黄曲霉毒素B1亚慢性暴露对雄性小鼠氧化、遗传系统的损伤及其作用机制

发布时间：2018-05-13 08:41

本文选题：黄曲霉毒素B_1 + 氧化损伤　；参考：《西南大学》2017年硕士论文

【摘要】：黄曲霉毒素B_1(Aflatoxin B_1,AFB_1)是目前已知的真菌毒素中毒性最强、对人类威胁最大的一种毒素。由于其极强的致癌性,并且其化学结构稳定高温下难以被破坏分解,AFB_1的暴露最终将会导致各类急性或亚慢性中毒的发生。虽然AFB_1的致癌作用及其相关机理已被证实,但其对生物体造成的氧化性、遗传性损伤及其相关机制的研究并不十分明确,这并不利于对黄曲霉毒素(Aflatoxins,AFT)的毒性机制的深入研究,同时也对AFB_1的防御体系建立造成了一定的阻碍。本实验对AFB_1产生的氧化性、遗传性毒害作用及相关分子作用机制进行了研究,为全面评估AFB_1的毒性同时为阐明其他类别的黄曲霉毒素的毒性机理提供一定的借鉴,同时也为黄曲霉毒素的降解脱毒或防御体内中毒提供更多的参考。本实验首先选用40只雄性小鼠,在亚慢性条件下进行连续50天3种不同剂量(0.09375、0.375、1.5mg/kg b.w./day)的AFB_1染毒,对血清及脏器中各氧化损伤指标、肝细胞凋亡及其相关信号通路等进行研究,以评估其造成的氧化性损伤及分子机制;对肝脏中DNA链断裂、交联,细胞微核及精子畸形等指标进行考察,以评估其遗传毒性及分子机制。并研究体外条件下AFB_1与DNA之间的相互作用机制及其与遗传毒性的相关性。具体的结果如下:(1)经过50天的AFB_1染毒后,AFB_1对小鼠机体能够造成明显的脏器损伤、血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)水平的显著性升高。各染毒组小鼠体重随染毒剂量的增加而降低,且肝脏、肾脏、睾丸三种器官重量也出现下降趋势,表明该三种脏器受到AFB_1染毒的影响。AFB_1对机体造成的病理性损伤具体为:低剂量时以炎症效应为主,高剂量时则导致肝细胞局部坏死、肾小球肿胀及肾小管上皮细胞坏死等情况。(2)亚慢性条件下,AFB_1能够对小鼠肝脏产生明显的氧化性损伤。不同剂量的染毒组均能够导致肝脏中活性氧(ROS)的升高,中、高剂量组肝脏中超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)水平下调表明其抗氧化系统受到损伤,无法启动清除过量的ROS,但低剂量组小鼠肝脏启动其抗氧化机制,免于机体受到过量ROS的损伤。中、高剂量组小鼠肝脏中脂质、蛋白、DNA均受到ROS的攻击,造成氧化性损伤,机体处于氧化应激状态,但低剂量AFB_1组小鼠体内的生物大分子物质并不受到显著性的损伤。(3)AFB_1产生的氧化性损伤除导致细胞坏死外,凋亡也是一种可能的方式,且肝细胞凋亡率随AFB_1浓度的增大而升高,在本试验范围内具有剂量-效应关系。低剂量时AFB_1对细胞造成的影响主要以炎症、局部细胞坏死为主,而较大剂量时则以细胞凋亡为主。AFB_1介导的肝细胞凋亡分子机制可能与上调Bax、Caspase-3、P53蛋白,下调Bcl-2蛋白的表达有关。AFB_1亚慢性染毒诱导的氧化性损伤中存在细胞凋亡的相关分子机制。这提示我们,若能从细胞凋亡的角度入手,更好的对其调控,将有可能为遏制AFB_1的氧化毒性提供新的途径。(4)亚慢性条件下的AFB_1暴露能够引起雄性小鼠肝细胞DNA链断裂、DNA-DNA交联、DNA-蛋白质交联形成,同时导致精子畸形率升高、微核形成及染色体损伤的增多。该五项指标除DNA-DNA交联外,其余各项指标均随AFB_1剂量的增大而升高,并且具有剂量-效应关系,这说明AFB_1不仅对体细胞而且对生殖细胞也具有遗传毒性,能产生致突变作用。(5)当较大剂量的AFB_1暴露于小鼠时(1.5mg/kg),DNA-DNA交联率并不随AFB_1浓度的增大而升高。这一结果表明,当AFB_1浓度增大到一定程度时(1.5mg/kg),其引发的各种DNA损伤形式中DNA-DNA交联的比例下降,推测其他形式的DNA损伤(如DNA链断裂、DNA-蛋白交联等)所占比重增大。AFB_1产生遗传毒性的机制可能是机体氧化应激所产生大量ROS等自由基直接对DNA造成攻击,或是ROS介导的氧化应激间接导致DNA氧化损伤所形成的。(6)AFB_1不需经过代谢,能够直接与DNA之间发生相互作用,形成二元复合物。同时DNA能够明显淬灭AFB_1的内源荧光,其淬灭类型为静态结合动态淬灭的方式。两者的结合常数为4.12×103 L/mol;相互作用力主要为氢键和疏水相互作用;结合模式为沟槽结合。AFB_1的结合导致DNA的构象有轻微的改变,但并没有对其构型造成较大的影响。分子对接结果说明,AFB_1与DNA的结合位于小沟槽区,且AFB_1的O6和O3原子能够和DA-17、DG16这两个碱基形成三个较强的氢键。这提示我们,AFB_1对DNA造成的损伤可能存在新的作用方式,这种方式可能与其毒作用机制有关。综上所述,亚慢性AFB_1的暴露能够对小鼠的氧化、遗传系统造成一定程度的损伤,并且其作用机制都与机体处于氧化应激状态、大量ROS的生成有关,这对于其致癌机制的研究与相关药物的开发具有指导意义。
[Abstract]:Aflatoxin B_1 (Aflatoxin B_1, AFB_1) is the most toxic and most dangerous toxin of the known mycotoxins at present. Because of its strong carcinogenicity and its chemical structure stable at high temperature, it is difficult to break down, and AFB_1 exposure will eventually lead to the occurrence of acute or subchronic poisoning in various classes, although AFB_1 causes it. The role of cancer and its related mechanism has been confirmed, but the study on the oxidation, hereditary damage and related mechanism of the organism is not very clear. This is not conducive to the deep study of the toxic mechanism of Aflatoxins (AFT) and the