T-2毒素降解菌株的筛选、鉴定与降解机制分析

为获得高效降解T-2毒素的微生物菌株并探究其降解机制,本研究以T-2毒素为底物从小麦样品中筛选降解微生物并进行鉴定,探究该菌株对T-2毒素的降解特性,利用超高效液相色谱-飞行时间质谱仪分析其代谢产物与降解机制。结果表明,从50份小麦样品中筛选到2株T-2毒素高效降解菌株AFJ-2和AFJ-3,通过形态学观察和16S rDNA序列分析,初步鉴定为短小杆菌属(Curtobacterium sp.)菌株和芽孢杆菌属(Bacillus sp.)菌株。菌株AFJ-2和AFJ-3分别能在7 h和12 h内将5μg/mL T-2毒素完全降解,2株菌的胞内酶均对T-2毒素具有显著降解效果(P<0.05),不存在吸附作用。菌株AFJ-2可将T-2毒素转化为HT-2毒素和T-2三醇,菌株AFJ-3降解T-2毒素产生新茄镰孢菌醇(neosolaniol,NEO),基于降解位点推测,2株菌共同作用于T-2毒素可产生新的产物4-脱乙酰-NEO。研究结果丰富了生物降解T-2毒素的菌种资源库,为T-2毒素降解酶的分离纯化及功能基因的挖掘提供一定参考依据。

T-2 toxin-induced apoptosis involving Fas,p53,Bcl-xL,Bcl-2,Bax and caspase-3 signaling pathways in human chondrocytes

Objective:To investigate the effects of T-2 toxin on expressions of Fas,p53,Bcl-xL,Bcl-2,Bax and caspase-3 on human chondrocytes.Methods:Human chondrocytes were treated with T-2 toxin(1～20 ng/ml)for 5 d.Fas,p53 and other apoptosis-related proteins such as Bax,Bcl-2,Bcl-xL,caspase-3 were determined by Western blot analysis and their mRNA expressions were determined by reverse transcriptase-polymerase chain reaction(RT-PCR).Results:Increases in Fas,p53 and the pro-apoptotic factor Bax protein and mRNA expressions and a decrease of the anti-apoptotic factor Bcl-xL were observed in a dose-dependent manner after exposures to 1～20 ng/ml T-2 toxin,while the expression of the anti-apoptotic factor Bcl-2 was unchanged.Meanwhile,T-2 toxin could also up-regulate the expressions of both pro-caspase-3 and caspase-3 in a dose-dependent manner.Conclusion:These data suggest a possible underlying molecular mechanism for T-2 toxin to induce the apoptosis sig- naling pathway in human chondrocytes by regulation of apoptosis-related proteins.

Promotion of the articular cartilage proteoglycan degradation by T-2 toxin and selenium protective effect

Objective: To identify the relationship between T-2 toxin and Kashin-Beck disease (KBD),the effects of T-2 toxin on aggrecan metabolism in human chondrocytes and cartilage were investigated in vitro. Methods: Chondrocytes were isolated from human articular cartilage and cultured in vitro. Hyaluronic acid (HA),soluble CD44 (sCD44),IL-1β and TNF-α levels in super-natants were measured by enzyme-linked immunosorbent assay (ELISA). CD44 content in chondrocyte membrane was deter-mined by flow cytometry (FCM). CD44,hyaluronic acid synthetase-2 (HAS-2) and aggrecanases mRNA levels in chondrocytes were determined using reverse transcription polymerase chain reaction (RT-PCR). Immunocytochemical method was used to investigate expressions of BC-13,3-B-3(-) and 2-B-6 epitopes in the cartilage reconstructed in vitro. Results: T-2 toxin inhibited CD44,HAS-2,and aggrecan mRNA expressions,but promoted aggrecanase-2 mRNA expression. Meanwhile,CD44 expression was found to be the lowest in the chondrocytes cultured with T-2 toxin and the highest in control plus selenium group. In addition,ELISA results indicated that there were higher sCD44,IL-1β and TNF-α levels in T-2 toxin group. Similarly,higher HA levels were also observed in T-2 toxin group using radioimmunoprecipitation assay (RIPA). Furthermore,using monoclonal antibodies BC-13,3-B-3 and 2-B-6,strong positive immunostaining was found in the reconstructed cartilage cultured with T-2 toxin,whereas no positive staining or very weak staining was observed in the cartilage cultured without T-2 toxin. Selenium could partly inhibit the effects of T-2 toxin above. Conclusion: T-2 toxin could inhibit aggrecan synthesis,promote aggrecanases and pro-inflammatory cytokines production,and consequently induce aggrecan degradation in chondrocytes. These will perturb metabolism balance between aggrecan synthesis and degradation in cartilage,inducing aggrecan loss in the end,which may be the initiation of the cartilage degradation.

