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Objective:To investigate the relationship between T-2 toxin, deoxynivalenol (DON) induced differentially expressed genes in human chondrocytes and Kashin-Beck disease (KBD), and to search for potential molecular markers of KBD.Methods:Gene microarray profiling was used to analyze the differentially expressed genes induced by T-2 toxin (0.01 μg/ml) and DON (1.0 μg/ml) in normal human chondrocytes, and the differences and similarities between them and the differentially expressed genes in KBD chondrocytes were compared. KEGG pathway enrichment analysis was performed on differentially expressed genes in each group. And the expression patterns of KBD susceptibility genes in T-2 toxin and DON induced human chondrocytes were further compared and analyzed.Results:Gene microarray profiling analysis showed that there were 882 (349 up-regulated genes, 533 down-regulated genes) and 2 118 differentially expressed genes (1 124 up-regulated genes, 994 down-regulated genes) in human chondrocytes induced by T-2 toxin and DON compared with normal control cells, respectively. Compared with differentially expressed genes in KBD chondrocytes, the genes with the same expression trend included B cell translocation gene 1 (BTG1), G protein signaling regulatory protein 5 (RGS5), fatty acid binding protein 4 (FABP4) and key protein senescence 1 (FBLN1), the same KEGG pathway including p53, extracellular matrix receptor interaction and phosphatidylinositol-3-kinase-protein kinase B (PI3K-Akt) signaling pathway. Both T-2 toxin and DON induced human chondrocytes to up-regulate the expression of KBD susceptibility gene growth differentiation factor 5 (GDF5) and down-regulate the expression of collagen type ⅨA1 (COL9A1).Conclusion:The BTG1, RGS5, FABP4, FBLN1, GDF5 and COL9A1 genes play an important role in the pathogenesis of KBD and can be used as potential molecular markers for the pathogenesis of KBD.
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As the main pollutant of food crops, T-2 toxin has toxic effects on human and animal digestive system, nervous system, reproductive development and so on. In Kashin-Beck disease related areas in China, the etiological substance of Kashin-Beck disease is the abnormal accumulation of T-2 toxin in the grain produced in the endemic area. The prevention and treatment of Kashin-Beck disease has achieved remarkable results through the comprehensive prevention and control measures, such as changing the production and life style, grain exchange, etc., but there are still pathogenic factors in the external environment of the disease area. In this paper, the metabolic kinetics of T-2 toxin is reviewed, and the physicochemical properties, distribution in the body, metabolic kinetics, biotransformation and damage of T-2 toxin and its metabolites to various organs are described, so as to provide new thinking for the study on the effects of T-2 toxin on various organs of the body.
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Objective:To investigate the effects of T-2 toxin on expression of inflammatory cytokines, human leukocyte differentiation antigen (CD) 44 and integrin in chondrocytes cultured in vitro. Methods:Primary chondrocytes from SPF Wistar rats aged 1 to 2 days were isolated and cultured in vitro, and the chondrocytes were identified by toluidine blue staining. The effects of different dose of T-2 toxin (0, 2, 4, 6, 10, 12, 20 ng/ml) on proliferation of chondrocytes for 24, 48 and 72 h were detected via the cell counting kit-8 (CCK-8) method. According to the cell survival rate, T-2 toxin concentrations of 0 (control), 2, 4, 6 and 8 ng/ml were selected for subsequent experiments, and the exposure time was 48 h. The contents of interleukin (IL)-6, IL-1β, tumor necrosis factors (TNF)-α and CD44 in cell supernatant were detected via the enzyme linked immunosorbent assay (ELISA). The protein expression levels of integrin α5 and integrin β1 in chondrocytes were detected by Western blotting. Results:At the same exposure time, there were significant differences of the survival rate of chondrocytes between different dose groups ( F = 130.759, 258.250, 123.337, P < 0.01). At 48 h after exposure, there were statistically significant differences in IL-6, IL-1β, TNF-α and CD44 contents in the culture supernatant of chondrocytes between different dose of T-2 toxin groups ( F = 10.613, 4.805, 2.943, 12.395, P < 0.01 or < 0.05); among them, the IL-6 levels of 2, 4, 6 and 8 ng/ml groups were higher than that of control group ( P < 0.05); the IL-1β levels of 6, 8 ng/ml groups were higher than that of control and 2 ng/ml groups, and the 6 ng/ml group was higher than that of 4 ng/ml group ( P < 0.05); the TNF-α levels of 6, 8 ng/ml groups were lower than that of control group ( P < 0.05); the CD44 levels of 2, 4, 6 and 8 ng/ml groups were lower than that of control group ( P < 0.05). At 48 h after exposure, there were statistically significant differences in integrin α5 and integrin β1 protein expression levels between different dose of T-2 toxin groups ( F = 4.635, 4.376, P < 0.05). Among them, the protein expression levels of integrin α5 in 6, 8 ng/ml groups were significantly lower than that of control group ( P < 0.05); the protein expression levels of integrin β1 in 6, 8 ng/ml groups were significantly higher than that of control group ( P < 0.05). Conclusion:T-2 toxin may up-regulate the expressions of IL-6, IL-1β and integrin β1, while down-regulate the expressions of TNF-α, CD44 and integrin α5, then disrupt the balance between chondrocytes and extracellular matrix, and cause chondrocytes damage.
