【作者】 熊飞；

【导师】 严红；

【作者基本信息】 华中科技大学 ， 公共卫生， 2012， 硕士

【摘要】 丙烯酰胺(Acrylamide,ACR)是一种从水合丙烯腈中提取出来的具有乙烯基的水溶性有机化合物，是国内外广泛应用于化工冶炼、污水处理、纺织加工及化妆品生产的一种化学原料。早在20世纪70年代就有职业性中毒的报道，主要表现为神经系统症状，除职业接触外，在饮用水、高温油炸食品和食品的包装材料中都可以检测到单体丙烯酰胺的存在，由于其可以直接经口进入人体，从而引起了社会的高度关注。有关流行病学调查和实验研究发现，丙烯酰胺对人和实验动物除了有生殖毒性、遗传毒性及致癌性等作用以外，还有明显的神经毒性。丙烯酰胺急性、亚急性中毒主要以损伤中枢神经系统为主，表现为神经精神症状和小脑共济失调，而慢性中毒则以损害周围神经系统为主，关于其机制目前尚不清楚。纹状体作为基底节最大的综合处理元件，包含着大量的多巴胺能神经元，在整个脑部中的多巴胺神经递质含量最高。在多巴胺能系统神经通路中多巴胺神经递质首先在突触前膜中进行合成、转运、释放、降解，以达到突出间隙中多巴胺神经递质的平衡，突触间隙中的多巴胺神经递质进入突触后膜发挥突触后效应，而多巴胺相关转运蛋白在整个过程中发挥着重要作用，维持突触间隙中多巴胺神经递质的平衡，当该平衡被打破后神经系统就会出现不同程度的病症，主要表现在感觉运动整合、躯体平衡运动及学习记忆等方面。以往对ACR的人群流行病学和实验研究时所出现的神经症状和体征，似乎很大程度上预示着与纹状体多巴胺神经通路中多巴胺神经递质平衡被打破有关。但直至目前，有关丙烯酰胺影响纹状体维持多巴胺神经递质平衡的相关转运蛋白的研究报道较少，其具体机制仍未完全明确。因此，本研究拟通过体内和体外实验的结合，检测丙烯酰胺对多巴胺转运体、单胺囊泡转运体及多巴胺合成酶—酪氨酸酸羟化酶的影响，探讨其引起神经毒性的可能机制，为防治丙烯酰胺中毒提供理论依据。第一部分丙烯酰胺对大鼠神经行为毒性的影响目的：探讨丙烯酰胺对大鼠神经行为毒性的影响。方法：健康成年雄性SD大鼠40只，体重230±20g，由华中科技大学同济医学院实验动物中心提供，合格证号：SCXK（鄂）2010-0007号。大鼠在10h：14h明暗光循环的动物房内，自由饮水、摄食，检疫观察7天后用于实验。将大鼠按体重随机分为4组，分别为对照组、低剂量(20mg/kg)组、中剂量(30mg/kg)组、高剂量（40mg/kg）组，每组10只，均单笼饲养。按照对应的染毒剂量对各ACR剂量组进行灌胃染毒，对照组则灌胃给予等容量的生理盐水，每周连续染毒5天间隔2天，总共19天，并于第0，6，13，19天称量体重并进行步态评分，于第0，19天测定大鼠的后肢支撑力和甩尾时间。末次给药24小时后，断头处死大鼠，迅速取出脑组织，用预冷PBS缓冲液洗净，称量大小脑重量，并于冰盘上迅速分离纹状体、大脑皮层及小脑，并用干净滤纸吸干置于﹣80℃冰箱中保存备用。取出大鼠心、肝、脾、肺、肾、睾丸等脏器，称量重量并记录。结果：（1）在整个染毒期内低剂量组大鼠虽然体重增长始终低于对照组，但差异无显著性；与对照组比较，中剂量组大鼠平均体重于第19d明显降低，差异有统计学意义(P<0.01)；高剂量组大鼠平均体重于第6d、13d、19d也明显下降，差异有统计学意义(P<0.01)。另外，与低、中剂量组相比，高剂量组大鼠平均体重于第19d明显降低，差异有统计学意义(P<0.01)。（2）脏器系数显示，高剂量组大小脑、心脏、肺脏、肾脏及睾丸脏器系数均有增加，且差异具有统计学意义（P<0.05或P<0.01）。（3）染毒第6d、13d、19d，与对照组比较，低、中、高剂量组步态评分均明显升高，差异有统计学意义(P<0.01)，且染毒结束后，其分值分别集中于1-2、2-3、3-4分之间，并呈现出时间—剂量—效应关系。另外，与低、中剂量组相比，高剂量组步态评分于第6d、13d、19d也明显增加，差异有统计学意义(P<0.05或P<0.01)。（4）染毒第19d，与对照组相比，低、中、高剂量组后肢支撑力指数均明显增加，差异有统计学意义(P<0.01)，且呈现出剂量—效应关系。另外，与低、中剂量组相比，高剂量组后肢支撑力指数也明显增加，差异有统计学意义(P<0.01)（5）染毒第19d高剂量组的甩尾时间比对照组明显缩短，差异有统计学意义(P<0.01)。结论：ACR亚急性染毒能导致大鼠体重下降；大小脑、心脏、肺脏、肾脏及睾丸脏器系数增加；步态评分增高；后肢支撑力指数增加；甩尾时间缩短。具有明显的神经毒性，主要引起感觉运动功能异常。第二部分丙烯酰胺对大鼠多巴胺相关转运蛋白mRNA和蛋白表达的影响目的：探讨丙烯酰胺对大鼠纹状体多巴胺转运体(DAT)、单胺囊泡转运体(VMAT2)和酪氨酸羟化酶（TH）mRNA表达和蛋白水平的影响。方法：动物分组及处理同第一部分。实时荧光定量PCR技术检测大脑皮层、小脑和纹状体内DAT、VMAT2、THmRNA表达水平，并检测纹状体中MAO-BmRNA表达水平；WesternBlot蛋白印迹技术检测纹状体内DAT、VMAT2、TH的蛋白表达水平。结果：（1）大脑皮层结果显示：与对照组比较，各剂量组VMAT2mRNA表达水平均明显降低，其中低、中剂量组分别降低了30%、29%，差异具有统计学意义(P<0.05)，而高剂量组则降低了56%，差异具有统计学意义(P<0.01)。DAT、TH各组间差异均无统计学意义。（2）小脑结果显示：与对照组比较，低、中、高剂量组DAT、VMAT2、THmRNA表达均明显降低，差异具有统计学意义(P<0.01)。（3）纹状体结果显示：DATmRNA表达与对照组相比，低、中、高剂量组均明显降低，差异具有统计学意义(P<0.01)；Westernblot结果显示，高剂量组的糖基化DAT蛋白表达明显降低，仅为对照组的78%，差异具有统计学意义(P<0.05)。与对照组相比，高剂量组VMAT2mRNA表达显著降低(P<0.01)，VMAT2蛋白表达也显著降低，差异具有统计学意义(P<0.05)。与对照组相比，高剂量组THmRNA和蛋白表达均明显升高，差异具有统计学意义(P<0.05)。与对照组相比，高剂量组MAO-BmRNA表达明显降低，差异具有统计学意义(P<0.01)。结论：ACR亚急性染毒不仅会使大鼠小脑中DAT、VMAT2、THmRNA表达降低，还会使纹状体中DAT、VMAT2、MAO-BmRNA和蛋白表达降低，THmRNA和蛋白表达增加，提示ACR亚急性染毒会导致多巴胺神经递质的合成和转运功能失常，引起胞质和突触间隙中多巴胺含量改变，从而导致多巴胺神经系统功能紊乱。第三部分丙烯酰胺对PC12细胞多巴胺相关转运蛋白的蛋白表达的影响目的：探讨丙烯酰胺对PC12细胞中多巴胺转运体(DAT)、单胺囊泡转运体(VMAT2)和酪氨酸羟化酶（TH）蛋白表达的影响。方法：取对数生长期的PC12细胞，分为1个对照组和6个染毒组，各染毒组ACR终浓度分别为0.1mmol/L，0.3mmol/L，0.6mmol/L，1.25mmol/L，2.5mmol/L，5mmol/L，染毒24h。用WesternBlot蛋白印迹技术检测PC12细胞中DAT、VMAT2、TH的蛋白表达水平。结果：（1）DAT蛋白定量结果显示：与对照组比较，0.6mmol/L和1.25mmol/L组PC12细胞中糖基化DAT明显降低，差异具有统计学意义(P<0.05)；与对照组比较，0.1mmol/L、0.6mmol/L、1.25mmol/L组非糖基化DAT也明显降低，差异具有统计学意义(P<0.05)。（2）VMAT2蛋白定量结果显示：与对照组比较，0.6mmol/L-5mmol/L组PC12细胞中VMAT2明显降低，差异具有统计学意义(P<0.01)；与0.1mmol/L和0.3mmol/L组比较，0.6mmol/L-5mmol/L组VMAT2也降低，差异具有统计学意义(P<0.5或P<0.01)。（3）TH蛋白定量结果显示：与对照组比较，2.5mmol/L、5mmol/L组PC12细胞中TH含量明显升高，差异具有统计学意义(P<0.05，P<0.01)。结论：ACR可以引起PC12细胞中DAT、VMAT2蛋白含量降低，同时还可以引起TH蛋白含量升高，提示ACR可以引起PC12细胞中多巴胺神经递质的合成和转运功能异常。综上所述，ACR亚急性染毒能导致大鼠体重下降，后肢支撑力指数增加，步态评分增高，甩尾时间缩短，说明其神经毒性作用比较明显；其作用于小脑和纹状体后，不仅会使大鼠小脑中DAT、VMAT2、THmRNA表达降低，还会使纹状体中DAT、VMAT2、MAO-BmRNA和蛋白表达降低及THmRNA和蛋白表达增加。另外，ACR还能引起PC12细胞中DAT、VMAT2蛋白含量降低，同时还可以造成TH蛋白含量升高。因此，推测纹状体DA系统功能紊乱可能与多巴胺相关转运蛋白受到影响有关。更多还原