The roles of carboxylesterase and CYP isozymes on the in vitro metabolism of T-2 toxin

Background: T-2 toxin poses a great threat to human health because it has the highest toxicity of the currently known trichothecene mycotoxins. To understand the in vivo toxicity and transformation mechanism of T-2 toxin, we investigated the role of two principal phase Ⅰ drug-metabolizing enzymes(cytochrome P450 [CYP450] enzymes) on the metabolism of T-2 toxin, which are crucial to the metabolism of endogenous substances and xenobiotics. We also investigated carboxylesterase, which also plays an important role in the metabolism of toxic substances.Methods: A chemical inhibition method and a recombinant method were employed to investigate the metabolism of the T-2 toxin by the CYP450 enzymes, and a chemical inhibition method was used to study carboxylesterase metabolism. Samples incubated with human liver microsomes were analyzed by high performance liquid chromatography-triple quadrupole mass spectrometry(HPLC- Qq Q MS) after a simple pretreatment.Results: In the presence of a carboxylesterase inhibitor, only 20% T-2 toxin was metabolized. When CYP enzyme inhibitors and a carboxylesterase inhibitor were both present, only 3% of the T-2 toxin was metabolized. The contributions of the CYP450 enzyme family to T-2 toxin metabolism followed the descending order CYP3A4, CYP2E1, CYP1A2, CYP2B6 or CYP2D6 or CYP2C19.Conclusions: Carboxylesterase and CYP450 enzymes are of great importance in T-2 toxin metabolism, in which carboxylesterase is predominant and CYP450 has a subordinate role. CYP3A4 is the principal member of the CYP450 enzyme family responsible for T-2 toxin metabolism. The metabolite produced by carboxylesterase is HT-2, and the metabolite produced by CYP 3A4 is 3'-OH T-2. The different metabolites show different toxicities. Our results will provide useful data concerning the toxic mechanism, the safety evaluation, and the health risk assessment of T-2 toxin.

Comparison of T-2 Toxin and HT-2 Toxin Distributed in the Skeletal System with That in Other Tissues of Rats by Acute Toxicity Test

Twelve healthy rats were divided into the T-2 toxin group receiving gavage of 1 mg/kg T-2 toxin and the control group receiving gavage of normal saline. Total relative concentrations of T-2 toxin and HT-2 toxin in the skeletal system（thighbone, knee joints, and costal cartilage） were significantly higher than those in the heart, liver, and kidneys（P 〈 0.05）. The relative concentrations of T-2 toxin and HT-2 toxin in the skeletal system（thighbone and costal cartilage） were also significantly higher than those in the heart, liver, and kidneys. The rats administered T-2 toxin showed rapid metabolism compared with that in rats administered HT-2 toxin, and the metabolic conversion rates in the different tissues were 68.20%-90.70%.