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Objective To understand the current environmental selenium and T-2 toxin levels in the critically ill areas of Kaschin-Beck disease in Qinghai Province.Methods In three historical villages with serious diseases of Kaschin-Beck disease,Xia,Xiemalang and Yaoshidao were selected;at the same time,three villages from non-diseased areas were selected as control villages including Damitan,Gongba and Deang in 2018.Six villages were used as survey sites.Collected hair samples of children aged 6-12 years old,to measure the hair selenium content in Xinghai and Guide survey sites [children's selenium content reference value was (0.60 ± 0.03)mg/kg].Ten samples of self-produced grain were collected from each survey site,selenium content [wheat selenium content reference value was (0.053 ± 0.007) mg/kg] and T-2 toxin content in grain was detected in the six survey sites.Ten soil samples were collected from each survey sites.Soil selenium content was detected in the six survey sites [soil selenium content reference value was (0.24 ± 0.03) mg/kg].Results The selenium contents of children's hair in the diseased and non-diseased areas of Xinghai County,Guide County were (0.252 ± 0.071),(0.296 ± 0.087);(0.225 ± 0.032),(0.238 ± 0.040) mg/kg,respectively.The selenium contents of wheat in the diseased and nondiseased areas of the three counties were 0.000 19,0.003 66;0.000 15,0.004 16;0.016 78,0.016 94 mg/kg.The soil selenium contents in the diseased and non-diseased areas of the three counties were (0.095 ± 0.015),(0.114 ± 0.014);(0.082 ± 0.013),(0.083 ± 0.018);(0.080 ± 0.005),(0.060 ± 0.013) mg/kg.The T-2 toxin contents of wheat in the diseased and non-diseased areas of the three counties were (3.173 ± 0.762),(3.100 ± 0.473);(2.506 ± 0.430),(3.186 ± 0.451);(2.416 ± 0.619),(2.879 ± 0.456) μg/kg.Conclusions The content of hair selenium of children is close to the normal reference value in the area of Kaschin-Beck disease in Qinghai Province.The contents of soil selenium and the main grain selenium of the residents are low in the diseased village.A certain amount of T-2 toxin is detected in the main grain of residential households.
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Objective To investigate the effects of different doses of T-2 toxin on the expression of cytokines cytokines and pathological changes in parental mice and their offspring. Methods One hundred female mice and 25 male mice (CD-1, SPF) were adapted for one week. After regular random mating, observation of vaginal suppository within the first 24 hours was as the 0th day of pregnancy. The pregnant rats were divided into high dose, medium dose, low dose and control groups according to body weight by a random number table(Feed: the doses of T-2 toxin were 1 200, 600, 300, and 0 μg/kg, respectively), with 16 - 18 rats in each group. The high, middle and low dose groups began to consume the poisoned feed on the 0th day of pregnancy, while the control group consumed the standard feed. After natural delivery, their offspring were continually treated the same way as their mother until the offspring reached adulthood. Serum levels of interleukin-1β (IL-1β) and interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), organ coefficient and pathological changes of articular cartilage were determined. Results The levels of IL-1β,IL-6 and TNF-α in the control, low, middle and high T-2 toxin groups during the pro-pregnancy of the middle-aged mice were [(219.56 ± 19.32), (136.89 ± 20.41), (210.49 ± 21.23), (207.41 ± 21.23); (192.73 ± 22.43), (136.25 ± 29.55), (187.43 ± 39.32), (232.48 ± 39.32); (1 303.02 ± 142.10), (1 072.60 ± 78.30), (1 065.03 ± 37.44), and (1 169.72 ± 104.18) ng/L], respectively. The differences between control and T-2 toxin treated groups were statistically significant (F = 17.124, 6.237, 7.670, P < 0.05). For further pairwise comparison,IL-1β and IL-6 in low dose group were significantly lower than those in control, middle and high dose groups (P < 0.05); TNF-α content in control group was significantly higher than those in low,middle and high dose groups(P<0.05).There were significant differences in the levels of IL-1β,IL-6 and TNF-α between the control group and the low,middle and high dose groups of offspring weanling mice[(142.36 ± 13.36),(113.01 ± 8.65), (102.13 ± 8.31), (123.42 ± 10.41); (109.92 ± 9.76), (100.26 ± 15.60), (85.25 ± 9.97), (100.21 ± 16.46);(1 308.45 ± 204.90), (1 248.60 ± 96.85), (1 081.09 ± 105.51), (1 204.87 ± 153.96) ng/L, F = 49.823, 10.530, 7.490, P < 0.05]. The levels of IL-1β and IL-6 in the control group were significantly higher than those in the low, middle and high dose groups(P < 0.05).The levels of TNF-α in the control group were significantly higher than those in the medium and high dose groups(P < 0.05).The levels of the three cytokines IL-1β, IL-6 and TNF-α in adult filial mice were significantly different [(69.71 ± 9.61), (61.31 ± 10.07), (63.07 ± 10.39), (58.56 ± 9.69); (172.55 ± 24.55),(146.91 ± 13.47),(151.02 ± 24.93), (157.21 ± 17.86); (1 136.87 ± 137.39), (1 002.22 ± 86.52), (987.12 ± 130.80),(1 047.21 ± 171.64)ng/L, F=4.670,5.636, 4.775, P < 0.05], the contents of the three cytokines in the poisoning groups were significantly lower than that of the control group (P < 0.05). The organ coefficients of thymus, spleen and liver in the second trimester were significantly different [(0.14 ± 0.03), (0.20 ± 0.06), (0.15 ± 0.02), (0.12 ± 0.03); (0.71 ± 0.16), (0.78 ± 0.14), (0.77 ± 0.15), (0.38 ± 0.10); (6.19 ± 0.43), (5.57 ± 0.57), (6.04 ± 0.32), (5.11 ± 0.29), F = 4.056, 11.064, 8.312, P < 0.05], and the thymus index was significantly increased in low dose group (P<0.05),spleen coefficient decreased significantly in high dose group (P < 0.05), and liver coefficients in low and high dose group were significantly decreased (P < 0.05). In the offspring, the midbrain coefficient of viscera showed significant changes [(3.45 ± 0.73), (3.11 ± 0.31), (2.98 ± 0.45), (3.04 ± 0.22), F = 7.529, P < 0.05], which was significantly decreased in the exposed rats(P<0.05).Both the mid-pregnant mice and filial mice showed varying degrees of changes in epiphyseal cartilage injury. The degree of epiphyseal cartilage injury became higher with increasing dosages of T-2 toxin in mid-pregnancy and post-weaning parental mice, and the injury was more serious in post-weaning mice. Conclusions Exposure to T-2 toxin can cause decrease of cytokines IL-1β, IL-6 and TNF-α in the blood of CD-1 pregnant and filial mice, and also cause the cartilage damage in mice, which are aggravated following increased doses of T-2 toxin and extension of exposure time.
