DCFH-DA assay kit (Beyotime, Jiangsu, China). In brief, cells were cultured in 6-well plates and exposed to T-2 toxin (10 ng/ml) or DON (800 ng/ml), alone and in combination for 24 h. DCFH-DA was added and cells were incubated at 37 °C for 30 min. Then, the cells were washed three times with serum-free medium and visualized by an inverted fluorescence microscope (Nikon, Tokyo, Japan). The fluorescence intensity was analyzed by Image-Pro Plus 6.0 software.
Mitochondrial membrane potential assay. Mitochondrial transmembrane potential (ΔΨm) was measured using a fluorescent dye JC-1 assay kit (Beyotime, Jiangsu, China). Following treatment with T-2 toxin (10 ng/ ml) or DON (800 ng/ml), alone or in combination in 6-well plates for 24 h, cells were collected and resuspended in 0.5 ml of fresh medium. After addition of 0.5 ml JC-1 work solution, the cells were incubated at 37 °C for 20 min. Then, cells were washed twice and re-suspended in JC-1 buffer solution. The fluorescence was measured using a Facscalibur Flow Cytometer (Becton Dickinson, Mountain View, CA, USA). Data were processed with the CellQuest software (BD Biosciences).
Experimental animals and groups. All animal protocols were approved by the Animal Ethics Committee of Xi'an Jiaotong University and were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Eighteen 3-week-old SD rats weighing 30~60 g were purchased from Animal Experimental Centre of Xi'an Jiaotong University and randomly divided into 3 groups. Group A (6 rats) were administered with T-2 toxin as a single dose at 2 mg/kg body weight (bw) by oral gavage. Group B (6 rats) were administered with DON as a single dose at 10 mg/kg bw by oral gavage. Group C (6 rats) were given double distilled water at 0.2 ml/kg bw by oral gavage, and they were used to verify the viability of the analytical method by recovery tests. After 8 h of exposure, rats were euthanized, and the liver, kidney, and knee joint were collected. The samples were stored at −20 °C prior to their analysis.

Measurement of T-2 toxin and DON by ELISA.
The T-2 toxin and DON concentrations in tissues were analyzed using a AgraQuant ® T-2 toxin ELISA kit (COKAQ6000, Romer Labs Singapore Pte Ltd., Singapore) and a RIDASCREEN ® DON ELISA kit (R5906, R-Biopharm, Darmstadt, Germany), respectively. The tissue homogenates were prepared, as described by Pestka et al. 36 . The assays were performed according to the manufacturer's protocols. After the reaction was stopped, the absorbance was measured with an automatic ELISA reader (Infinite M200, Tecan, Switzerland) at 450 nm for both T-2 toxin and DON. The concentrations of T-2 toxin and DON were quantified according to a standard curve. Data were reported as T-2 toxin or DON equivalents per g of organ tissue.
Statistical analyses. Data were expressed as mean ± standard error (SEM) or standard deviation (SD), as calculated by SPSS software. Statistical analyses were performed by one-way ANOVA among groups, and the Student's t test was employed to determine the significant differences between two groups. P < 0.05 was considered to be significant.

Results
Cytotoxicity of mycotoxins T-2 toxin and/or DON. The C28/I2, L-02 and HK-2 cells were evaluated for cell viability by MTT assay after exposure to T-2 toxin or DON alone or in combination. As shown in Figs 1, 2, and 3, cell proliferation, assessed as % of untreated control set at 100%, decreased in a time-and concentration-dependent manner in all three cell lines.
The relative cytotoxic effects were evaluated by calculating the concentration of mycotoxin that produced 50% inhibition of cellular proliferation (IC50). The IC50 values of both mycotoxins decreased with the increased exposure time (Table 1). According to the IC50 values, T-2 toxin was less cytotoxic on C28/I2 and HK-2 cells than on L-02 cells, and DON was less cytotoxic on C28/I2 cells than on L-02 and HK-2 cells (Table 1). T-2 toxin was also much more efficient inhibitor of proliferation than DON (Table 1).

Cell ultrastructure changes induced by T-2 toxin and/or DON. To visualize morphological changes
in C28/I2, L-02, and HK-2 cells after exposure to mycotoxins for 24 h, the cellular ultramicroscopic structures were view by TEM. Compared to untreated control cells, the electron density in nucleus and cytoplasm of each cell line decreased to a different extent (Fig. 4a), and swelling, vacuolar degeneration, and increased density of mitochondria were also observed after exposure all three cell lines to T-2 toxin or DON alone or in combination (Fig. 4b).  Fig. 5a. For C28/I2 cells, T-2 toxin significantly induced accumulation of cells in the G0/G1 phase, while DON alone or in combination with T2-toxin significantly induced accumulation of cells in the G2/M phase when compared to untreated control cells (Fig. 5b). For L-02 cells, T-2 toxin significantly induced accumulation of cells in the G0/G1 phase, but cell cycle arrest in the presence of DON alone or together with T2-toxin was not significantly from that in untreated control cells (Fig. 5c). For HK-2 cells, T-2 toxin and/or DON caused a significant arrest in the G0/G1 phase, which was accompanied by decreased accumulation of cells in the S and G2/M phase when compared to untreated control cells (Fig. 5d).