Increased Chondrocyte Apoptosis in Kashin-Beck Disease and Rats Induced by T-2 Toxin and Selenium Deficiency

Objective 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. Methods 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 d UTP 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 m RNA levels were determined by real-time reverse transcription polymerase chain reaction. Results 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 m RNA 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. Conclusion 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.

Experimental Haematobiochemical Alterations in Broiler Chickens Fed with T-2 Toxin and Co-Infected with IBV

The purpose of this experimental study was to evaluate and record the effects of T-2 toxicity alone and in association with IBV infection on haematobiochemical parameters. A total of 128 one-week-old chicks were divided into four groups of 32 birds each and were treated respectively with T-2 toxin alone, IBV alone, T-2 toxin and co-infected with IBV, and no treatment (control) for a period of 6 weeks. Haematologically, the birds treated with T-2 toxin developed anaemia as indicated by significant decrease in haemoglobin levels, total erythrocyte counts and packed cell volume values;leucopenia, lymphocytopenia heterophilia and thrombocytopenia. The IBV infected birds exhibited lymphocytophilia and heteropoenia;the degrees of severity of leucopenia, lymphocytopenia heterophilia and thrombocytopenia were more pronounced in T-2+IBV groups. The serum biochemistry revealed hypoproteinemia and hypoalbuminemia in all the treated groups consistently. Besides, hypoglobulinemia and increased levels of alanine aminotransferase in T-2+IBV, and increased levels of alkaline phosphatase in toxin group alone were recorded. The changes in biochemical parameters were more in magnitude in the combination treatment group and their severity increased with duration of treatment. It was concluded that T-2 toxin made the birds more susceptible to IBV infection.

Evaluation of Gojiextract and Charcoal as Antioxidant on T-2 Toxin Administration Onliver Male Mice

With a view to study the effects of exposure to T-2 toxin and their amelioration by Goji extract or charcoal, male mice were treated with a sublethal dose of T-2 toxin (200 μg/kg B.W) intraperitoneally. T-2 Toxin showed an increase (P ≤ 0.05) in blood of ALT, ALP, Total Lipids, TAS, and TNF. These were decreased by Goji extracts or charcoal, and were improved partially by the two treatments. It is concluded that the treatment of rats with Goji extract or charcoal ameliorated the adverse effects of toxins but the results suggest that Goji extracts may be used as antioxidant and antidote rather than charcoal for T-2 Toxin in mice.

Efficacy of Biological Control and Cultivar Resistance on <i>Fusarium</i>Head Blight and T-2 Toxin Contamination in Wheat

Laboratory and green house experiments were carried out to evaluate the efficacy of fungicides, biological agents and host resistance in managing FHB and the associated T-2 toxin. In vitro activity of fungicides and antagonists was determined by paired culture method. Effect of microbial agents on FHB severity and mycotoxin content was determined by co-inoculating F. graminearum and F. poae with Alternaria spp., Epicoccum spp. and Trichoderma spp. Fungicides Pearl? (500 g/L carbendazim), Cotaf? (50 g/L hexaconacole), Thiovit? (micronised sulphur 80% w/w) and Folicur? (430 g/L tebuconazole) were the standard checks. Host resistance was determined by inoculating F. poae and F. graminearum to four wheat cultivars and fifteen lines in pot ex-periments. Fungicides resulted in 100% inhibition of pathogen radial growth in in vitro while microbial agents suppressed pathogen growth by up to 53%. Thiovit? and Trichoderma were the most effective in reducing FHB severity in green house pot experiments. The wheat cultivars and lines varied in susceptibility with cultivar Njoro BW II showing least susceptibility while line R1104, cv. Mbuni and cv. KIBIS were most susceptible. All the wheat cultivars and lines accumulated T-2 toxin by up to 5 to 28 μg/kg. The results indicated that neither fungicides nor antagonists can solely be relied on in managing FHB and toxin accumulation. Therefore, integration of biocontrol agents, fungicides and further breeding efforts to improve lines and cultivars with promising resistance to FHB and T2-toxin contamina-tion is recommended.