【Abstract】 Acrylamide (ACR) is vinyl water-soluble organic compound prepared on an industrial scale by the hydrolysis of acrylonitrile by nitrile hydratase. ACR is used in chemical refining, waste water treatment, textile processing and the production of cosmetics. It can be detected in the drinking water, fried food and food packaging matetials. As early as the1970s, the neurotoxicity of ACR was reported in occupational poisoning population. Because of mostly entering the body by mouth, it has raised widespread concern in societies. Epidemiological investigations and experimental studies have shown ACR to be neurotoxic in both animals and human, and also to have reproductive toxicity, genetic toxicity and carcinogenicity. Acute and sub-acute poisoning of ACR cause damage to the central nervous system, manifested as neuropsychiatric symptoms and cerebellar ataxia, while chronic poisoning cause damage to the periphetal nervous system, but its mechanism is not clear.The striatum is the largest integrated processing elements in the basal ganglia, and contains a large number of dopaminergic neurons. Dopamine neurotransmitter is mainly distributed in it. Dopamine in the neural pathways of dopaminergic system is firstly biosynthesized, transported, released, and degraded on the presynaptic membrane, in order to maintain the balance of prominent gap in the dopamine neurotransmitter. The dopamine neurotransmitter from the saynaptic cleft to the postsynaptic membrane play a postsynaptic effect, while dopamine transporter protein in the whole process play an important role in maintaining the balance of the neurotransmitter dopamine in the synaptic cleft. When this balance is broken, the nervous system can experience different symptoms, which are mainly in the sensory-motor integration, body balance movement and learning and memory. In past studies with ACR, population epidemiological and experimental studies have largely seemed to indicate that neurological signs and symptoms are related with break in the balance of dopamine neurotransmitter.Up to now, there are only a few reported about the affects of ACR on the dopamine transport-related proteins which maintain the balance of dopamine neurotransmitter in the striatum, and the exact mechanism is not yet clear. Hence, the need to investigate the potential mechanisms of neurotoxicity of ACR, and explore the impact of acrylamide on dopamine transporter, vesicular monoamine transporter and tyrosine hydroxylase, in vivo and vitro. The aim of this study is to provide a theoretical basis for the treatment of acrylamide poisoning.Part I Neurobehavioral Toxicity of Acrylamide in RatsObjective:The aim of this part is to investigate the neurobehaviaral toxicity of ACR in rats.Methods:Forty healthy adult SD male rats which were provided by