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Objective To study the effect of T-2 toxin on proliferation and cell cycle of rat chondrocytes,in order to provide a new idea in molecular mechanism of T-2 toxin-induced chondrocyte damage.Methods Primary chondrocytes of neonatal Wistar rats were isolated and stained by toluidine blue staining and type Ⅱ collagen immunofluorescence staining.The effects of different concentrations of T-2 toxin [0 (control),1,5,10,20,50,100 μg/L)] on proliferation of chondrocytes for 24 h were detected by cell counting kit-8 (CCK-8) method,and control,1 (low dose),5 (medium dose),and 10 μg/L (high dose) T-2 toxin were selected for subsequent experiment;cell cycle changes were detected by flow cytometry;Real-time PCR and Western blotting were used to detect the effects of T-2 toxin on mRNA and protein expressions of proliferating cell nuclear antigen (PCNA) and Cyclin D1 in chondrocytes.Results With increase of T-2 toxin concentration (control,1,5,10,20,50,100 μg/L),the cell survival rates [(100.00 ± 0.00)％,(93.12 ± 1.66)％,(77.12 ± 1.11)％,(59.44 ± 4.09)％,(46.64 ± 3.86)％,(38.15 ± 3.37)％,(33.79 ± 0.99)％] were decreased,and the differences were statistically significant (F =139.21,P ＜0.05).The percentages of quiescent phase/pre-DNA synthesis phase (G0/G1 phase) ceils in 1,5,10 μg/L T-2 toxin groups [(22.03 ± 0.42)％,(30.54 ± 2.61)％,(36.01 ± 1.51)％] were significantly higher than that in control group [(13.79 ± 1.65)％,P ＜ 0.05];the percentages of DNA synthesis phase (S phase) cells [(60.27 ± 3.53)％,(53.88 ±4.38)％,(49.55 ± 2.49)％] were significantly lower than that in control group [(76.72 ± 4.24)％,P ＜ 0.05].The differences of mRNA levels of PCNA and Cyclin D1 between groups were statistically significant (F =46.80,17.97,P ＜ 0.05),and 5,10 μg/L T-2 toxin groups (0.77 ± 0.13,0.79 ± 0.08,0.60 ± 0.07,0.56 ± 0.05) were lower than the control group (0.99 ± 0.02,1.01 ± 0.01,P ＜ 0.05).The expressions of PCNA protein in 5,10 μg/L T-2 toxin groups (0.69 ± 0.03,0.49 ± 0.03) were lower than that in control group (0.92 ± 0.05,P ＜ 0.05);the expressions of Cyclin D1 protein in 1,5,10 μg/L T-2 toxin groups (0.80 ± 0.06,0.60 ± 0.07,0.33 ± 0.13) were lower than that in control group (0.95 ± 0.07,P ＜ 0.05).Conclusion T-2 toxin can inhibit the proliferation of chondrocytes,which may be worked through influencing the expression of cell cycle protein,causing cell cycle arrest,thereby inhibiting DNA synthesis.
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Objective To study the effects of different doses of T-2 toxin on organs and bones of BALB/c pregnant mice and their offspring mice.Methods Sixty female SPF BALB/c mice at the age of 6-weeks were mated with 30 6-week-old male SPF BALB/c mice.Female mice were divided into control,low dose,medium dose and high dose groups according to body weight via the random digital table method,with 15 mice in each group.After mating with male mice,the pregnant mice in each group were 14,10,9 and 15,respectively.Nutritional interventions (feed:the doses of T-2 toxin were 0,600,1 200 and 2 400 ng/g,respectively) were initiated from the gestation day 0 until the first generation mice were grown up (6-weeks-old).The growth status and organ coefficient (heart,liver,kidney,thymus,spleen and brain) of the two generations in each period were recorded.Skeletal X-ray photographs of the two generations were taken by digital radiography.The histopathological changes in the organs (liver,thymus,spleen and epiphyseal cartilage) of the two generations were observed under light microscope.Results Among the pregnant mice,there were no significant differences in organ coefficients (P ＞ 0.05).No abnormalities were observed in each group of skeletal X-ray photographs.In the female generation mice,there were no significant differences in the coefficients of heart,liver and kidney (F =0.233,2.196,0.430,P ＞ 0.05),while there were significant differences in the coefficients of thymus,spleen and brain (F=3.683,3.148,4.498,P ＜ 0.05),and the thymus coefficient of medium dose group was higher than that of control group;the thymus coefficient of high dose group was lower than that of other three groups;the spleen coefficients of the three dose groups were higher than that of control group;the brain coefficients of the three dose groups were lower than that of control group (P ＜ 0.05).In the male generation mice,there were no significant differences in the coefficients of thymus and brain (F =2.447,1.620,P ＞ 0.05),while there were significant differences in the coefficients of heart,liver,kidney and spleen (F =5.339,2.738,11.435,2.872,P ＜ 0.05),and the heart coefficient of high dose group was lower than that of control group and low dose group;the coefficients of liver and kidney in medium dose and high dose groups were lower than those in control and low dose groups;the spleen coefficients of the three dose groups were higher than that of control group (P ＜ 0.05).The pathological changes of liver,thymus,spleen and epiphyseal cartilage were found in dose exposure groups;epiphyseal hyperplasia was found in the skeletal X-ray photographs of medium and high dose groups.Conclusion T-2 toxin has no significant effects on pregnant mice,but it could cause damage to the organs and epiphyseal plate cartilage of the first generation,and the location of the injury is related to gender.
