【作者】 杨炀；

【导师】 常重杰；

【作者基本信息】 河南师范大学 ， 遗传学， 2012， 硕士

【摘要】 随着我国经济的不断发展，人们对食品质量的要求标准也在不断的提高。近年来食品安全问题越来越突出，由于苏丹红在食品添加上所引起的恐慌，使人们越来越关注人工合成染料这类非食用食品添加剂对人体的危害。因此，通过毒理学和分子生物学的原理方法来研究非法食品添加剂对动物的毒理学效应，以及动物食用后对动物遗传物质DNA损伤程度的研究，对于当前制定非食用添加剂的使用安全指标具有非常重要的理论和现实意义。本研究根据评价外源物质毒性作用的毒理学标准，从急性毒性试验、生理毒性试验、遗传毒性试验（微核及核异常试验）、胚胎及仔鱼毒性试验、DNA损伤的RAPD分析五个方面初步探讨了苏丹红Ⅳ（Sudan Ⅳ）对两种泥鳅（大鳞副泥鳅和泥鳅）的毒性效用及DNA损伤效应。获得了如下结果：1．急性毒性试验表明，苏丹红Ⅳ（Sudan Ⅳ）对大鳞副泥鳅的24h半数致死浓度（median lethal concentration，LC50）为2.125g.L^-1，48h半数致死浓度（median lethalconcentration，LC50）为1.925g.L^-1，安全浓度（safe concentration，Sc）为0.474g.L^-1；苏丹红Ⅳ （Sudan Ⅳ）对泥鳅的24h半数致死浓度为2.225g.L^-1，48h半数致死浓度为2.025g.L^-1，安全浓度为0.503g.L^-1。结果表明泥鳅相对于大鳞副泥鳅对苏丹红Ⅳ具有较高的耐受性。2．在急性毒性试验结果的基础上，设置不同浓度梯度，进一步探讨了苏丹红Ⅳ对血细胞微核率及核异常率的影响。苏丹红Ⅳ （Sudan Ⅳ）对大鳞副泥鳅的设计染毒浓度分别为A(0.025g.L^-1)、B(0.050g.L^-1)、C(0.100g.L^-1)、D(0.200g.L^-1)、E(0.400g.L^-1)以及空白对照组；苏丹红Ⅳ （Sudan Ⅳ）对泥鳅的设计染毒浓度分别为A(0.030g.L^-1)、B(0.060g.L^-1)、C(0.120g.L^-1)、D(0.240g.L^-1)、E(0.480g.L^-1)以及空白对照组。染毒后1、2、3、4、5天分别取材，观察、计算微核率。结果显示，两种泥鳅红细胞微核率及核异常率与苏丹红Ⅳ浓度大体上呈正相关，同时也有一定的时间效应。3．以两种泥鳅肝组织中谷丙转氨酶（GPT）、谷草转氨酶（GOT）的活性变化为指标，检测了苏丹红Ⅳ对两种泥鳅的生理毒性效用。实验数据显示受试动物肝脏GOT和GPT活性随着染毒时间的延长呈现出明显的波动，具体表现为先上升再下降最后一天又开始上升。这种特殊的现象也许表明不仅苏丹红Ⅳ本身能够影响泥鳅的生理功能，其次级代谢物对其生理功能也有明显的影响。4．从50条10个碱基序列的随机引物中筛选出8条能产生较多扩增条带的随机引物，以染毒泥鳅和大鳞副泥鳅基因组DNA为模板，分析基因组DNA的RAPD分子标记谱带变化规律并与空白对照进行比较，进一步探究苏丹红Ⅳ对两种泥鳅基因组DNA的损伤情况。分析结果发现，在不同浓度不同时间染毒基因池中，苏丹红Ⅳ能不同程度地诱使RAPD谱带的增加、缺失以及带的亮度强弱的变化。结果表明苏丹红Ⅳ能够诱发基因组DNA的部分碱基发生突变。另外，本研究还对染毒后部分新产生的RAPD谱带进行了克隆测序和序列分析，这些研究结果对于揭示苏丹红毒性的分子机理具有重要的参考意义。5．本研究还进一步分析了苏丹红Ⅳ对两种泥鳅胚胎和仔鱼发育分化的影响。以两种泥鳅的胚胎孵化率和畸形率为指标，在两种泥鳅受精卵发育至原肠胚中期时开始进行染毒，为开始处理时间进行染毒培养，探讨其对胚胎毒性效应。实验结果表明，苏丹红Ⅳ能不同程度地诱使两种泥鳅受精卵孵化率降低、畸形率升高，对胚胎发育致畸、致死作用明显。同时，两种泥鳅的胚胎对苏丹红Ⅳ的敏感程度也存在一定的差异。以上结果表明，苏丹红Ⅳ对两种泥鳅的胚胎发育、抗氧化酶的活性、遗传物质的稳定性具有较明显的毒性作用，应当严格禁止其作为添加剂出现在食品中。更多还原

