【作者】 吴庆华；

【导师】 袁宗辉；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2014， 博士

【摘要】 T-2毒素由镰刀菌(Fusarium)产生,是单端孢霉烯族毒素类中毒性最强的毒素。粮食作物,尤其小麦、大麦、燕麦和玉米是T-2毒素主要的污染对象。通过污染饲料,T-2毒素对动物和动物性食品消费者健康造成危害。T-2毒素与核糖体结合或破坏线粒体功能来抑制细胞的蛋白质和核酸的合成,通过激活MAPK、JAK/STAT等信号转导通路,导致炎性细胞因子的表达,诱导细胞凋亡和坏死。T-2毒素的代谢途径与毒性产生密切相关。T-2毒素进入动物体内被代谢成各种产物,其毒性差异变化巨大。HT-2毒素、3’-OH-T-2毒素和Neosolaniol(NEO)等是T-2毒素在动物体内的主要代谢产物。与T-2毒素相比,一些代谢产物毒性降低,但有些代谢物的毒性升高。研究清楚T-2毒素的代谢途径和主要代谢产物是揭示其在动物体内如何发挥毒性的重要前提。所以,揭示T-2毒素在动物中,尤其是食品动物中的代谢途径,对毒性机理研究的开展和食品安全以及人类健康等方面至关重要。然而,迄今为止,人们尚未清楚T-2毒素在食品动物中的整体代谢特点,尤其是不同种属动物对T-2毒素代谢转化存在着何种异同,尚无系统研究。T-2毒素引起核糖体和线粒体的损伤并最终导致细胞凋亡与T-2毒素迅速激活MAPK信号通路密切相关。MAPK被证明是单端孢霉烯族毒素发挥细胞毒性的重要信号通路之一。MAPK的激活迅速而短暂,但与细胞凋亡密切相关的炎性细胞因子,如IL-6、TNF-α和IL-1β等的激活却是持续而缓慢的,所以MAPK通路下游很可能存在其它信号通路激活炎性因子。本实验室前期工作已经初步发现JAK/STAT是单端孢霉烯族毒素的下游信号通路之一,并与毒素的免疫毒性密切相关。然而,目前与T-2毒素毒性密切相关的MAPK和JAK/STAT信号通路的上下游关系仍是未知,两者的激活场所在哪里?哪些因子连接两者的传递?尚未有报道。已有研究表明肝脏和肠道是代谢单端孢霉烯族毒素的主要场所。所以,本课题系统研究了T-2毒素在猪、鸡、鱼和大鼠肝微粒体、肝胞液以及肝细胞中的代谢情况。包括分析鉴定主要的代谢产物,讨论主要的代谢途径以及种属间代谢的异同。此外,为进一步完善T-2毒素在动物体内的代谢,本文以猪为例探讨了T-2毒素在猪盲肠道模型中的代谢和降解情况,从而与肝脏的代谢相呼应。在毒性机理研究上,本研究着重从T-2毒素作用后MAPK和JAK/STAT信号通路的相互交联转导关系,以及某些重要基因和蛋白的功能去揭示T-2毒素细胞毒性的潜在毒性机理。为此,本研究通过信号通路特异性抑制剂、免疫荧光/激光共聚焦和透射电镜等方法和手段研究了MAPK和JAK/STAT信号通路的交联转导关系,毒素的胞内毒性靶标,并深入探讨了JNK1和STAT3交联关系和功能。本研究对于寻找毒素的毒性化合物和残留标示物提供了重要的靶标信息,对阐释动物对毒素的耐受性机理、毒素的防控和人类以及动物疾病的预防有重要意义。毒作用信号转导研究也对深入认识T-2毒素的毒理机制以及信号在胞内外传递并行使功能研究有重要参考价值。1.T-2毒素在动物肝脏中比较代谢研究本研究首先制备了猪、鸡、大鼠、鲤和草鱼肝微粒体、肝胞液和肝细胞,加入NADPH生产系统,在37℃(鱼28℃)下与T-2毒素孵育2h后,样品经过沉淀蛋白、高速离心和过滤膜等前处理过程,采用HPLC-MS-IT-TOF检测鉴定代谢物,探讨T-2毒素在动物肝脏中的代谢途径,比较T-2毒素在不同动物种属间代谢的异同。结果表明,T-2毒素在肝微粒体中主要被代谢成5种产物(MT1-5),分别为HT-2毒素(MT1、Neosolaniol (NEO)(MT2)、3’-OH-T-2毒素(MT3)、3’-OH-HT-2毒素(MT4)和T-2triol (MT5),同时还发现T-2毒素原形。在猪和鸡中发现HT-2毒素(MT1). NEO(MT2)、3’-OH-T-2毒素(MT3)和3’-OH-HT-2毒素(MT4)。大鼠肝微粒体中除发现上述4种代谢物外,还检测到T-2triol,证明大鼠肝脏代谢T-2毒素能力强于其他动物。与陆地动物显著不同,水生动物鲤肝微粒体中只检测到HT-2毒素、NEO、3’-OH-T-2毒素和微量3’-OH-HT-2毒素代谢物。草鱼中检测到3’-OH-T-2和3’-OH-HT-2毒素,但并未检测到HT-2毒素。3’-OH-T-2毒素和NEO是鱼类的主要代谢产物。陆地动物(猪、鸡和大鼠)生成HT-2毒素的相对含量比鱼类显著高p<0.05),但3’-OH-T-2的含量比鱼类显著低(p<0.05)。可见,水解反应生成HT-2毒素是陆地动物肝脏微粒体的主要代谢方式,羟基化反应是鱼类肝脏代谢T-2毒素的主要方式。肝胞液中发现了HT-2毒素(MT1)、NEO (MT2)和3’-OH-T-2毒素(MT3),但并未发现3’-OH-HT-2毒素,且3’-OH-T-2毒素的相对含量也非常低。鲤中未发现羟基化代谢物,因此肝胞液中代谢酶的羟基化能力低于肝微粒体。猪、鸡和大鼠肝细胞中,除T-2毒素外,均发现了水解代谢产物HT-2毒素和NEO,且HT-2毒素所占的比例高于NEO。猪代谢产生HT-2毒素能力最强,鸡最弱。肝细胞中未发现羟基化代谢物。上述研究表明,T-2毒素在动物肝脏中的代谢存在相似性和差异性。水解(生成HT-2毒素、NEO和T-2triol)和羟基化(3’-OH-T-2和3’-OH-HT-2)是T-2毒素在动物肝脏中的主要代谢方式。猪、鸡和大鼠中常见代谢物HT-2毒素在草鱼和鲤肝脏中很少产生,鱼类中3’-OH-T-2占主导地位。水解生成HT-2毒素是陆地动物猪、鸡和大鼠的共同主要代谢途径,而鱼类中羟基化是主要的代谢方式,表明鱼类对T-2毒素的代谢与陆地动物存在较大差异。鱼类和陆地动物肝脏中CYP450单加氧酶和羧酸酯酶的种类,以及对羟基化和水解催化能力的差异可能是造成T-2毒素代谢途径不同的重要原因。本研究进一步完善了T-2毒素在肝脏中的代谢机制,对T-2毒素的残留监控和残留标示物的确定以及毒理机制研究具有重要的指导意义。2.T-2毒素在猪盲肠模型中代谢和降解研究将T-2毒素与来自1头猪的盲肠内容物(Cecum)在厌氧环境下培养20min、40min、1h、2h、4h、8h和24h,采用槲皮素(Quercetin)检测微生物的正常活性,试验重复4头猪(Cecum1-4)。样品通过QuEChERS方法进行前处理,采用Agilent1100HPLC串联API4000QTrap质谱定量T-2毒素的降解和代谢物。潜在代谢物的鉴定采用精确质谱Thermo HPLC串联LTQ Orbitrap XL-HESI检测。本研究与肝脏的数据结合,将更好的揭示T-2毒素的代谢途径和生物利用度。与盲肠Cecum4降解T-2毒素能力相比,来自另外3头猪盲肠(Cecum1-3)微生物降解T-2毒素的能力显著低(p<0.05)。在盲肠Cecum1-3中,T-2毒素降解在31.1-45.9%之间。