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T-2 toxin is a secondary fungal metabolite that belongs to the trichothecene mycotoxin family.T-2 toxin can lead to the structural and functional changes of cartilage cells and cartilage cell degeneration and necrosis.With strong cytotoxicity,T-2 toxin can cause definite damage to cartilage cells.In this paper,we reviewed recent studies on the effects of T-2 toxin on chondrocyte apoptosis,ultra structural changes and extracellular matrix in vitro.
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Objective To investigate the death of chondrocytes in rats which feed with T-2 toxin under selenium (Se) deficient conditions.Methods Thirty two healthy male SD rats were divided into two groups by weight which were normal diet group and Se deficiency diet group,16 rats in each group.Rats in normal diet group were fed with Se 101.5 μg/kg diet,and rats in Se deficiency diet group were fed with Se 1.1 μg/kg diet for 30 d.Normal diet group was divided into control group and T-2 toxin group,and Se deliciency diet group was randomly divided into Se-deficiency group and Se-deficiency plus T-2 toxin group,8 rats in each group.After that,rats in T-2 toxin and Se-deficiency plus T-2 toxin groups were administrated intragastrically with T-2 toxin (100 μg/kg) everyday for 30 d.Rats were put to death,the left knee was taken and stained with hematoxylin-eosin and SafraninFast green,pathological changes of rat's knee joint cartilage were observed under light microscopy,expression levels of active caspase-3 and receptor interacting protein 3 (RIP3) in rat's articular cartilage cells were determined via the immunohistochemical method.The apoptosis was also detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL).Results Red ghost outlines of chondrocyte and multiple chondral cell clusters surrounded the non-cell areas in deep zone of articular cartilage of knee joint stained with hematoxylineosin were seen in Se-deficiency plus T-2 toxin group under light microscope.In the superficial zone of cartilage,the positive percent of TUNEL and active caspase-3 in Se-deficiency plus T-2 toxin group was higher than those of control group,Se-deficiency group and T-2 toxin group [(7.47-± 0.34)％ vs (4.68 ± 0.54)％,(2.67-± 0.64)％,(2.56 ±0.54)％;(4.75 ± 0.67)％ vs (1.24 ± 0.25)％,(0.00 ± 0.00)％,(0.00 ± 0.00)％,P ＜ 0.05].In the middle zone of cartilage,the positive percent of TUNEL,active caspase-3 and RIP3 in Se-deficiency plus T-2 toxin group was significantly higher than those of control group,T-2 toxin group and Se-deficiency group [(72.06 ± 6.15)％ vs (16.10 ± 3.00)％,(19.57 ± 3.49)％,(19.33 ± 5.19)％;(51.13 ± 4.18)％ vs (10.97-± 3.01)％,(15.36 ± 4.37)％,(15.23 ± 3.13)％;(25.91 ± 13.39)％ vs (1.59 ± 1.14)％,(4.32 ± 2.91)％,(7.50 ± 5.00)％,P ＜ 0.05].The positive percents of TUNEL,active caspase-3 and RIP3 were not significantly different in the deep zone (P ＞ 0.05).Conclusion The death of the middle zone in the rat cartilage induced by T-2 toxin under selenium deficient conditions isapoptosis and necroptosis.
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Objective: To investigate the inhibitory effect of T-2 toxin on proliferation of the human hepatocellular carcinoma HepG2 cells and its promotion effect of apoptosis.Methods:The HepG2 cells in the logarithmic phase were selected and divided into control group (without T-2 toxin)and experimental groups (given 0.25,2.50,25.00,250.00 and 2500.00 μg·L-1 T-2 toxin).After 24 h treatment,the morphology of cells was observed under inverted microscope;the inhibitory rate of proliferation of cells was determined by MTT assay;the cell cycle and apoptotic rate of cells were analyzed by flow cytometry;Hochest 33258 staining was used to observe the apoptotic morphology of cells;the activity of caspase-3 in HepG2 cells was detected.Results:After treated for 24 h,the inverted microscope observation results showed that the number of the cells in experimental groups was decreased significantly and the cells shrank and deformed.The MTT results showed that compared with control group,the inhibitory rates of proliferation of the cells in experimental groups were increased (P <0.01).The flow cytometry results showed that compared with control group, the percentage of the cells in SubG1 phase in experimental group was significantly increased,and the apoptotic rates of the cells in experimental groups were significantly increased.The Hoechest 33258 staining results showed that the chromatin condensation was observed in the cells in experimental groups,and the nuclei were dense and stained.Compared with control group,the activities of intracellular caspase-3 of the cells in experimental groups were significantly increased (P < 0.01 ). Conclusion:T-2 can inhibit the proliferation of human hepatocellular carcinoma HepG2 cells,and induce the apoptosis.
