【作者】 王慧姣；

【导师】 刘毅； 彭双清；

【作者基本信息】 湖南农业大学 ， 动物医学， 2004， 硕士

【摘要】 镰刀菌毒素在自然界分布较为广泛，是最危险的食品污染物之一，对人类健康以及畜牧业的危害十分严重。丁烯酸内酯(Butenolide，BUT)是镰刀菌毒素的一种，它是由三线镰刀菌、雪腐镰刀菌、木贼镰刀菌、拟枝孢镰刀菌和梨孢镰刀菌等产生的一种镰刀菌毒素。其化学名为4-乙酰氨基-4-羟基-2-丁烯酸-．-内酯(4-Acetamido-4-hydroxy-2-butenoic acid-．-lactone)。该毒素与流行于新西兰、澳大利亚、美国和意大利等国家和地区放牧牛群烂蹄病的发生有关。目前尚未见丁烯酸内酯引起人类中毒的文献报道。对BUT的急性中毒特点、靶器官效应及毒性作用机制研究甚少，有许多问题尚待阐明。本课题研究采用生物化学、病理学、毒理学等多学科现有的基础理论和实验方法，以大鼠和鸡胚作为动物模型，进行体内外实验，研究丁烯酸内酯的毒性效应及其作用机理，特别是导致脂质过氧化的作用机制以及硒化合物和其他抗氧化剂的保护作用，最终为阐明镰刀菌毒素与地方性克山病和大骨节病的关系提供依据。本研究分为三个部分。 第一部分 丁烯酸内酯所致损伤的病理学特点 本实验用BUT对大鼠急性毒性和亚急性毒性实验进行病理研究，观察了大鼠BUT中毒后其血浆生化指标及组织病理形态的变化。 大鼠BUT急性毒性实验结果表明，在较短的时间内就可以引起多个脏器毒性病理变化，主要表现为肝肾细胞空泡变性、肺出血以及空肠局部糜烂。随着中毒时间的延长，大鼠中毒12h、24 h后，其病理变化反而有所减轻。脏器系数的变化以中毒后6h心脏、肝脏和脾脏的变化最为明显。 大鼠BUT亚急性毒性实验结果表明，碱性磷酸酶(ALP)、肌酐(CREA)明显升高，白蛋白(ALB)、尿素氮(BUN)、甘油三脂(TG)、总胆固醇(TCHOL)、乳酸脱氢酶(LDH)、血糖(GLU)显著性降低。 结果提示，大鼠BUT中毒起效迅速，此毒素不仅对肝功能和肾功能产生严重影响，而且也造成呼吸系统的损伤，说明该毒素引起的毒性损伤作用是多器官的、综合性的。 第二部分 丁烯酸内酯脂质过氧化效应 本部分中，为了确定丁烯酸内酯的脂质过氧化效应，我们观察了体内、外条件下丁烯酸内酯对动物主要脏器各生化指标的影响。结果表明，给孵化11d的鸡胚注射丁烯酸内酯10、50、100μg/egg，继续孵育8d后剖杀并测定心、肝、肾的各生化指标，发现心脏、肝脏和肾脏的脂质过氧化产物丙二醛(MDA)含量均有所升高，并与丁烯酸内酯呈剂量效应关系；巯基(-SH)和NO含量以及谷胱甘肽过氧化物酶(GSH-Px)活性明显降低，并与丁烯酸内酯呈剂量效应关系。结果提示，丁烯酸内酯可以引发脂质过氧化反应，使脂质过氧化产物蓄积，巯基和多种酶类损耗，从而使机体的抗氧化能力降低。 用BuT分两个不同剂量10mg.kg一’和20mg.kg一’给大鼠连续灌胃50d后，大鼠血浆、肝脏和肾脏MDA含量均明显升高，并与丁烯酸内酷剂量呈正相关关系。BUT对大鼠血浆、肝脏和肾脏的一SH和NO含量以及GSH一Px活性明显降低，且与BUT的剂量呈负相关效应，其中对肝脏琉基含量的影响更为明显。 BUT在体外对大鼠肝匀浆MDA含量同样具有升高效应;对一SH和NO含量以及GSH一Px活性也有明显降低作用。第三部分硒化合物及其他抗氧化剂对丁烯酸内醋所致脂质过氧化效应的保护作用 为了探讨抗氧化剂在丁烯酸内酷致脂质过氧化效应中的作用，也为寻找丁烯酸内酷致机体损伤的防治措施，本部分应用硒化合物及其他抗氧化剂，如还原型谷耽甘肤(GSH)，维生素C(viaC)，维生素E(virE)，超氧化物歧化酶(SOD)等，通过第二部分所述手段(各生化指标的的测定)，观察其对丁烯酸内酷致机体毒性损伤的影响。从而判断丁烯酸内醋引起脂质过氧化的可能途径以及与机体损伤的关系。结果表明，在鸡胚实验中，给孵化lld的每枚鸡胚注射100陀BUT+5陀抗氧化剂，发现以上各氧化剂除对肾脏的MDA值几乎没有影响外，其他指标均有不同程度的变化。在大鼠肝匀浆体外实验中，还原型谷肤甘肤(GSH)，维生素C(vitc)，维生素E(VitE)对丁烯酸内酷致各生化指标的改变均有明显的影响，而硒和SOD没有影响。结果提示，酶类(SOD)抗氧化剂只有在动物机体代谢过程中，才能够发挥不同程度的抗氧化作用;硒是谷肤甘肤过氧化物酶的组成成分，它参与机体的许多生物学过程，也只有通过动物机体的代谢过程才能保护机体免受一系列外来化合物的损害。其他非酶类抗氧化剂在体内外条下都能够发挥其抗氧化作用。更多还原