Apoptosis induced by T-2 toxin and/or DON.
Changes in the nuclei of C28/I2, L-02, and HK-2 cells due to T-2 toxin and/or DON exposure were studied by Hoechst 33342 staining. All three cell lines treated with T-2 toxin or DON alone or in combination for 24 h exhibited typical morphologic changes of apoptotic cells, with the appearance of irregularly shaped nuclei and fragmented chromatin (Fig. 6a). Flow cytometry analysis showed that the proportions of apoptotic cells were significantly increased when cells exposed to mycotoxins for 24 h compared to untreated cells (Fig. 6b and c). HK-2 cells were most resistant against apoptosis.

Oxidative stress induced by T-2 toxin and/or DON.
Oxidative stress is involved in the apoptosis induced by mycotoxins. In the present study, we found that the ROS production in C28/I2, L-02, and HK-2 cells was significantly increased by mycotoxin treatment for 24 h compared to untreated controls ( Fig. 7a and b).   Fig. 1. Each data point represents the mean ± SEM from three independent experiments with replicate samples, *p < 0.05 indicates significant differences between the mixture and T-2 toxin alone, # p < 0.05 represents significant differences between the mixture and DON alone.
The distribution of T-2 toxin and DON in knee joint, liver and kidney. The recovery rates of T-2 toxin and DON from tissues were 66%~114% and 65%~115%, respectively. Following 8 h of the acute contamination of SD rats, the concentrations of T-2 toxin were 14.34 ± 8.69, 10.41 ± 7.74, 11.66 ± 9.07 ng/g of organ tissue in knee joint, liver and kidney, respectively. The differences were not significant among the three tissues (Fig. 8a) Fig. 1. Each data point represents the mean ± SEM from three independent experiments with replicate samples, *p < 0.05 indicates significant differences between the mixture and T-2 toxin alone, # p < 0.05 represents significant differences between the mixture and DON alone.  Table 2. Dose-effect relationship parameters and mean combination index (CI) values of T-2 toxin or DON alone or in combination on C28/I2 cells. The parameters m, D m , and r are the slope, antilog of the x-intercept, and the linear correlation coefficient of the median-effect plot, which signifies the shape of the dose-effect curve, the potency (IC50), and the conformity of the data to the mass-action law, respectively. IC10, IC25, IC50, IC75, and IC90 are the doses required to inhibit proliferation by 10%, 25%, 50%, 75% and 90%, respectively. Ant indicates antagonistic effect.  Table 3. Dose-effect relationship parameters and mean combination index (CI) values of T-2 toxin or DON alone or in combination on L-02 cells. The parameters m, D m , and r are the slope, antilog of the x-intercept, and the linear correlation coefficient of the median-effect plot, which signifies the shape of the dose-effect curve, the potency (IC50), and the conformity of the data to the mass-action law, respectively. IC10, IC25, IC50, IC75, and IC90 are the doses required to inhibit proliferation by 10%, 25%, 50%, 75% and 90%, respectively. Ant indicates antagonistic effect.  Table 4. Dose-effect relationship parameters and mean combination index (CI) values of T-2 toxin or DON alone or in combination on HK-2 cells. The parameters m, D m , and r are the slope, antilog of the x-intercept, and the linear correlation coefficient of the median-effect plot, which signifies the shape of the dose-effect curve, the potency (IC50), and the conformity of the data to the mass-action law, respectively. IC10, IC25, IC50, IC75, and IC90 are the doses required to inhibit proliferation by 10%, 25%, 50%, 75% and 90%, respectively. Ant and Syn indicate antagonistic and synergistic effect, respectively. knee joint, liver and kidney, respectively. Notably, the concentrations in knee joint and liver were significantly lower than that in kidney (Fig. 8b).