Effects of T-2 Toxin Exposure on Bone Metabolism and Bone Development of Mice

T-2 toxin is the most widespread mycotoxin in crops,feed and food,which poses a serious threat to body health.Bone is the main target tissue for T-2 toxin accumulation.Ingestion of food contaminated by T-2 toxin is the main cause of Kashin-Beck disease.However,the specific mechanism of bone damage caused by T-2 toxin is still unclear.In this study,a total of 40 male C57BL/6N mice were divided into four groups and orally treated with 0,0.5,1.0 and 2.0 mg·kg^(-1) body weight T-2 toxin for 28 days.The results showed that exposure to T-2 toxin led to weight loss,bone mineral density reduction and femoral structural damage of mice.In addition,osteoblast-mediated bone formation was inhibited,and osteoclast-mediated bone resorption was enhanced.Meanwhile,the levels of bone metabolism-related hormones including parathyroid hormone,calcitonin and 1,25-dihydroxyvitamin D3 were reduced.More importantly,it was found that the level of neuropeptide Y(a neurohormone)was decreased.These results provided a new perspetive for understanding the osteotoxicity of T-2 toxin.

T-2毒素的高灵敏时间分辨荧光免疫分析

以时间分辨荧光免疫分析(TRFIA)方法建立快速灵敏的T-2毒素的全自动检测方法。采用T-2-BSA包被96孔板作为固相抗原,与游离的T-2竞争有限的抗T-2单克隆抗体,以Eu3+标记的羊抗鼠抗体示踪,采用间接竞争免疫分析方法在解离增强荧光免疫分析体系中建立T-2-TRFIA。同时对这一方法的稳定性、灵敏度、回收率和特异性进行考核。此方法灵敏度为0.2μg/L,测量范围0.2～500μg/L,批内变异为7.5%,批间变异为12.7%,平均回收率为89.6%。与赭曲霉毒素A、黄曲霉毒素B1、牛血清白蛋白无交叉反应。10条不同时间进行的间接竞争T-2-TRFIA的效应点均值ED80、ED50和ED20分别为2.66,6.97,29.11μg/L。同时,用TRFIA和ELISA试剂盒同时检测T-2毒素,在ELISA和TRFIA产品的共同可测范围之内,两者的相关系数为0.926。

固相萃取-超高效液相色谱-质谱联用技术检测大鼠血浆中T-2毒素及其主要代谢产物

应用固相萃取-超高效液相色谱-三重四极杆-线性离子阱串联质谱技术,建立了高灵敏度的大鼠血浆中单端孢霉烯族(T-2)毒素及其5种主要代谢物的分析方法。在优化的固相萃取条件下,大鼠血浆经HLB固相萃取柱净化富集后,以XDB-C18色谱柱(50 mm×4.6 mm,1.8μm)分离,以5 mmol/L乙酸铵溶液和甲醇溶液为流动相进行梯度洗脱,以玉米赤霉酮为内标,在正离子多反应监测模式下对各分析物进行定性和定量分析。各分析物在0.1～500.0μg/L范围内线性良好(R2>0.997);检出限介于0.02～0.3μg/L之间;加标回收率为93.5%～120.3%;相对标准偏差均小于13%。本方法快速、重现性好、灵敏度高,已成功应用于T-2毒素中毒的溯源性研究。

宽口径毛细管柱GC测定鸡粪中T-2毒素代谢产物的方法

目的测定鸡粪中Ｔ－２四醇、Ｔ－２三醇、Ｔ－２毒素、ＨＴ－２毒素的含量。方法人工染毒，化学提取，ＴＢＴ衍生，宽田径毛细管柱ＧＣ测定，投予剂量不同回收率略有差异。结果该方法的回收率 ２００ｎｇＴ－２四醇８３．１％～９２． ８％；Ｔ－２三醇 ８３． ６％～９０． ６％；Ｔ－２毒素、ＨＴ－２毒素 ７９． ６％～９０． ６％。结论方法的回收率高，操作简单，可供研究Ｔ－２毒素代谢机制应用。