而在盲肠Cecum4中,T-2毒素降解迅速,8h后T-2毒素剩余26.6±0.6%,24h后仅有3.0±0.1%的T-2毒素被检测到。所以来自不同猪的盲肠对T-2毒素的降解存在很大个体差异性。孵育24h后代谢物除HT-2毒素外,未检测到其它代谢物。在盲肠Cecum4中,孵育8h后HT-2毒素并未进一步降解代谢,但在24h时间点相对低的回收率可能预示着HT-2毒素被代谢成其它化合物,但生成的量低于HPLC-MS/MS仪器的最低检测限(LOD)。T-2毒素对猪的毒性很有可能是由T-2和HT-2毒素的混合毒性造成。盲肠孵育中代谢物只发现了HT-2毒素,其他脱环氧等代谢物均未发现。因此,当研究T-2毒素的毒性时,HT-2毒素的机体吸收也不容忽视。当T-2毒素到达大肠后,代谢成HT-2毒素也可被机体吸收并造成危害。T-2毒素在盲肠中的代谢和降解研究,进一步补充了肝脏中的代谢数据,丰富了T-2毒素的肝肠代谢信息,为T-2毒素的体内代谢和毒性研究提供重要参考。3.T-2毒素介导的MAPK和JAK/STAT信号通路交联转导研究为深入研究T-2毒素介导的MAPK和JAK/STAT信号通路之间的交联转导关系,将T-2毒素(14nM)与RAW264.7细胞共同孵育不同时间(0.5、1、2、4、8、12和24h),通过信号通路特异性抑制剂研究信号通路中p38/MAPK和JAK、 STAT之间的相互转导关系,此外还关注了连接两个信号通路之间可能的结点,并通过透射电镜观测了胞内细胞器在T-2毒素影响下超显微结构病变,寻找T-2毒素胞内潜在的毒性靶标,从而为分子毒理研究提供更直观的证据。通过特异性抑制剂、免疫荧光/激光共聚焦和流式细胞仪等方法和手段,本文初步探讨了JNK和STAT基因的功能。最后,关注了T-2毒素的毒性与胞内代谢和浓度的相关关系。研究结果发现,ERK、JNK1和p38MAPK基因能够被T-2毒素在1-2h内迅速激活,随后又快速沉寂,说明MAPK为一个快速反应通道。JAK2和STAT3基因被T-2毒素激活后并上调,但与MAPK信号通路不同,JAK/STAT信号通路激活过程缓慢,在12h才出现显著的上调。因此,在RAW264.7细胞中JAK/STAT是MAPK通路的一个下游通路,可能传递着来自MAPK的信号。IL-6在众多炎性细胞因子中基因上调最为显著,2h后上调30.43倍,12h后上调48.47倍。因此,IL-6在MAPK和JAK/STAT的信号传递中,可能发挥中至关重要的作用。为更好的研究信号通路之间的上下游关系,通过相应的特异性抑制剂干预,观测其他基因及其蛋白磷酸化的表达。加入JNK1特异性抑制剂SP600125后,T-2毒素诱导的JAK2、ERK1/2和K-Ras的基因表达均受到显著抑制(p<0.05)。STAT3mRNA表达活性也受到显著抑制(p<0.05),但STAT1mRNA并未出现显著抑制效应。T-2毒素诱导IL-6基因表达在12h出现了显著的抑制p<0.05)。T-2毒素激活JNK1后,信号通过JAK2传导,随后通过STAT3传递,其中IL-6和K-Ras在MAPK和JAK/STAT信号通路中发挥着纽带传递作用。阻断JNK1活性后,CIS、SOCS1和SOCS2基因显著上调表达(p<0.05),说明JNK1对此SOCS的3个亚家族进行负调控,但阻断JNK1后SOCS3基因下调,表明JNK1对SOCS3为正调控关系。RAW264.7细胞中T-2毒素诱导后,JNK1信号经JAK2、STAT3、ERK和p38传导,激活的JAK2、STAT3、ERK和p38基因又可调控JNK1基因表达。阻断STAT3表达后,T-2毒素诱导的IL-6基因在12h受到显著抑制(p<0.05)。综合研究结果,IL-6激活JAK2/STAT3通路后,STAT3又可继续激活IL-6后续基因表达,进入下一循环,继续发挥作用。经T-2毒素诱导后JNK1蛋白迅速磷酸化,并在1h开始转入细胞核内,随后会从核内返回胞质,但仍会继续进入核内。T-2毒素引起的p-JNK1穿梭入核至少持续12h。阻断JNK1活性后,STAT3磷酸化和入核均受到抑制,但STAT1未受影响。表明RAW264.7细胞中,T-2毒素诱导的STAT3磷酸化和入核发挥功能均受JNK1调控。透射电镜观测结果显示,RAW264.7细胞与T-2毒素(14nM)孵育12h后,内质网扩张、线粒体肿大、核质边缘化,表明细胞出现了凋亡症状。28nMT-2毒素作用细胞12h后,核质完全边缘化,内质网扩张甚至形成空泡,线粒体肿胀,粗面内质网上核糖体出现脱粒,出现严重凋亡症状。观测结果表明T-2毒素主要作用于核糖体和线粒体。阻断JNK1表达后,线粒体出现了明显肿胀,细胞凋亡率显著上升,基因Bcl-2/Bax和Bcl-xL/Bax比值均出现显著下降p<0.05),Caspase-3和Caspase-9基因水平显著上调,凋亡数据进一步验证了细胞超显微结构病变,表明JNK1具有保护RAW264.7细胞线粒体功能。阻断STAT3活性后,Bcl-2/Bax和Bcl-xL/Bax比值也出现明显下调p<0.05),进一步证明JNK1-STAT3为一条细胞保护通路,此通路可能通过保护线粒体的正常功能来抑制T-2毒素诱导的细胞凋亡。另一方面,本研究也证明T-2毒素是一把双刃剑,具有两面性,这一点也进一步丰富了对T-2毒素细胞毒性机理的认识。液相质谱检测结果显示,在12h时细胞内T-2毒素含量显著高于2h时的含量。电镜超显微结构也观测到12h时细胞受损比2h时更加严重,表明T-2毒素长时间接触RAW264.7细胞,对其产生更高的毒性。2h后检测到的代谢物有HT-2毒素和3’-OH-T-2毒素,12h为3’-OH-T-2毒素,但含量甚低,T-2毒素始终是细胞内主要的毒性物质。RAW264.7细胞中T-2毒素刺激后,MAPK和JAK/STAT信号通路之间存在错综复杂的交联转导关系。本研究揭示了IL-6和K-Ras在连接MAPK和JAK/STAT信号转导中发挥重要功能。[NK1-STAT3信号通路受T-2毒素刺激后发挥了保护线粒体,抑制细胞凋亡的功能。T-2毒素激活凋亡通路的同时,也能激活细胞的保护通路,抑制细胞的凋亡。T-2毒素靶向性损害RAW264.7细胞的线粒体和核糖体。T-2毒素进入细胞后,虽被代谢成多种物质,但T-2毒素仍占主导地位调控信号转导。综上所述,本文首先研究了T-2毒素在肝脏和盲肠中的代谢,阐明了T-2毒素在不同种属动物中的代谢途径和特点,鉴定出5种主要代谢产物,充分完善了T-2毒素的代谢机制,对T-2毒素的残留监控及残留标示物的确定具有重要的指导意义。T-2毒素诱导下MAPK和JAK/STAT信号通路交联转导关系研究,进一步揭示了T-2毒素的毒性机制,为毒素防控、疾病预防、癌症治疗和新药开发提供了重要参考。更多还原

