【作者】 赵立；

【导师】 屠康；

【作者基本信息】 南京农业大学 ， 食品科学， 2006， 博士

【摘要】 铅是一种无处不在的重金属，被称为三大毒害金属之一，受到人们广泛的关注。随着现代工业的飞速发展，在环境体系和生态系统中的任何一个环节都可以检测出。铅被广泛使用，尤其近20年来，铅的使用量超过了以往所有年中的使用，大量的使用可以导致当地的和全球的空气，烟尘以及土壤污染。铅是对人体危害极大的一种重金属，它对神经系统、骨骼造血系统、消化系统、生殖系统等均有危害。国内外对于人类和小鼠等哺乳动物铅中毒方面的研究很多，鸟类的研究多集中于野生种类，对于从饲料中添加铅来探讨铅的沉积及其对鸡胚胎毒性的研究很少。因此，本文以饲料为污染源，以集约化饲养的家禽鸡为实验动物，研究了饲料中铅在种蛋中的沉积情况以及铅在胚胎各组织器官中的沉积，了解铅沉积的器官特异性；通过组织病理学和细胞超微结构的观察来了解铅对肝、肾细胞结构的影响；同时通过对胚胎肝、肾抗氧化酶的影响探讨铅对禽类胚胎的毒性作用机制，为进一步研究铅的毒性作用机理提供理论依据。具体研究内容和结果如下：1．重金属在鲜蛋中的沉积情况本论文对江苏南京地区市售各产地和鸡种的鸡蛋进行随机采样，根据国家无公害标准检测重金属残留情况，结果表明，不同产地、不同品种的鸡蛋各种重金属污染的程度有所不同，但无论蛋清、蛋黄还是全蛋，无论是产地还是品种，除无公害蛋以外，铅都毫无例外的超过无公害标准，铅沉积量蛋清在0.2054～0.5817mg·kg^-1之间，蛋黄在0.3788～1.2918 mg·kg^-1之间，全蛋在0.3365～0.8787 mg·kg^-1之间，蛋黄中沉积最多。因此，铅在鲜蛋中的超标问题及相应的解决措施应引起足够重视。2．饲料中不同含量铅在种蛋中的沉积及其影响通过在伊莎褐（ISA）种鸡饲料中添加不同剂量的铅，研究铅在种蛋各成分中的沉积以及铅对种蛋品质的影响，实验分为四组，分别为对照组(铅含量为3.55mg·kg^-1)、15mg·kg^-1、30mg·kg^-1、60mg·kg^-1组，实验期为30天，分别于实验第0、5、10、15、20、25、30天测定种蛋各成分中的铅含量。结果表明，饲料中铅的添加和沉积时间影响其在种蛋各成分中的沉积，同时影响蛋的品质。在短时间内，铅对蛋重无影响，对蛋形指数有影响但无规律性，说明铅可能引起蛋的畸型。铅主要沉积在蛋壳中，其次沉积于蛋黄中，其含量都显著高于蛋清和全蛋。蛋壳铅的沉积使蛋壳厚度变薄，蛋壳强度无差异，同时破蛋率显著下降。当沉积到第25天时各成分铅含量达到平衡，蛋壳铅量与全蛋铅量之间相关系数为0.857，达到了显著的水平，逐步回归结果为y=1.845*10^-2a+0.265（y为全蛋铅含量，a为蛋壳铅含量），提示可用蛋壳铅含量预测全蛋铅含量。饲料中铅含量在15mg·kg^-1以下时并未显示出与对照组的显著差异。饲喂第25天的种蛋作为后期实验的采样时间。3．饲料中不同含量铅在不同胚龄鸡胚各器官中的沉积情况取饲喂至第25天的种蛋进行孵化，在孵化至第9、14和19天时，取鸡胚胎进行各器官的铅沉积的测定，探讨了饲料中不同含量铅在鸡不同胚龄各器官中的沉积情况。结果表明，鸡胚生长到14天时各器官铅的沉积量最多，沉积量大小顺序依次为肾脏、肝脏、肌肉。各组肌肉铅含量都小于国家标准。在饲料中添加铅至30mg·kg^-1时，铅在鸡胚中的沉积已达极限，30mg·kg^-1组14天鸡胚肾脏、肝脏和肌肉的铅含量分别达到了1.2193mg·kg^-1，0.2714mg·kg^-1，0.1446mg·kg^-1。随着饲料铅含量的增加，并未出现鸡胚各组织中铅沉积量的增加。饲料铅含量与14天和19天鸡胚肾脏铅含量相关性具有统计学上的显著性，相关系数分别达到了0.574和0.380。4．饲料中不同含量铅对不同胚龄鸡胚肝、肾病理组织学及超微结构的影响组织病理学的分析和重金属沉积相关数据的结合对有关重金属对活体组织影响的研究可以提供更有价值的信息。本文通过对不同胚龄鸡胚肝肾组织病理学和细胞超微结构的观察，探讨了饲料中不同含量的铅对三个胚龄鸡胚病理组织学的影响。结果表明，当鸡胚生长到9天时，铅对肝脏的组织病理学并未产生明显的影响。鸡胚9天时的肝脏细胞器较生长到14天和19天时种类少，数量少。同时，肾细胞中的细胞器较肝细胞中的丰富，种类多。对于肝脏，当鸡胚生长到14天和19天，饲料铅含量达到15mg·kg^-1时，可引起肝细胞轻微病变，而饲料铅含量达到30mg·kg^-1时，肝脏组织结构才可发现明显病变现象。肝脏亚细胞对铅更为敏感。对于肾脏，无论是14天还是19天的鸡胚，当饲料铅达到15mg·kg^-1时，肾脏组织和肾细胞内细胞器结构均可见明显的变化，因此，肾脏对铅的毒性较肝脏更为明显。同时发现，对于两种器官，饲料铅含量为30mg·kg^-1和60mg·kg^-1的损伤程度几乎相同，说明饲料铅为30mg·kg^-1已使肝肾的损伤达到最大。鸡胚期肝肾细胞变性主要表现为肝细胞变形、细胞间隙增加、核膜肿胀、线粒体肿胀，内部嵴不清晰，嵴断裂溶解，线粒体发生溶解以及炎症细胞的产生等。5．饲料中不同含量铅对不同胚龄鸡胚肝、肾抗氧化能力影响的研究通过鸡胚肝肾抗氧化酶活力的测定，部分的探讨了铅对鸡胚毒性机理。结果表明，在整个生长发育期，各实验组，随着鸡胚的生长，肝肾的抗氧化能力有所变化，肝脏和肾脏中的MDA含量都随着鸡胚的生长而减少。肝脏抗氧化酶中SOD、GST活力降低，CAT和GR活力增加，而肾脏中所有的抗氧化酶活力都随着鸡胚的生长而降低。而对于饲料中铅的添加，当鸡胚生长到第9天时，肝脏中只有GST活力下降，而对于14天的鸡胚，只有肾脏CAT活力下降，其它酶都没有变化，只有当鸡胚生长到19天时所有的抗氧化酶活力才受到影响，随着铅的添加而下降。以上说明，随着胚胎的生长，铅对鸡胚肝脏抗氧化酶活力的影响并不一致，而肾脏的抗氧化能力却显著下降。脂质过氧化反应并不是铅对鸡胚胎毒性的一个主要标志。铅对抗氧化酶的抑制是铅对鸡胚胎毒性的一个机制。同时结果表明肾脏是调节鸡胚抗氧化体系一个主要器官。更多还原

【Abstract】 Lead is ubiquitously a kind of heavy metal that was regarded as one of the three mostnoxious metals, so it is widespread paid attention. Lead can be determined during any partof environmental and ecological system with the fast development of modem industry.Lead was widespread used, especially in the recent two decades, the quantity exceeded thatof the all past years that lead to the local and global pollution of the air, smoking and soil.Lead is a huge harmful kind of heavy metal for the body that can lead to the dysfunction onthe nerve system, hematogenic system, digestive system and reproductive system, etc. Wecan find many studies of the lead toxicology on the mammal of human and rat, meantime,the research on bird were mainly focused on wild kinds. Few data was found out about thelead deposition and the effect of lead on the embryo toxicology of chick in the way offeedstuff. In the paper, the lead was added in the way of feedstuff and the experimentalanimal was the poultry chicks that were feed on a large scale. The lead deposition ofhatching eggs and the tissue and organ of the chick embryos was researched in