胶束电动毛细管色谱法分离定量T-2毒素代谢物

目的 建立一种测定 T- 2毒素代谢物 T- 2四醇 (T- 2 - 4- ol)、3′-羟基 - HT- 2毒素 (3′- OH- HT- 2 )、3′-羟基 - T- 2毒素 (3′- OH- T- 2 )、T- 2四醇四醋酸 (T- 2 - 4- ol- 4- ac)、T- 2三醇 (T- 2 - 3- ol)、HT- 2毒素和 T- 2毒素的方法。方法 采用胶束电动毛细管色谱法 (MECC)。结果 研究了不同浓度的 SDS和硼砂、不同体积分数的乙腈及不同温度对灵敏度和分离度的影响。发现柱温 30℃ ,所用缓冲液中 SDS的浓度为 6 0 m mol/ L,硼砂浓度为 10mm ol/ L,乙睛的体积分数为 15 %时 ,灵敏度和分离度最高。气压进样 10 s,信号噪音比为 3:1时 ,7种 T- 2毒素代谢物的检测极限 3′- oh- ht- 2是 4.7m g/ L 其余全部为 9.4mg/ L。在上诉条件下 ,所有分析物的浓度与峰面积之间均具有良好的线形关系。结论 MECC可以用来分离定量 7种 T- 2毒素代谢物 ,为测定人或动物体液中的 T-

Tracer GC/FTIR分析T-2毒素及其代谢产物

用TracerGC/FTIR系统测定了T-2毒素及其四种主要代谢产物(HT-2、T-2triol、T-2tetraol及NEO)的硅烷化衍生物的凝聚态红外谱图,在优化样品沉淀片移动速率的基础上,建立了五种毒素的气-红联用检测方法。用此方法分析染毒大白鼠全血样品,结果表明方法的适用性很好。

T-2毒素的危害及脱毒研究进展

T-2毒素是由镰刀属菌产生的一种次级代谢产物,属于单端孢霉烯族常见的A型真菌毒素。T-2毒素具有强毒性,它不仅会污染田间作物和库存谷物造成粮食产后损失,还会对人畜的健康造成巨大损害,是近年来全球范围内粮食行业和畜牧业防治的重点。目前关于T-2毒素生物毒性的研究比较深入,但关于T-2毒素脱毒,尤其是生物脱毒的研究较少。综述了T-2毒素的危害致毒机理及物理、化学、生物等脱毒方法。将获取高效脱毒酶和开发脱毒工艺作为研究重点,利于我国粮食、畜牧、副产物深加工等行业的良性发展。

Effective protective agents against the organ toxicity of T-2 toxin and corresponding detoxification mechanisms:A narrative review

T-2 toxin is one of the most widespread and toxic fungal toxins in food and feed.It can cause gastrointestinal toxicity,hepatotoxicity,immunotoxicity,reproductive toxicity,neurotoxicity,and nephrotoxicity in humans and animals.T-2 toxin is physicochemically stable and does not readily degrade during food and feed processing.Therefore,suppressing T-2 toxin-induced organ toxicity through antidotes is an urgent issue.Protective agents against the organ toxicity of T-2 toxin have been recorded widely in the literature,but these protective agents and their molecular mechanisms of detoxification have not been comprehensively summarized.In this review,we provide an overview of the various protective agents to T-2 toxin and the molecular mechanisms underlying the detoxification effects.Targeting appropriate targets to antagonize T-2 toxin toxicity is also an important option.This review will provide essential guidance and strategies for the better application and development of T-2 toxin antidotes specific for organ toxicity in the future.