【Abstract】 As one of the primary members of type-A trichothecenes, T-2toxin is produced mainly by Fusarium genus. T-2toxin is the greatest toxic one for animals among trichothecenes, and it could contamiant cereals such as wheat, barley, oats, and maize. T-2toxin is making a great harmful on animals and humans through feed contaimination. Importantly, T-2toxin could also inhibit the sysnthesis of protein, DNA and RNA through conjugating with ribosomes as well as the damage of mitochondria. This toxin could also activate MAPK and JAK/STAT signaling pathways as well as cytokine expression and induce cell apoptosis and immune dysfunction.Metabolism of T-2toxoin has a close relationship with its toxicities. HT-2toxin,3’-OH-T-2, and neosolaniol (NEO) are the major metabolites of T-2toixn in animals. The toxicities change greatly once T-2toxin is biotransformed into different productes. The toxicity of most metabolites such as NEO and T-2triol are decreased. However, the toxicities of some metabolites such as3’-OH-T-2, are not decreased significantly, or even display a slightly higher toxicity. Thus, elucidation of the metabolic pathways and identifying the major metabolites of T-2toxin in animals are prerequisite to reveal its toxicity in animals, and are also crucial for food safety and human health. However, up to date, a global metabolism of T-2toxin in food producing animals is still unclear; especially the metabolic profiles are never compared in different species.T-2toxin can cause damage on ribosomes and mitochondria and ultimately lead to cell apoptosis. MAPK signaling pathway is proved to be an important toxicological pathway of trichothecenes and plays critical roles in toxicity. T-2toxin can active this pathway rapidly and transiently. It is known that animals are normally exposed to trichothecenes slowly. The effects of inflammatory cytokines on cells are performed slowly and continually. For example, IL-6, IL-1β, and TNF-a were found to be activated after12h by trichothecenes in RAW264.7cells. MAPK is proved to be an important target signaling pathway of trichothecenes and could be activated in a rapid and transient way. Thus, a contradiction rises when trying to explain the rapid MAPK activation but slow activation of inflammatory factors. We suspect that some other signal pathways might exist at the downstream to transmit the signals from MAPKs and activate the inflammatory factors. Indeed, it is already proved from our previous work that JAK/STAT pathway are the downstream targets of trichothecenes and played important roles in regulation of proinflammatory cytokines as well as apoptosis. However, the upstream and downstream interrelationship between MAPK and JAK/STAT signal pathways is still unclear. Where do they peform their fuctions once they are activated, in nucleus or cytoplasm? What are the messengers to connect the two pathways and transmite the signals? All these questions need to be answered.It is already known that T-2toxin can be well biotransformed in liver and intestine. Thus, we