order tounderstand the organ specificity of lead deposition. With the help of histopathology and cellultrastructure, we realized the effect of lead on the hepatic and renal cell structure; in thesame time, we discussed the change of antioxidant enzymes of liver and kidney in order tounderstand the lead toxicity mechanism for chick embryos, which provided a theory basisabout the further research of lead toxicology.The contents and results were as follows.1. The deposition of heavy metals in fresh eggs In this paper, the chick eggs saled in NanJing City JiangSu Province were randomlysampled to examine the deposition of heavy metals according as the national harmlessstandards, the results was showed that the deposition of heavy metals in chick eggs fromdifferent places and breeds varied. The content of lead in all eggs （egg white, egg yolk andwhole egg） except for harmless eggs unlimitedly exceeded that of the standard. The contentof lead in egg white, egg yolk and whole egg was 0.2054～0.5817mg·kg^-1, 0.3788～1.2918mg·kg^-1, 0.3365～0.8787mg·kg^-1, respectively. The deposition in egg yolk was maxim. So,the super-concentration of lead in fresh eggs and relative resolvent should be paid attention.2. The deposition and effect of different contents of lead in feedstuffon the hatching eggsThe lead of different contents were added into the laying chick （ISA） feedstuff toresearch the deposition in hatching eggs and the effect of lead on the quality of eggs. Theexperiment was divided into four groups as follows: ck group, adding lead of 15mg·kg^-1group, 30 mg·kg^-1 group, 60 mg·kg^-1 group. The duration of whole experiment was 30 days.Content of components in hatching eggs was determined in the 0, 5th, 10th, 15th, 20th, 25th,30th day, respectively. The results were showed that the deposition of lead in components ofhatching eggs and the quality of eggs was influenced by the adding lead into feedstuff anddeposition period. In the short period, lead did not influence the egg weight; the eggshape index was affected without order according to the lead residue level which could leadto the malformation of the eggs and harm the development of embryo. For all componentsof eggs, lead was mainly deposited into eggshell, then egg yolk, all of which weresignificantly higher than that of egg white and whole eggs. The deposition of eggshell madethe shell thin, but the eggshell strength was not influenced and the percentage of crackedeggs significantly dropped.In the 25th day of the experiment period, the deposition of components was up to themaximum. At the same time, the correlation coefficient between the content of eggshell andwhole egg was to remarkable level, 0.857. The stepwise regression equation wasy=1.845*10^-2a+0.265 （y indicated lead content in whole egg, a indicated lead content ineggshell）, which gave a clue of that lead content in eggshell could be examined to forecastthat in whole eggs. When the content in feedstuff was lower 15 mg·kg^-1, the deposition ineggs was not significantly different compared with ck group. The 25th hatching eggs wereselected as the sampling period in the later experiment. 3. The deposition and effect of different content of lead in feedstuffon tissue and organs of chick embryo at different growing periodThe hatching eggs in the 25th day were hatched. At the 9th, 14th,19th day of hatchingperiod, tissue and organs of chick embryos were gained to determine the content of lead inorder to discuss the effect of deposition of lead on chick embryo. The results were showedthat the period of maximum deposition was the 14th chick embryos. At this time, the orderof content in tissue and organs was kidney, liver, the lowest muscle. The