Discussion
Grain contamination with mycotoxins has been convincingly associated with KBD in the past decades. However, contamination with mycotoxins in food is a common problem existing in many countries. Whether KBD is simply an environmentally related disease or directly caused by mycotoxins remains controversial because KBD is found only in a limited regions, including Northeast to Southwest China, Southeastern Siberia, and North Korea 13,14 . The present study compared the effects of individual and combined treatments with T-2 toxin and DON using three cell lines derived from kidney, liver, and cartilage and suggested that the damaging effects mycotoxins on cartilage in KBD are not specific to chondrocytes. The toxic effects of T-2 toxin and DON on cartilage are well established and previous studies have demonstrated the possible relationship between these mycotoxins and the risk of KBD 37,38 . Cartilage is the main anatomical sites affected by KBD in patients. However, a previous study showed that the effects of fusarochromanone and T-2 toxin on articular chondrocytes were not specific 39 . The results of our study also showed that T-2 toxin or DON, alone or in combination, could inhibit cell proliferation of cell lines derived from cartilage (C28/I2), liver (L-02), and kidney (HK-2) cells in a time-and dose-dependent manner. This suggests that the damaging effects of T-2 toxin and DON are not specific for chondrocytes. In addition, the cytotoxicity of T-2 toxin and DON on L-02 and HK-2 cells were greater than on chondrocytes, which suggests that liver and kidney cells may be more sensitive than chondrocytes to these mycotoxin. Morphological examination showed decreased electron density in the nucleus and cytoplasm and altered mitochondria in the three in all three cell lines after T-2 toxin and/or DON exposure.
Mycotoxins coexist naturally in feed and food worldwide. Thus, it is necessary to take into account the toxicity of mycotoxin mixtures. The cytotoxicity of T-2 toxin and DON alone or in combination, has been investigated in Chinese hamster ovary (CHO-K1) and mammalian kidney epithelial (Vero) cells, and antagonistic effects were Apoptosis is a form of programmed cell death that occurs in aging or damaged cells 40 . T-2 toxin and DON have been shown to induce apoptosis in many cell types 41 . Apoptosis has been assumed to be a crucial pathological change in chondrocytes of KBD patients, and recent studies have indeed shown that T-2 toxin induced apoptosis in human chondrocytes. This finding suggests a relationship between T-2 toxin and risk of KBD 32,42 . However, apoptosis is not the primary pathological feature in chondrocytes of KBD patients, and apoptotic chondrocytes and similar changes in expression of apoptosis-related genes were observed in the cartilage from patients with primary osteoarthritis 43 . Based on the results of our present study, T-2 toxin and DON individually or together can induce apoptosis in cell lines derived from tissues other than cartilage, suggesting that apoptosis induced by these mycotoxins is not specific to human chondrocytes.
Cell cycle arrest is one of the most important toxic effects of many mycotoxins. Previous studies showed that T-2 toxin induced G0/G1 phase arrest in differentiated murine embryonic stem cells 7 , and DON induced G2/M phase arrest in human umbilical vein endothelial cells and human epithelial cells 44,45 . Our study showed that T-2   toxin resulted in G0/G1 phase arrest in C28/I2, L-02 and HK-2 cells, while significant G2/M phase arrest induced by DON was observed only in C28/I2 cells. However, the limitation of this data is that we analysed cell cycle only using a single dose for 24 h.
Several studies have shown that oxidative stress is involved in the apoptosis induced by T-2 toxin and DON 41 . Our study showed that T-2 toxin or DON, either individually or combined, can significantly increase the ROS generation in C28/I2, L-02, and HK-2 cells. It is widely recognized that the induction of ROS can modulate the mitochondrial membrane potential, triggering the release of cytochrome c into the cytoplasm to initiate the apoptotic pathway 46,47 . Moreover, many reports have shown that T-2 toxin and DON induce apoptosis through ROS-mediated mitochondrial pathway in different cell types 7,[48][49][50] . In the present study, we observed swelling, vacuolar degeneration and increased density of mitochondria in all three cell types after treatment with T-2 toxin and/or DON. In addition, a simultaneous decrease of mitochondrial membrane potential was also observed. These results suggest that ROS-mediated mitochondrial pathway was possibly involved in the T-2 toxin and/or DON-induced apoptosis of C28/I2, L-02 and HK-2 cells, but it need to be further investigated when blocking of ROS accumulation.
T-2 toxin and DON are rapidly absorbed and distributed in animal tissues after exposure. Previous studies showed that concentrations of T-2 toxin and DON in plasma and most tissues in mice peaked within 15 min following oral administration, and then rapidly declined in a biphasic pattern 36,51 . In the present study, we analyzed the concentrations of T-2 toxin and DON in the knee joint, liver, and kidney of SD rats at 8 h after administration with a single dose of these two mycotoxins based on previous studies 36,52,53 , with consideration of the absorption and clearance features of these two toxins in animal bodies after exposure, and the sensitivity of the ELISA kits used in this study. The study showed that the concentrations of T-2 toxin in the knee joint, liver, and kidney were not significantly different, but the concentrations of DON in knee joint and liver were significantly lower than in the kidney. Our results suggest that T-2 toxin and DON do not preferentially accumulate in joint tissues.
In conclusion, using cell lines derived from cartilage, liver, and kidney as models, we found that treatment with T-2 toxin and/or DON showed time-and concentration-dependent cytotoxic effects on cell viability. The combination of T-2 toxin and DON exhibited a similar antagonistic effect. Exposure to T-2 toxin or DON, alone or in combination could also induce cell ultrastructural changes, especially in the cytoplasm and mitochondria, increased oxidative stress with ROS generation, and decreased mitochondrial membrane potential, accompanied by cell cycle arrest and increased apoptosis in all three culture models of C28/I2, L-02 and HK-2 cell lines. In addition, T-2 toxin and DON accumulate in joint tissues to a similar extent as they do in liver and kidney. Our findings suggest, therefore, that the damaging effects of T-2 toxin and/or DON on chondrocytes were not specific in vitro. Whether T-2 toxin and DON contribute directly to the etiology of KBD and why these mycotoxins selectively damage the cartilage in KBD patients will deserve further study.