【Abstract】 With the continuous growth of the national economy, people’s food quality standards graduallyimprove. In recent years as Sudan red in food adds the panic, people pay more and more attention to thiskind of synthetic dyes non edible food additive which is harmful to the human body. Therefore, thestudy of toxicology and molecular biology principle method, used to study the toxic substances onanimal DNA injury severity, monitoring food toxic substance in action, has very important theoreticaland practical significance for the current formulation of additives in food safety in use indicator.This study based on toxicological evaluation of exogenous substances toxic effects, is divided intofive parts: acute toxicity test, physiological toxicity test, genetic toxicity test （micronucleus test）,embryonic and larval toxicity test, DNA injury: a RAPD analysis. We preliminary discussed the toxicityand molecular biological effects of Sudan red IV （Sudan IV） on two kinds of Loach （Misgurnusanguillicaudatus,）. On the basis of the toxicology and molecular biology experimental results on SudanIV （Sudan IV） toxicity, make scientific assessment, providing digital basis for reasonable formulationof food in Sudan IV （Sudan IV） use safety indicators, in order to serve our country food safetyproduction better.Through acute toxicity test, the Sudan red IV （Sudan IV） on Loach24h median lethal concentration（median lethal concentration, LC50）2.125g.L^-1,48h median lethal concentration （median lethalconcentration, LC50）1.925g.L^-1, safe concentration （safe concentration, Sc）0.474g.L^-1; Sudan IV（Sudan IV） on the24h median lethal concentration （median lethal concentration, LC50）2.225g.L^-1,48h median lethal concentration （median lethal concentration, LC50）2.025g.L^-1, safe concentration（safe concentration, Sc）0.503g.L^-1.Based on the results of acute toxicity test, the genetic toxicity test （of micronuclei and nuclearanomalies test）. With blood cell micronucleus rate as an index, designed each test group concentration.Sudan IV （Sudan IV） on Loach exposure concentrations were A (0.025g.L^-1), B (0.050g.L^-1), C(0.100g.L^-1), D (0.200g.L^-1), E (0.400g.L^-1),0（control group）, a total of6groups; Sudan IV （Sudan IV） on Loach exposure concentrations were A (0.030g.L^-1), B (0.060g.L^-1), C (0.120g.L^-1),D (0.240g.L^-1), E (0.480g.L^-1),0（control group）, divided into6groups. Processing time in exposureafter1,2,3,4, and5days respectively with a material, observation, calculation. The results showed,in two species of loaches erythrocyte micronucleus and abnormal nucleus and Sudan IV concentrationgenerally positively correlated, but also have a certain time effect.According to the genetic toxicity test do the physiological toxicity test, with aminotransferase （GPT）,aspartate aminotransferase （GOT） activity of the two kinds of Loach Liver alanine as indicator,.Experimental data shows the liver GOT and GPT activity increased with the prolongation of time, thenfall, however, on the last day, it began to rise again, descripting loach liver has the detoxificationfunction itself. As time goes on, the GOT and GPT activity will gradually periodicly changes. Theinhibition degree of the enzyme varies from one kind of loach to another.In order to further explore the Sudan red IV （Sudan IV） in two species of loaches in the level of DNAdamage, screening8primers from50random primers, performed a RAPD （random amplifiedpolymorphic DNA） molecular markers on Loach, analysising the DNA damage of the two kinds ofloaches before and after the exposure. The analysis results show that, the Sudan red IV （Sudan IV） canto a different degree induce the addtion, deletion of the DNA fragment of the two species of loaches,and alteration of band brightness, obviously shown in the level of DNA damage effect.At the embryo toxicity test, with two kinds of loach embryo hatching rate and abnormal rate asindexes, studied the toxicity effec of the Sudan red IV （Sudan IV） on the embryo of the two kinds ofloaches. When the loaches were in the embryo gastrulation period, they were exposed to medium forcultivation, experimental results show that, Sudan IV （Sudan IV） to different degrees can reduce twokinds of loaches embryo hatching rate, increase deformity rate, leading to teratogenic, lethal effecton the embryonic development level to some extent. The degree of sensitivity varies from one kind ofloach embryo to another.更多还原