Occurrence of <i>Fusarium</i>species and associated T2-toxin in Kenyan wheat

Survey covering 120 wheat fields was conducted in three wheat-growing districts of Kenya during the 2008 cropping season to determine the incidence of Fusarium head blight (FHB) and T2-toxin contamination in grain. FHB incidence was determined as the number of blighted ears per 10m2. Information gathered included wheat production practices, rainfall and temperature data. Fungal pathogens were isolated from wheat stems, heads, straw, grains and soil and identified based on cultural and morphological characteristics. Wheat grain samples were analyzed for T2-toxin by competitive Enzyme Linked Immunosorbent Assay (ELISA). High FHB incidences of up to 88% were recorded. Fungal genera isolated included Fusarium, Epicoccum, Trichoderma, Alternaria and Penicilium. Wheat plant parts with high infection with Alternaria and Epicoccum had corresponding low levels of Fusarium spp. Whereas Fusarium spp. were the most common fungal pathogens in stems, heads and soil, Epicoccum was frequently isolated from straw and grains. Fusarium speciesisolated included F. poae, F. graminearum, F. stilboides, F. verticilloides, F. fusarioides, F. tricinctum and F. heterosporum with F. poae and F. graminearum accounting for approximately 40% of all Fusarium infections. T-2 toxin was detected in all the grain samples and varied from 3 to 22 ppb. The study showed that FHB and T2-toxin are prevalent in the study districts and the high diversity of Fusarium species implies a challenge in FHB management as well as a risk of chronic T2-toxin exposure to humans and livestock.

THE EXPERIMENTAL STUDY ON THE EFFECTS OF SIX MYCOTOXINS ON THE CULTURAL CHONDROCYTES

The effects of deoxynlvalenol (DON), T--2 toxin, nivalenol (NIv), hutenolide (BuT).alternariol methyl ether(AME) and monlliformin (cON ) on rabbit articular chondrocytes were observed by using the method of chondrocyte monolayer culture. The amounts or DNA in chondrocytesand glucuronate in matrix were measured. And the chondrocytes were observed by inversion microscope and transmission electron microscope (TEM). The results showed that the cultured chondrocytes were damaged by all the six mycotoxinsl and the synthesis of DNA and the divided reproductionof chondrocytes were restrained; the damage errect was more evident, esl,ecially in the early stage ofculturel the higher concentration or toxin in the media was used, the lower density of the culturalckondrocytes was observed; the cells were even round damaged and dead, so long as the media contolued toxin. When the six mycotoxins arrected the cultural chondrocytes r.spectively, three dirfereut kinds or ultrastructural changes in ckondrocytes were seen by TEa. The relationship betweenmycotoxiu and KBD was preliminarily discussed, and some problems still need further investigation..

T-2 toxin induces developmental toxicity and apoptosis in zebrafish embryos

T-2 toxin is one of the most important trichothecene mycotoxins occurring in various agriculture products. The developmental toxicity of T-2 toxin and the exact mechanism of action at early life stages are not understood precisely. Zebrafish embryos were exposed to different concentrations of the toxin at 4-6 hours post fertilization （hpf） stage of development, and were observed for different developmental toxic effects at 24, 48, 72, and 144 hpf. Exposure to 0.20 Ixmol/L or higher concentrations of T-2 toxin significantly increased the mortality and malformation rate such as tail deformities, cardiovascular defects and behavioral changes in early developmental stages of zebrafish. T-2 toxin exposure resulted in significant increases in reactive oxygen species （ROS） production and cell apoptosis, mainly in the tall areas, as revealed by Acridine Orange staining at 24 hpf. In addition, T-2 toxin-induced severe tail deformities could be attenuated by co-exposure to reduced glutathione （GSH）. T-2 toxin and GSH co-exposure induced a significant decrease of ROS production in the embryos. The overall results demonstrate that T-2 toxin is able to produce oxidative stress and induce apoptosis, which are involved in the developmental toxicity of T-2 toxin in zebrafish embryos.