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OBJECTIVE To compare the species difference of T-2 toxin metabolism in liver micro-somes of different animals. METHODS T-2 toxin was incubated with liver microsomes from mice, rats,Beagle dogs, monkeys and humans, respectively, at 37℃ for some time. Then, the incubation liquid was detected by high liquid chromatography-mass spectrometry method after albumen precipitation. RESULTS The half-life (t1/2) of T-2 toxin was less than 1 min, 2-4 min in mouse and monkey liver microsomes, 13 min in dog liver microsomes, and 39 min in rat liver microsomes. The hepatic clear-ance (Clh) of T-2 toxin was divided into three groups among the five species of animals:humans, dogs and rats were in one group, monkeys a second group, and mice in another group. The Clh of mouse group was 3-4 times that of the human, dog and rat group. The affinity to T-2 toxin was different between the liver microsomes of these five species. The affinity of mouse liver microsomes was the strongest, followed by that of humans, dogs, rats and monkeys. The enzyme transfer rate of T-2 toxin was the highest in monkey liver microsomes followed by that of rats and dogs. It was one million times higher in monkey liver microsomes than in human and mouse liver microsomes. The major metabolites were 3′-hydroxyl-T-2 toxin and neosolaniol. T-2 triol and HT-2 toxins were the major metabolites in human and rat liver microsomes. HT-2 toxin and 3′-OH-T-2 toxin were the dominating metabolites in dog liver microsomes and T-2 triol and 3′-OH-T-2 toxin in mouse liver microsomes. T-2 toxin metabolited by hydrolysis effect in mouse, rat, dog and human liver microsomes, but through hydroxylation in monkey liver microsomes. CONCLUSION There are species differences in metabolic parameters, metabolites, amounts of metabolites, metabolic pathways of T-2 toxin in mouse, rat, dog, monkey and human liver microsomes.
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<p><b>OBJECTIVE</b>To investigate chondrocyte apoptosis and the expression of biochemical markers associated with apoptosis in Kashin-Beck disease (KBD) and in an established T-2 toxin- and selenium (Se) deficiency-induced rat model.</p><p><b>METHODS</b>Cartilages were collected from the hand phalanges of five patients with KBD and five healthy children. Sprague-Dawley rats were administered a selenium-deficient diet for 4 weeks prior to T-2 toxin exposure. The apoptotic chondrocytes were observed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Caspase-3, p53, Bcl-2, and Bax proteins in the cartilages were visualized by immunohistochemistry, their protein levels were determined by Western blotting, and mRNA levels were determined by real-time reverse transcription polymerase chain reaction.</p><p><b>RESULTS</b>Increased chondrocyte apoptosis was observed in the cartilages of children with KBD. Increased apoptotic and caspase-3-stained cells were observed in the cartilages of rats fed with normal and Se-deficient diets plus T-2 toxin exposure compared to those in rats fed with normal and Se-deficient diets. Caspase-3, p53, and Bax proteins and mRNA levels were higher, whereas Bcl-2 levels were lower in rats fed with normal or Se-deficiency diets supplemented with T-2 toxin than the corresponding levels in rats fed with normal diet.</p><p><b>CONCLUSION</b>T-2 toxin under a selenium-deficient nutritional status induces chondrocyte death, which emphasizes the role of chondrocyte apoptosis in cartilage damage and progression of KBD.</p>
Subject(s)
Adolescent , Animals , Child , Female , Humans , Male , Rats , Apoptosis , Biomarkers , Cartilage, Articular , Chondrocytes , Physiology , Kashin-Beck Disease , Matrilin Proteins , Genetics , Metabolism , Models, Animal , Random Allocation , Rats, Sprague-Dawley , Selenium , T-2 Toxin , PharmacologyABSTRACT
Objective To search for the metabolites that associated with articular cartilage damage in the urine of rat model with articular cartilage destruction induced by T-2 toxin.Methods Thirty healthy male Wistar rats aged 4-5 weeks were numbered by weight,randomly divided into two groups (n =15 per group),namely the control group and the model group.The rats in the control group were fed with standard rat diets,and those in the model group were given diets contaminated by T-2-toxin (300 μg/kg).Throughout the experiment,all animals were given free access to distilled water and diets.After continuous treatment for 3 months,all the rats were sacrificed.The changes of articular cartilage in rat knee joints were observed by histopathological method,the metabolic profile of rats' urine was determined by ultra performance liquid chromatography/quadrupole time of flight-mass spectrometry (UPLC/QTOF-MS) technique.Combined with multivariate statistical analysis,database searching was applied to explore and confirm the different metabolites associated with cartilage damage.Results Light microscope showed that rats' articular chondrocytes in the control group presented cells in neat rows and eumorphism,rats' articular chondrocytes in the model group presented extensive areas of chondrocyte degeneration,necrosis and loss.In rats' urine metabolic profiles,5 different metabolites associated with cartilage destruction were detected,such as 4-hydroxynonenal (HNE),trans-4,5-epoxy-2(E)-decenal (EDE),5-methyldeoxycytidine,and 5-L-glutamyl-glycine and prolyl-valine.Compared with the control group (mass spectrum peak area:65820 ± 5200,22080 ± 3538,4292 ± 3520,3277 ± 2025,1104 ± 990),all of them increased in the model group (mass speetrum peak area:90240 ± 18863,25610 ± 5071,9702 ± 6562,6029 ± 3905,4144 ± 5322,t =-3.903,-2.209,-2.814,-2.424,-2.174,all P ＜ 0.05).Conclusions The articular cartilage destruction induced by T-2 toxin could cause the changes of related metabolites in the urine;the 5 kinds of changed metabolites in urine are related to articular cartilage destruction.