experimental animal center of Tongji Medical College, Huazhong University of Science and Technology, all of them were randomly divided into four groups,10per group: control group, low dose group (20mg/kg), middle dose group (30mg/kg), high dose group (40mg/kg), which were single-caged. Rats were exposed to ACR solution though oral gavage in corresponding dose and control group were given equal volume of saline. Rats were continuous exposed for5days and rest two days, total nineteen days. The body weight and gait score were measured on day0,6,13and19, the force index of hindlimb legs and tail-drift time were measured on day0and19.24hours after the last administration, all rats were decapitated and the whole brain tissue was immediately removed and washed by pre-cooling PBS. After weighed the brain and cerebellum, striatum, cerebral cortex and cerebellum were stripped immediately and stored at-80℃for determination. Heart, liver, spleen, lung, kidney and testicle were weighed and recorded.Results:(1) Weight of the low dose group was lower than that of the control group, but the difference was not significant. Compared with the control group, the weight of the middle dose group reduced on day19(P<0.01) and the weight of high dose group also reduced on day6,13d,19(P<0.01). Compared with the low dose group and middle dose group, the weight of high dose group was lower on day19significantly (P<0.01).(2) Organ coefficient:brain, cerebella, heart, lung, kidney and testicle of high dose group increased significantly (P<0.05or P<0.01).(3) Compared with the control group, gait score increased (P<0.01) on day6,13and19At the end of this experiment, their scores were concentrated between1-2,2-3,3-4and showed a time-dose-effect relationship. In addition, compared with the control group, gait score of high dose group on day6,13and19significantly increased (P<0.05or P<0.01).(4) Compared with the control group, the force index of hindlimb legs was significantly increased in all groups (P<0.01). Compared with the low and middle dose group, the force index of hindlimb legs was increased significantly in the high dose group (P<0.01).(5) On day19, the tail-drift time of high dose group was lower than that of the control group (P<0.01).Conclusions:The body weight of rats were decreased, the organ coefficient of brain; cerebella, heart, lung, kidney and testicle increased; gait score increased; the force index of hindlimb legs and tail-drift time increased in the sub-acute exposure of ACR. ACR showed obvious neurotoxicity, and mainly caused sensory-motor dysfunction. Part Ⅱ The impact of ACR on dopamine transport-related proteins mRNA and protein expressions in ratsObjective:To investigate the impact of ACR on dopamine transporter (DAT), monoamine vesicle transporter (VMAT2) and tyrosine hydroxylase (TH) mRNA expression and protein levels in the rat striatum.Methods:The animal grouping and treatment were the same as the part one. The mRNA expression of DA^VMAT2、TH mRNA were measured by real-time PCR in the striatum, cerebral cortex and cerebellum, and the mRNA expression of MAO-B were measured in the striatum. Moreover, western blot were used to estimate the protein expression of DA、VMAT2、TH in the striatum.Results:(1) the result of the cerebral cortex:Compared with the control group, the expression of VMAT2mRNA reduced by30%(P<0.05),29%(P<0.05),56%(P<0.01) in low, middle and high dose group.