aimed to study the metabolism of T-2toxin in hepatic subcellular fractions (microsomes and cytosol) of pigs, chickens, rats, and carp (common carp and grass carp), as well as hepatocytes of rats, piglets and chickens. Based on the results, we discussed the metabolic difference of T-2toxin in different species. In addition, we also studied the degradation and metabolism of T-2toxin in pig cecum model. This study combined with the data in liver will further elucidate the metabolic fates of T-2toxin in animals. We also aim to study the cross talk between MAPK and JAK/STAT signaling pathways, which is induced by trichothecene T-2toxin. The cross talk between JNK1and STAT3were especially discussed. Specific inhibitors are useful tools to elucidate the signaling cross talk. In addition to the cross talk, we also monitored the typical ultrastructural changes of RAW264.7cells caused by T-2toxin and found out the potential toxicological targets of T-2toxin. The observation provides crucial visualized evidence for the molecular targets of trichothecenes. Finally, relationship between the toxic profiles and T-2toxin contents in intracellular in RAW264.7cells were also addressed. The results from this study will provide important data for explaning the sensitivity difference to T-2toxin in different animals, and is also important for identifying the residue markers as well as the prevention of disease. The study of signaling pathways will provide further information for the understanding of toxic mechanism of trichothecenes as well as the interrelationship between MAPK and JAK/STAT signaling pathways.1. A comparation of hepatic metabolism of T-2toxin in pigs, chickens, carp and ratsMetabolic profiles of T-2toxin in the hepatocytes of food producing animals, pigs and chickens; rats-the major experimental model were compared. Metabolic pathways of T-2toxin in liver microsomes and cytosol of pigs, chickens, common carp, grass carp, and rats were also especially concerned. The metabolites were identified by HPLC-MS-IT-TOF.In liver microsomes, five metabolites (MT1-5) were detected and identified. T-2toxin was mainly metabolized to HT-2toxin (MT1), neosolaniol (NEO)(MT2),3’-OH-T-2(MT3),3’-OH-HT-2(MT4), and T-2triol (MT5). T-2triol was only detectable in rat liver microsomes, implying that rat liver microsomes have stronger metabolic capability of T-2toxin than other species. In carp liver microsomes, HT-2toxin, NEO, and3’-OH-T-2were detected. Different with land animals, there was only trance amount of3’-OH-HT-2in carp liver microsomes. In grass carp,3’-OH-HT-2and3’-OH-HT-2, but not HT-2toxin were detected. The relative amount of HT-2toxin in land animals (pig, chicken, and rat) was much higher than that in carp (p<0.05). Hydrolysis to form HT-2toxin was the major metabolic pathway of T-2toxin in land animals (pig, chicken and rat). Different with land animals, HT-2toxin was not found in the liver microsomes of grass carp. In