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T-2 toxin is a secondary fungal metabolite that belongs to the trichothecene mycotoxin family,it is the most toxic in class A trichothecene mycotoxin.T-2 toxin can induce apoptosis in cells bearing high proliferating activity.The mycotoxin readily passes the placenta and is distributed to embryo tissues,which results in fetal brain damage,bone malformation,thymic atrophy and even death.This article reviewed the effects of T-2 toxin on immune system,blood system and cell toxicities in pregnant mice and generation mice.
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Objective To study the change of rats serum malondialdehyde (MDA) and the expression levels of 4-hydroxy acid nonene (4-HNE) and 8-hydroxy uridine (8-OHdG) of articular cartilage under low selenium (Se) and T-2 toxin poisoning,to explore oxidative damage of articular cartilage in rats.Methods Thirtytwo healthy male SD rats were divided into two groups by weight which were normal diet group and Se-deficiency group,16 rats in each group.Rats in normal diet group was fed with selenium 101.5 μg/kg diet,and rats in Sedeficiency group was fed with selenium 1.1 μg/kg diet for 30 d.Normal diet group was divided into control group and T-2 toxin group,and low selenium diet group was randomly divided into Se-deficiency group and Se-deficiency plus T-2 toxin group,8 rats in each group.After that,rats in T-2 toxin and Se-deficiency plus T-2 toxin groups were administrated intragastrically with T-2 toxin (100 mg/kg) everyday for 30 d.Rats were put to death,the left knee was taken and stained with hematoxylin-eosin and Safranin-Fast green,pathological changes of rat's knee joint cartilage were observed under light microscopy,expression levels of 8-OHdG and 4-HNE in rat's articular cartilage cells were determined by immunohistochemical method and rat's MDA content was determined by glucosinolates barbituric acid method.Results Chondronecrosis in deep zone of articular cartilage of knee joint stained with hematoxylin-eosin was seen in Se-deficient plus T-2 toxin diet group under light microscope.Significantly less Safranin-Fast green staining was observed in the cartilage of knee joints in the Se-deficient plus T-2 toxin diet group compared to the control group.Compared with control group [(3.41 ± 2.48)％,(2.28 ± 1.74)％],8-OHdG and 4-HNE in Se-deficient plus T-2 toxin group [(62.61 + 10.97)％,(75.03 ± 7.92) ％] positive expression rate increased significantly (F =16.24,18.61,all P ＜ 0.05).Comparison of serum MDA content in each group,the difference was statistically significant (F =4.32,P ＜ 0.05).The Se-deficiency group [(2.803 ± 0.163) μmol/L] was compared with control group [(1.873 ± 0.475) μmol/L] that the contents of serum MDA were increased.The T-2 toxin group [(2.890 ± 0.453) μmol/L] was compared with control group [(1.873 ± 0.475) μmol/L] that the content of serum MDA was increased (P ＜ 0.05).The Se-deficiency plus T-2 toxin group [(3.521 ± 0.292) μmol/L] was compared with Sedeficiency group and control group that the contents of serum MDA were increased (all P ＜ 0.05).Conclusions The marker of peroxidation products are increased in articular cartilage of SD rats under the condition of Sedeficiency and T-2 toxin poisoning.The cartilage damage and chondronecrosis due to Se-deficiency and T-2 toxin poisoning are related to oxidative damage.
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Objective Through observing the effect of low-dose T-2 toxin on chondrocyte,to study the molecular mechanism of cartilage damage.Methods The primary chondrocytes were isolated from articular cartilage of d 1-2 Wistar neonate rats through enzymatic digestion.Different doses (0.005,0.010,0.100 μg/L) of T-2 toxin were added after 24 h in vitro culture.The survival rate of chondrocytes was detected with Trypan blue staining.Echylosis (matrix metalloproteinase,MMP1) was analyzed by immunohistochemistry.The damage of articular chondrocyte was observed by transmission electron microscope.Results ①Cell morphology of in vitro cultured chondrocyte:the newly isolated chondrocytes were spherical.After 24 hours,the adherent cells gradually began to stretch the triangle or polygon;the nucleus was large and round;the cell was clear and transparent,containing secretory granules.②Cell proliferation:T-2 toxin had a significant inhibitory effect on chondrocyte proliferation,the higher the concentration of T-2 toxin,the significant the inhibitory effects [0.000 μg/L (control) group:3.45 × 108/L,0.005 μg/L T-2 toxin group:3.45 × 108/L,0.010 μg/L T-2 toxin group:2.06 × 108/L,x2 =9.554,P ＜ 0.05].③Immunohistochemical observation:dysplasia,nucleus condensation and membrane rupture were observed in T-2 toxin treated group,brown staining was observed in all groups at varying degrees.The deepest staining was in 0.005 μg/L T-2 toxin group,with the strongest secretion of MMP1;with increasing doses of the toxin,the damage to cartilage cells was severe,MMP1 secretion was less,staining was weak,and the weakest staining was in the 0.100 μg/L T-toxin group.④Under transmission electron microscopy:in control group,cytoplasm was rich in rough endoplasmic reticulum,nuclear membrane and cell membrane were clear;in 0.005 μg/L T-2 toxin group,the cell nucleus showed pyknosis,organelles were decreased in cytoplasm;in 0.100 μg/L T-2 toxin group,the microvilli was dropped out of cartilage surface,nuclear changes were obvious,and mitochondria was myeloid degeneration;rough endoplasmic reticulum was degranulation and expansion into cystiform,chondrocytes were apoptosis occasionally,the cell nucleus showed pyknosis,and the formation of high-density plaque.Conclusion Low dose of T-2 toxin could damage the primary cultured articular chondrocyte in vitro.The results have showed that there are damaged cytostasis,chondrocyte degeneration,necrosis and apoptosis.