(2) the result of the cerebellum:Compared with the control group, the mRNA expression of DA、 VMAT2、 TH mRNA reduced significantly (P<0.01).(3) the result of the striatum:Compared with the control group, in the low, middle and high dose group DAT mRNA reduced significantly (P<0.01), and protein quantification results showed that the expression of the glycosylated DAT protein in the high dose group was decreased significantly (P<0.05). Compared with the control group, VMAT2mRNA and protein expression in high dose group was decreased significantly (P<0.01), Compared with the control group, in the high dose group TH mRNA and protein significantly increased (P<0.05). Compared with the control group, the expression of MAO-B mRNA in the high dose group significantly reduced (P<0.01).Conclusion:Sub-acute poisoning of ACR led to VMAT2-mRNA expression in rat cerebral cortex and cerebellum decreased; DAT, VMAT2,MA0-B mRNA and protein expression decreased in the striatum; TH mRNA and protein expression was increased in the striatum. These finding suggest that sub-acute poisoning of ACR led to the synthesis and transport function of the dopamine neurotransmitter disorders, caused by the change of dopamine content in the cytoplasm and synaptic cleft, resulting in dysfunction of the dopaminergic systerm.Part III The impact of ACR on dopamine transport-related proteins expressions in PC12cellsObjective:To investigate the impact of ACR on dopamine transport-related proteins expressions in PCI2cells.Methods:Taking the logarithmic growth phase PC12cells, divided into one control group and six ACR groups for24h, final concentration was0.1mmol/L,0.3mmol/L,0.6mmo1/L,1.25mmol/L,2.5mmol/L,5mmol/L respectively. Western blot were used to estimate the protein expression of DAT, VMAT2, TH in PC12cellsResults:(1)DAT protein quantification results showed that:compared with the control group,0.6mmol/L and1.25mmol/L group of the glycosylated DAT was decreased significantly(P<0.05); Compared with the control group,0.1mmol/L,0.6mmol/L and1.25mmol/L group of the non-glycosylated DAT was also decreased significantly (P<0.05).(2)VMAT2protein quantitative results show that: compared with the control group,0.6mmol/L,1.25mmol/L,2.5mmol/L and5mmol/L group of VMAT2protein was decreased significantly (P<0.0\).(3) TH protein quantification results showed that:the2.5mmol/L and5mmol/L groups of TH protein were more than the control group significantly{P<0.05,(P<0.01).Conclusions:ACR could decrease DAT, VMAT2-protein expression, elevate TH protein expression in PC12cells. This study suggested that ACR could cause abnormal synthesis and transport function of the neurotransmitter dopamine in PC12cells. In summary, the ACR sub-acute exposure could decrease body weight, increase gait score and the force index of hindlimb legs, decrease tail-drift time, which suggested that ACR showed neurotoxic effects. ACR could decrease DAT, VMAT2gene and protein expression, increase TH gene and protein expression in vivo and in vitro. These results suggested that the striatum-DA system dysfunction may be related to the dopamine transporter protein更多还原