grass carp,3’-OH-T-2showed a relatively high amount, and followed with common carp. But in land animals, the capacity of transforming T-2toxin to3’-OH-T-2was relatively weak.3’-OH-HT-2was not found in liver cytosol and the amount of3’-OH-T-2was also very low. Especially in common carp, the hydroxyl products were not found. Thus, we conclude that the hydroxyl capacity in liver cytosol is weaker than that in liver microsomes.In hepatocytes, HT-2toxin and NEO were detectable. The amount of T-2toxin was much higher than NEO. Pigs produced a higher amount of HT-2toxin, chickens were the weakest. The hydroxyl products and C-8-deisovaleryl metabolites were not detected.Taken together, there was a similarity in the metabolic pathways of T-2toxin among different species, but an interesting and different profile was also monitored. T-2toxin can be metabolized rapidly in liver. Hydrolysis (HT-2, NEO, and T-2triol) and hydroxylation (3’-OH-T-2and3’-OH-HT-2) were the major metabolic pathways in the liver from these species. Hydrolysis and forming HT-2toxin was the same pathway in these land animals. As compared with hydrolysis reaction, the hydroxylation was much weaker in land animals. But carp showed an opposite metabolic characteristic. HT-2, the very common metabolite in land animals, was rarely found in carp, but3’-OH-T-2was the major one. This result implies that the metabolism of T-2toxin in fish has some differences with land animals. The different characteristic of CYP450monooxygenase and carboxylesterase as well as the differece cataltic abilty in hydroxyaiton and hydrolysis actions by these enzymes between carp and land animals are possibly the reasons for the different metabolic profiles of T-2toxin. The findings of this study further improves the metabolic mechanism of T-2toxin and is also important for residue determination, residue marker identification as well as the toxic mechanism of trichothecenes.2. Degradation and metabolism of T-2toxin in pig cecum modelTo further provide the information of metabolism of T-2toxin in animals, a pig cecum model was produced to investigate the fate and degradation of T-2in animal intestines. The data combineding with liver metabolism will better uncover the metabolic pathways and bioavailbility of T-2toxin in animals.T-2toxin was incubated with pig cecum at anaerobic conditions for20min,40min,1h,2h,4h,8h, and24h. Quercetin was used to monitor the activity of the bacterial in the pig cecum. The extraction of incubated samples was performed by a modified QuEChERS method. An Agilent1100series HPLC was linked to an API4000QTrap mass spectrometer. Heated Electrospray Ionization (HESI) coupled with a Thermo HPLC system was used to detect the potential metabolites.Four different cecums were analyzed in comparison to the sterilized control. The degradation by the microbiota in Cecum1-3was much slower compared to Cecum4. In Cecum1-3, the degradation ranged31.1-45.9%of the originally