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Objective To investigate the expressions of matrix metalloproteinases(MMPs) in Kashin-Beck disease(KBD) cartilage as well as in a KBD rat model of T-2 toxin poisoning under selenium deficient conditions, and to investigate the effect of T-2 toxin on MMP-13 expression in human chondrocytes in vitro in order to determine a possible mechanism underlying KBD. Methods Samples of articular cartilage were divided into 2 groups:controls(samples from 5 normal children, traffic accident or operation), and KBD(samples from 5 children with KBD, auctopsy). Thirty-two Sprague-Dawley rats were divided into two groups by body weight using random number table: normal diet group(n = 16) and selenium-deficient diet group(n=16). The selenium level in normal diet was 101.500μg/kg, and in selenium-deficient diet was 1.118μg/kg. Rats were fed for 4 weeks with selenium-deficient or normal diet, respectively. After successful build up of the low selenium rat model, normal diet group was then subdivided into 2 sub-groups: normal group(n = 8) and normal diet plus low T-2 toxin group(n = 8);and selenium-deficient diet group was also subdivided into 2 sub-groups: selenium-deficient group ( n = 8 ) and selenium-deficient diet plus T-2 toxin group ( n = 8 ) . T-2 toxin of 100 μg·kg-1·d-1 was administered by intragastric administration for 30 days. Then the rats were sacrificed, and their knee joints were processed for histopathological evaluation. MMP-1 and MMP-13 locations in cartilages were performed by inmmunohistochemistry. Human chondrocytes C28/I2 were cultured in vitro. The experiment was divided into 4 groups: empty vector plasmid group, MMP-13 promoter plasmid group, MMP-13 promoter plasmid plus 20 μg/L T-2 toxin group and MMP-13 promoter plasmid plus 40 μg/L T-2 toxin group. MMP-13-luciferase reporter plasmid and vector plasmid were transiently transfected into C28/I2 cells for 24 hours, and then treated with 20 - 40 μg/L T-2 toxin for 24 hours. Transactivation of human MMP-13 promoter was analyzed using luciferase reporter constructs containing sequences spanning-1602 to+20 bp in C28/I2 chondrocytes. Results The percentages of chondrocytes staining for MMP-1 in the superficial and middle zones of KBD samples [(29.73 ± 10.12)%, (28.27 ± 0.91)%] were significantly higher than those of controls[(2.47 ± 0.11)%, (0.00 ± 0.00)%, all P < 0.05]. The percentages of chondrocytes staining for MMP-13 in the superficial and middle zones of KBD samples [(13.21 ± 4.32)%, (41.85 ± 6.32)%] were significantly higher than those of controls[(5.72 ± 0.31)%, (0.00 ± 0.00)%, all P<0.05]. The percentages of chondrocytes staining for MMP-13 in the superficial and middle zones of rats fed with selenium-deficient diet plus T-2 toxin group[(13.21 ± 4.32)%, (61.85 ± 8.68)%] were significantly higher than those of the normal and selenium-deficient groups[(2.43 ± 0.22)%, (5.89 ± 0.69)%, (3.03 ± 0.29)%, (25.99 ± 0.57)%, all P < 0.05]. Moreover, T-2 toxin activated the MMP-13 promoter detected with luciferase reporter assays in C28/I2 cells. The luciferase activities in MMP-13 promoter plasmid plus 20 μg/L T-2 toxin group and MMP-13 promoter plasmid plus 40μg/L T-2 toxin group(0.082 78 ± 0.008 40, 0.103 35 ± 0.013 19) were significantly higher than those in empty vector plasmid group and MMP-13 promoter plasmid group(0.024 19 ± 0.000 96, 0.040 32 ± 0.003 56, all P < 0.05). Conclusions These data suggest that T-2 toxin induces cartilage matrix degradation through up-regulation of MMP-13 promoter expression. Increased MMPs staining intensity in KBD cartilage and the rat KBD model of T-2 toxin poisoning under selenium deficient conditions suggest that matrix degradation appear to be driven by MMPs activity.