content of lead inmuscle in every group was lower than that regulated in national standard. When the contentof lead in feedstuff was up to 30mg·kg^-1, the deposition in embryos was maximum; at thistime, for the 14th chick embryos, the content of kidney, liver and muscle was up to1.2193mg·kg^-1, 0.2714mg·kg^-1, 0.1446mg·kg^-1, respectively. The correlative coefficientsbetween content of lead in feedstuff and that of kidney of the 14th and 19th embryos werestatistically significant, 0.574 and 0.380.4 The effect of the different content of lead in feedstuff onhistopathology and ultrastructure of liver and kidney of chick embryos atdifferent growing periodThe combinative analyse of histopathology and the deposition status of heavy metalscould provide the most valuable information on the effect of heavy metals on the organismtissue. In this paper, the observation on the histopathology and ultrastructure of liver andkidney of chick embryos at different growing period was carded out to discuss the effect oflead on the histopathology of chick embryos. The results were showed that lead did notsignificantly influence the histopathology of liver. In the 9th day of chick embryos, the kindsand quantity of organelle of liver were fewer than that in the 14th and 19th day. Meantime,the kinds and quantity of organelle of kidney were more than that of liver.For the liver of the 14th and 19th day of embryos, lead of 15mg·kg^-1 in feedstuff couldlead to the slight pathological changes of hepatic cells, and when the content of lead was upto 30mg·kg^-1, the pathological changes of liver was obvious. The subcellular structure ofliver was most sensitive to lead. For the kidney, the content of 15mg·kg^-1 of the lead infeedstuff could find the obvious pathological changes in renal tissue and organelle, so, theeffect of lead on kidney was significantly higher than that on liver. We also found that30mg·kg^-1 and 60mg·kg^-1 of lead destroyed the two organs to the same extent, which showedthat 30mg·kg^-1 of lead was the maximum of damaging the liver and kidney. The histopathological changes of hepatic and renal cells were represented in the way oftransmutation of cells, the largement of cell interspace, the swollen karyotheca andmitochondrion, unclearness, breakage and dissolution of inner cristae, and the production ofinflammation.5. The effect of the different content of lead in feedstuff on thehepatic and renal antioxidant capacity of chicken embryos at differentgrowing periodThe hepatic and renal antioxidant enzyme activities of chick embryos were determinedto discuss the toxicology mechanism of lead on chick embryo. The results were showed thatwith the development of chick embryos, the hepatic and renal MDA content decreased;hepatic SOD and GST activities dropped; CAT and GR activities increased. But all of renalantioxidant enzymes fell with the development of chick embryos. For the 9th day chickembryos, hepatic GST activity decreased with the adding of lead; all of antioxidantenzymes activities except for descendent CAT activity did not change for the 14th day chickembryos. Only for the 19th day chick embryos, all antioxidant enzymes decreased with theadding of lead in the feedstuff. The above results were showed that the effect of lead on thehepatic antioxidant capacity did not obvious, but lead decreased the renal antioxidantcapacity with the development of chick embryos. Lipoxidantion reaction was not a majorindicator of lead toxicology. A mechanism of lead toxicology on chick embryo was thatlead inhibited the antioxidant enzyme activities. And the results were showed that kidney isa major organ that regulated the antioxidant system of chick embryo.更多还原