incubated amount of T-2toxin. Only a small increase of the degradation between8and24h incubation time was detectable, e.g., only0.8%of T-2toxin was further degraded between8and24h in Cecum1. However, a very strong degradation was observed in Cecum4. About26±0.6%of T-2toxin were left after incubation for8h. Only3.0±0.1%of T-2toxin was detectable after24h.Besides HT-2toxin, other metabolites, such as deepoxy-HT-2, were not detected. In Cecum4, the formed HT-2toxin was not further metabolized after a further incubation for8h. However, the relatively low recovery after24h incubation might indicate that HT-2toxin was possibly further metabolized to other products to an extent which was lower than the LOD of the HPLC-MS/MS equipment. Thus, we suspect that the toxic effects of T-2toxin in pigs are possibly afforded by the combination of T-2toxin and HT-2toxin.In conclusion, T-2toxin is metabolized to HT-2toxin as the main metabolite by the intestinal microbiota of pigs with large interindividual difference. In one out of four analyzed cecums, T-2was nearly totally metabolized to HT-2toxin, whereas the other three cecums showed a degradation of T-2toxin up to46%. Besides HT-2toxin, no other metabolites were detectable in the incubated samples. For toxicity evaluations of T-2toxin in pigs, the combination of T-2and its major metabolite HT-2has to be considered, as both compounds show a similar cytotoxicity and absorption. The dagradation and metabolism study of T-2toxin in pig cecum further complemented the data of liver metaboism of T-2toxin and enriched the enterohepatic metabolic information; it also provides important references for the in vivo metabolism and toxic mechanism of T-2toxin.3. The cross talk between MAPK and JAK/STAT signaling pathways induced by T-2toxin in RAW264.7cellsIn order to study the cross talk between MAPK and JAK/STAT signaling pathway which is induced by T-2toxin, T-2toxin was incubated with RAW264.7cells for indicated time and the interrelationship between JNK, ERK, p38MAPK and JAK, STAT were investigated using the specific inhibitors. Furthermore, we studied the potential messengers between the two pathways. Besides the cross talk, we also monitored the typical ultrastructural changes of RAW264.7cells, which was caused by T-2toxin, and found out the potential toxicological targets of T-2toxin. This observation provides crucial visualized evidence for the molecular targets of trichothecenes. Moreover, the function of JNK1and STAT3were discussed using the inhibitors, immunoflourenscence and flow cytomery. Finally, the link between the toxic profiles and intracellular T-2toxin contents were addressed.Results showed that the genes of ERK, JNK1and p38MAPK were activated within1-2h. However, they ceased quickly, implying that MAPK is a rapid signaling pathway. JAK2and STAT3mRNA were up regulated significantly, but they were slow and the highest peak was observed at12h. It is very possible that JAK/STAT