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OBJECTIVE ToexplorethepossiblemechanismoractiontargetsofT-2toxinembryo toxicity by observing the effect of T-2 toxin on mitochondrial function of differentiated murine e mbryonic stemcells(mESCs).METHODS Duringdifferentiationat24,72and120h,ESCswereexposedto T-2 toxin 0.5 μg·L-1 .Meanwhile,mESCs were pre-treated with antioxidant Trolox (200 μmol·L-1 )for 30 min and exposed to T-2 toxin (0.5 μg·L-1 )for 72 h.The mitochondrial ultrasture of differentiated mESCs was observed under a transi mission electrical microscope (TEM).The differentiated ESC mito-chondrial function,including respiratory control ratio (RCR),ATP synthase activity and mitochondrial membranepotential(MMP),wasmeasuredat144hafterdifferentiation.RESULTS Significant decrease of the mitochondrial number,deformation of mitochondrial structure,and lack of complete mito-chodrial crest were observed through TEM in the groups of T-2 toxin exposed for 72 and 1 20 h,respec-tively.Compared with the normal control group,RCR declined by 49.5% and 55.1%,ATP synthase activity decreased by 84.9% and 89.3%,and MMP decreased by 23.2% and 35.2% in T-2 toxin 0.5 μg·L-1 exposure 72 and 1 20 h group,respectively.However,the inhibition of mitochondrial function by T-2 toxin in differentiated mESCs recovered significantly in the presence of the antioxidant Trolox. CONCLUSION T-2toxininducesoxidativestressandinhibitsmESCsmitochondrialfunctionindifferenti-ated mESCs,and ROS-induced mitochondrial malfunction plays an i mportant role in T-2 toxin e mbryonic toxicity mechanis m.
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Objective To investigate the present prevalence state of children's Kaschin-Beck disease (KBD) in Xinghai county of Qinghai province,a relative active KBD area in 2009,and to investigate their nutritional selenium level of local children and the T-2 toxin contamination level in their staple food.Methods Right hand X-ray photographs of children aged 7 - 12 in Shang,Zhong and Xia villages of Tangnaihai countryside in Xinghai county were taken.X-ray diagnosis was carried out according to the Diagnostic Criteria of Kashin Beck Disease (GB 16003-1995 ).Selected samples (children's hair,drinking water and their staple food) were collected according to X-ray film taken.Selenium contents in hair,drinking water and staple food samples were measured by 2,3-diaminonaphthalene fluorescence,and T-2 toxin in staple food sample was detected with enzyme-linked immunosorbent assay(ELISA) kits.ResultsTotal X-ray detection rate of children KBD was 12.20％(31/254) and KBD positive rate of children in Xia village was up to 14.97％(22/147),Shang village was up to 9.52％(6/63),and Zhong village was up to 6.82％ (3/44).The selenium level in children's body and outer environment was very low,namely,the selenium content in hair,drinking water,wheat and flour was (0.250 ± 0.136)mg/kg,(0.156 ± 0.046)μg/L,(0.0045 ± 0.0030)mg/kg,and (0.0067 ± 0.0116)mg/kg,respectively.The T-2 toxin level was relatively high in children's staple food,which was (78.91 ± 46.17)μg/kg in wheat and (47.47 ± 46.47)μg/kg in flour.Conclusions In relative active KBD areas of Xinghai county of Qinghai province,the children's selenium nutritional level is low,and the T-2 toxin contamination level in their staple food is relatively high,which is consistent with the distribution of local children's KBD.
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Objective To determine effects of T-2 toxin and selenium on expression of aggrecanase in human chondrocyte.Methods Human chondrocytes were treated with T-2 toxin(0,1,10,20 μg/L),and/or sodium selenite(final concentration of selenium 0,0.1 mg/L) for 5 days.Aggrecan expression was determined by Western blotting,aggrecanase-1 and aggrecanase-2 mRNA levels were measured by RT-PCR.ResultsSelenium and T-2 toxin had effects on both aggrecan protein levels and its aggrecanases(include aggrecanase-1 and aggrecanase-2 ) mRNA levels(F =0.294,27.71 for aggrecan,F =107.45,362.25 for aggrecanase-l,F =34.68,22.26 for aggrecanase-2,respectively,all P ＜ 0.05),and there was interaction between selenium and T-2 toxin on aggrecan,aggrecanase-1 and aggrecanase-2 expression(F =79.99,230.76,388.33,all P ＜ 0.05).Furthermore,selenium presented significant antagonism to T-2 toxin on aggrecan,aggrecanase-1 and aggrecanase-2 expression.Aggrecan expression levels(0.278 ± 0.015,0.235 ± 0.029,0.195 ± 0.028,0.399 ± 0.028,0.299 ± 0.020,0.263 ±0.019) induced by both 1,10,20 μg/L T-2 toxin and 0,0.1 mg/L selenium were significantly decreased than the levels(0.472 ± 0.0358,0.197 ± 0.018,all P ＜ 0.05) in control group(0 mg/L toxin).Selenium partially blocked the effects induced by 1,10,and 20 μg/L T-2 toxin(all P＜ 0.05).One,10,20 μg/L T-2 toxin and 0,0.1 mg/L selenium increased both aggrecanase-1 mRNA levels(0.535 ± 0.033,1.071 ± 0.043,1.454 ± 0.058,1.057 ±0.048,0.555 ± 0.036,0.902 ± 0.045) and aggrecanase-2 mRNA levels(0.596 ± 0.038,0.656 ± 0.033,0.949 ±0.049,0.600 ± 0.040,0.453 ± 0.031,0.164 ± 0.011),when compared with control(0.481 ± 0.023,0.346 ±0.020 for aggrecanase-1 and 0.387 ± 0.020,1.021 ± 0.046 for aggrecanase-2,respectively,all P ＜ 0.05).Selenium partially blocked 10,20 μg/L T-2 toxins induced upregulation of aggrecanase-1 (all P ＜ 0.05) and aggrecanase-2 (all P ＜ 0.05 ).Conclusions These data suggest a possible molecular mechanism that T-2 toxin could induce cartilage matrix degradation through the upregulation of aggrecanases expression and enzyme activities.Trace element selenium has some protective effect on cartilage proteoglycan degradation induced by T-2 toxins.