establishment of the defense system of AFB_1. This experiment is on AFB_1 The oxidation, genetic toxicity and related molecular mechanisms were studied to provide a reference for the comprehensive assessment of the toxicity of AFB_1 and to elucidate the toxicity mechanism of other types of aflatoxin, and to provide more reference for the detoxification of aflatoxin or intoxication in vivo. 40 male mice were treated with 3 different doses of AFB_1 (0.09375,0.375,1.5mg/kg b.w./day) for 50 days under subchronic conditions. The oxidative damage and molecular mechanism caused by the oxidative damage and the related signaling pathway were evaluated for the oxidative damage and the related signaling pathways in the serum and the organs of the organs in order to evaluate the oxidative damage and the molecular mechanism. The DNA strand breaks in the liver. Cross-linking, cell micronucleus and sperm malformation were investigated to assess their genotoxicity and molecular mechanisms. The interaction mechanism between AFB_1 and DNA in vitro and the correlation with genetic toxicity were studied. The specific results are as follows: (1) after 50 days of AFB_1 exposure, AFB_1 can cause obvious organ damage to the mice The weight of ALT and AST increased significantly in the serum, and the weight of the three organs in the liver, kidney and testis also declined, indicating that the three organs were infected by AFB_1, and the pathological damage caused by.AFB_1 was low. In the high dose, the local necrosis of the hepatocytes, the swelling of the glomeruli and the necrosis of the renal tubular epithelial cells. (2) under subchronic conditions, AFB_1 can produce obvious oxidative damage to the liver of mice. The active oxygen (ROS) in the liver can be increased by the different doses of the infected group, and the high dose group of the liver is in the high dose group. The downregulation of superoxide dismutase (SOD) and reduced glutathione (GSH) showed that the antioxidant system was damaged and could not start to remove excess ROS, but the low dose group of mice liver started its antioxidant mechanism, so that the body was immune to excessive ROS. The lipid, protein, and DNA in the liver of the high dose group were attacked by ROS. In the oxidative stress, the body is in the oxidative stress state, but the biological macromolecules in the low dose AFB_1 mice are not significantly damaged. (3) the oxidative damage produced by AFB_1 is also a possible way of apoptosis in addition to cell necrosis, and the liver cell withering rate increases with the increase of AFB_1 concentration. There is a dose effect relationship within the circumference. The effects of AFB_1 on the cells at low doses are mainly inflammation and local cell necrosis, while the mechanism of apoptosis induced by apoptosis mainly mediated by.AFB_1 may be associated with