pathway is the downstream singling of MAPK. IL-6among the studied cytokines showed the relatively highest up-regulation level, which was up regulated30.43-fold at2h and48.47-fold at12h. Thus, we suspected that IL-6possibly plays a very important role in the cross talk between MAPK and JAK/STAT signaling pathways. In addition, K-Ras had an important function in the cross talking, the up regulation after1and12h implying that K-Ras also plays roles in connecting the cross talk between MAPK and JAK/STAT. In order to study the up-and down stream relationships, we used the specific inhibitors and monitored the gene levels and protein phosphorylation. When JNK1gene expression was blocked by its inhibitor SP600125, both the mRNA expression and protein phosphorylation of JAK2, and K-Ras response to T-2toxin were significantly suppressed. The gene expression and protein phosphorylation of STAT3, but not STAT1were significantly decreased. Interestingly, the gene expression of IL-6induced by T-2toxin was suppressed by SP600125at12h but not at2h. These results implies that the activated signals induced by T-2toxin could transfer from JNK1to JAK2/STAT3. Talk from JNK1to STAT3was possibly connected by IL-6. SOCS family is an important negatively regulator of JAK/STAT signaling pathway, we studied the relationships between JNK1and SOCS1,2,3, and CIS through blocking JNK1activity and found out that the mRNA expressions of CIS, SOCS1, and SOCS2were increased significantly, whereas the mRNA expression of SOCS3was decreased markedly. This observation implies that JNK1could regulate CIS, SOCS1, and SOCS2negatively, but positively regulate SOCS3.Once induced by T-2toxin, signal of JNK1could transmit to JAK2, STAT3, ERK, and p38. Moreover, these activated genes can reversely regulate JNK1activity. Blocking STAT3activity, IL-6gene expression at12was suppressed remarkedly. Thus, we suspected that STAT3would further activate IL-6after the activation of JAK2/STAT3by IL-6and join in the next circulations.JNK1was phosphorylated rapidly and imported into the nucleus, but the nucleus translocation is in a dynamic way and will later export to cytoplasm. When JNK1activity was blocked, the phosphorylation and nucleus translocation of STAT3were inhibited, but STAT1was not affected. This observation implies that phosphorylation and nucleus translocation of STAT3is JNK1-dependent.When examined by transmission electron microscopy, mitochondrial swelling and the rough endoplasmic reticulum dilation were clearly visible when treated with T-2toxin at the level of14nM for12h. Once the toxin was increased to28nM, more serious morphological changes were observed. Besides the swelling mitochondria and the dilation of rough endoplasmic reticulum, polysomes on rough endoplasmic reticulum were also breakdown and degranulated. In addition, condensation and marginalization of chromatin aggregation were also monitored, which suggests that cells are induced to