【Abstract】 Fusarium toxins are distributed extensivelly in nature, which are the most dangerous contaminations to food and have very serious danger to human health and stockbreeding. Butenolide (BUT) is a kind of Fusarium toxin extracted from F. tricinctum NRRL3249 , Fusarium nivale , Fusarium equseti , Fusarium sporotrichiodes and Fusarium poae,etc. The common name of this toxin is butenolide (4- Acetamido-4-hydroxy-2-butenoicacid acid . -lactone). It was believed that fescue foot was caused by BUT. However, the mechanism of BUT toxicity is unclear and there are no reports about BUT poisonning in mankind at present yet. In this dissertation, we carried experiments to study the toxic effect of BUT and its toxicity mechanism in rats and chicken embryos both in vivo and in vitro. The effect of lipid peroxidation induced by BUT and the protecting function of selenium and other antioxidants were observed. The present study offered the basis for expound relation of Fusarium toxins and local Keshan disease and Kaschin-Beck disease finally. This research is divided into three parts as follow.Part 1: Pathological characteristics in visceras caused by BUTWe carried on acute toxic and subacute toxic experiment in rats to observe the biochemical and pathological changes in rat visceras after poisoned by BUT.The acute toxic experimental results in rats indicated that BUT can cause a lot of damaging pathological changes in visceras within short time. The changes included the vacuole denaturalization in liver and kidney cells, the lung bleeding and debaucling. When the rats were poisonned after 12h and 24h, the pathologicalchanges lightened to some extent instead. Changes of visceras were most obvious in heart, liver and spleen of which was poisonned after 6h.The results from subacute toxic experiment in rats (treated with BUT for 50 days) indicated that ALP and CREA in the BUT group is obviously higher than that of the control group, ALB , BUN , TG , TCHOL, LDH and GLU of the BUT group is obviously lower than that of the control group.The results showed that the acute poisoning effect in rats caused by BUT is rapidly. The toxin can not only exert a serious influence to liver function and kidney function, but also caused respiratory system damages, which proved that this toxicity can cause comprehensive damages.Part 2: lipid peroxidation effect caused by BUTIn order to confirm that BUT can cause lipid peroxidation, we have observed biochemical changes induced by BUT in rat visceras. We injected BUT at 10 , 50 , 100 . g for each egg to chicken embryo hatched for 11 days, killed and examined the biochemical indexs of heart, liver and kidneys after continuing hatching for 8 days. The results indicated that the content of MDA in heart, liver and kidneys rose to some extent, in contrast, the content of - SH, GSH-Px and NO reduced obviously in dosage dependent manner. The results suggest that BUT was able to induce lipid peroxidation, cause -SH and enzyme lost consequently reduced the anti-oxidant ability of the organism.Part 3: Selenium and other antioxidants antagonized to lipid peroxidation caused by BUTTo probe into antagonizing of antioxidant to lipid peroxidation which induced by BUT, and look for methodes to prevent the lipid peroxidation cauced by BUT, we used selenium compound and other antioxidants such as GSH, VitC, VitE, and SOD etc. with means as stated in second part (surveyed biochemical indexs) to observed the influence of antioxidants to lipid peroxidation caused by BUT. So we can judgethe possible ways of lipid peroxidation and relations between BUT and organism damages.We injected 100u.g BUT and 5 u.g antioxidant to chicken embryo hatched for 11 days, other operations were the same as in the second part. Results from experiment in chicken embryo indicated antioxidants can affect the changes of MDA caused by BUT in heart and liver except in kidney. In vitro, GSH, VitC and VitE can obviously influence biochemical changes which caused by BUT, but the selenium and SOD had not effect.Briefly, the profe更多还原