the up regulation of Bax, Caspase-3, P53 protein, and the expression of Bcl-2 protein in.AFB_1 subchronic exposure There is a molecular mechanism of apoptosis in oxidative damage, which suggests that if we can regulate it from the angle of apoptosis, it will be possible to provide a new way to prevent the oxidative toxicity of AFB_1. (4) AFB_1 exposure in subchronic conditions can cause DNA strand breaks in male mice liver cells, DNA-DNA crosslinking, DNA- protein The formation of mass crosslinking resulted in the increase of sperm abnormality, the formation of micronucleus and the increase of chromosome damage. The five indexes, except for DNA-DNA cross-linking, were increased with the increase of the dose of AFB_1, and had a dose effect relationship, which indicated that AFB_1 not only had genetic toxicity to the somatic cells but also to the germ cells. Mutation. (5) when a large dose of AFB_1 was exposed to mice (1.5mg/kg), the cross-linking rate of DNA-DNA did not increase with the increase of AFB_1 concentration. This result showed that when the concentration of AFB_1 increased to a certain degree (1.5mg/kg), the proportion of DNA-DNA crosslinking in various forms of DNA damage caused by the AFB_1 decreased, and other forms of DNA damage (such as the DNA chain broken) were conjectured. The mechanism of increasing the proportion of.AFB_1 to increase the genetic toxicity of DNA- protein may be caused by a large number of free radicals such as ROS produced by the oxidative stress of the body, or the oxidative stress mediated by ROS, which indirectly leads to the oxidative damage of DNA. (6) AFB_1 does not need to be metabolizing and can interact directly with DNA. The two element composite is formed. At the same time, the internal fluorescence of AFB_1 can be quenched by DNA, and the quenching type is a static and dynamic quenching method. The binding constant of the two is 4.12 x 103 L/mol; the interaction force is mainly hydrogen bond and hydrophobic interaction, and the binding mode is a slight change in the conformation of DNA with the combination of groove and.AFB_1. The molecular docking results show that the combination of AFB_1 and DNA is located in the small groove area, and the O6 and O3 atoms of AFB_1 can form three strong hydrogen bonds with the two bases of DA-17 and DG16. This suggests that AFB_1 may have a new way of action on DNA caused by damage, which may be associated with its toxic agent. In summary, the exposure of subchronic AFB_1 can cause a certain degree of damage to the oxidation of mice and the genetic system, and its mechanism is related to the oxidative stress of the body and the production of a large number of ROS, which is of guiding significance for the research of its carcinogenic mechanism and the development of related drugs.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

【参考文献】