apoptosis by T-2toxin.Interestingly, when the gene expression of JNK1was blocked, the apoptotic ratio was increased significantly than the blank or toxin treated group (p<0.05). This result demonstrates that JNK1induced by T-2toxin has an anti-apoptotic function in RAW264.7cells. The ratios of Bcl-xL/Bax and Bcl-2/Bax were both decreased after blocking JNK1or STAT3activity. The mRNA expression of Caspase-3and Caspas-9were markedly increased (p<0.05) when blocking JNK1and STAT3activities. These results further prove that the apoptosis induced by T-2toxin is a mitochondria-dependent caspase pathway. JNK1-STAT3pathway could inhibit apoptosis and its function is very possibly mediated by regulating the function of mitochondria. JNK1-STAT3is newly proved to be a cell survival pathway, and T-2toxin is shown to have a Janus face. This study adds to our further understanding of the toxic mechanism of trichothecenes.Finally, the intracellular content of T-2toxin in different time points was detected. The content of T-2toxin was increased significantly at12h than at2h, indicating that intracellular content of T-2toxin can be accumulated with time increase. Moreover, T-2toxin can be biotransformed to HT-2toxin and3’-OH-T-2toxin at2h, whereas3’-OH-T-2toxin was the sole metabolite at12h in RAW264.7cells. But at the two time points, T-2toxin was the major product to perform the toxic effects and induced gene expression.Taken together, a complicated cross talk between MAPK and JAK/STAT signaling pathways mediated by T-2toxin is reported for the first time. K-Ras and IL-6are proved to be critical messengers for the cross talk between JNK1and STAT3. Importantly, T-2toxin has a Janus face, which not only induces cell apoptosis, cell survival/defense pathways, such as JNK1-STAT3, could also be activated simultaneously. However, the cell defense signaling is possibly not strong and was swamped in the cell death signals, which makes it easily to be ignored. On the other hand, this study also reveals that cells could up regulate some defense signaling pathways when they are exposed to dangerous environment. This work also showes that ribosome and mitochondria are two major toxic targets of T-2toxin. The findings add to our further understanding of the toxic mechanisms of trichothecenes and the cross talk between MAPK and JAK/STAT signaling.In summary, we first investigated the metabolism of T-2toxin in liver and intestine, and dentified5metabolites as well as the major metabolic pathways, also the metabolic profiles in different speices were compared. This study consummated the metabolic mechanism of T-2toxin, and provided crucial information for residue mornitoring and determination of residue marker of T-2toxin. The cross talk between MAPK and JAK/STAT signaling pathway further uncovered the toxic mechanism of T-2toxin and provide an important referece for toxin controlling, disease prevention, cancer treatment and drug development.更多还原