【作者】 何林；

【导师】 吴赤蓬；

【作者基本信息】 暨南大学 ， 流行病与卫生统计学， 2008， 硕士

【摘要】 目的:研究一些常用食品添加剂对机体遗传物质的损伤作用,并探讨食品添加剂之间可能存在的联合毒性作用,为正确评价这些食品添加剂的安全使用剂量提供参考依据。方法:首先用MTT法检测七种食品添加剂（日落黄、柠檬黄、苋菜红、亮蓝、复合绿、复合紫、亚硝酸钠）对中国仓鼠肺成纤维细胞（CHL细胞）的细胞毒性,并分别计算出它们对CHL细胞的IC₅₀。根据各添加物的IC₅₀确定染色体畸变试验、单细胞凝胶电泳（SCGE）试验、凋亡和细胞周期试验的染毒剂量。通过上述的实验,筛选了日落黄、柠檬黄、复合绿和亚硝酸钠四种遗传毒性较明显的添加剂做联合毒性试验,由于日落黄、柠檬黄、复合绿都是色素,而亚硝酸钠是一种防腐剂,现实中色素和防腐剂联用的情况比较普遍,所以用亚硝酸钠和三种色素分别作两两联合遗传毒性试验,它们之间联合遗传毒性通过SCGE试验和细胞凋亡试验来检测。结果:1.MTT法检测受试物的细胞毒性:复合绿、柠檬黄、日落黄、亚硝酸钠随剂量增高,对CHL细胞生长抑制作用明显增强,细胞毒性较大,而苋菜红、亮蓝、复合紫、在不同剂量对CHL细胞生长抑制作用差别不大,细胞毒性不明显;受试物对CHL细胞生长半数抑制浓度(IC₅₀)分别为:复合绿（0.37g/L）、柠檬黄（0.50g/L）、日落黄（0.70g/L）、亚硝酸钠（1.14g/L）、苋菜红（1.42g/L）、复合紫（1.77g/L）、亮蓝（7.48g/L）;其中以复合绿、柠檬黄和日落黄毒性较大。2.SCGE检测受试物对DNA损伤:各受试物对CHL细胞的DNA均有一定程度的损伤作用,其中以复合绿、亚硝酸钠、柠檬黄、和日落黄的遗传毒性作用最为明显,分别在0.1g/L、0.1g/L、0.1g/L和0.037g/L剂量下可引起细胞拖尾率明显高于空白对照组（P＜0.05）,并有明显的剂量-反应关系。3.染色体畸变试验检测DNA损伤:对七种受试物进行的染色体畸变分析发现复合绿、日落黄、亚硝酸钠和柠檬黄致细胞染色体畸变作用明显,与空白对照组相比较差异有统计学意义（P＜0.05）,分别在0.020g/L、0.037g/L、0.128g/L、0.080 g/L和剂量下就能明显引起染色体畸变率的增加（畸变率在10%以上）,而其他三种色素的致染色体畸变作用则不明显。4.受试物的细胞凋亡和周期试验:在七种受试物中,只有亚硝酸钠（1.5g/L）和复合绿（0.1g/L）有较明显的促进CHL细胞凋亡的作用,与空白对照组比较差异有统计学意义（P＜0.05）,而其他受试物的促凋亡作用不显著。在细胞周期试验中,各受试物与空白组比较,除复合绿GREEN外,S期细胞比例均增加,表现为一定的S期阻滞;而复合绿则表现为G₁期、G₂期比率增加,S期下降,说明细胞的DNA受到一定程度的损伤。5.联合作用分析:用SCGE试验分别研究亚硝酸钠分别与日落黄、柠檬黄、复合绿三种合成色素的联合毒性作用,结果表明亚硝酸钠和柠檬黄之间存在联合作用（P=0.015）,而且随着受试物剂量的增加,细胞拖尾率增加,表现为协同作用;亚硝酸钠和复合绿（GREEN）之间联合作用不明显（P=0.387）,可能只表现为简单的相加作用;而亚硝酸钠和日落黄之间也存在联合作用（P=0.010）,同样表现为协同作用。除以上形态学分析（拖尾率）外,对彗星试验的距离指标的分析也得出类似的结果。结论:1.各受试物均有一定的细胞毒性,且剂量反应关系明显,以复合绿、柠檬黄和日落黄毒性最大。2.亚硝酸钠表现为一定的遗传毒性（浓度为0.1g/L时可引起染色体畸变率和细胞拖尾率增加,0.15g/L时引起细胞凋亡率增高）,复合绿、柠檬黄和日落黄均表现出明显的遗传毒性。3.亚硝酸钠和柠檬黄之间存在联合作用,亚硝酸钠和日落黄之间也存在联合作用,均表现为协同作用。更多还原

【Abstract】 Objective:To investigate the injury of some commonly used food additives onto the genetic material,and research for the joint action among them more.Hoping that can provide the reference of how to judge the safe food additives dosage.Methods:There are seven kinds of food additives（they are Sunset Yellow FCF,Tartrazine, Green complex,Purple complex,Sodium Nitrite,Brilliant Blue FCF and Amarante ）. Firstly,we must make sure the cells toxicity of these additives onto the CHL cells by MTT,then we can count out the IC₅₀ respectively.According to the IC₅₀ of the additives, we can sure the poisoned dosage of additives in chromosome aberration test,SCGE test, apoptosis and of cell cycle.According the above test,it is better for using these four genotoxicity-obvious additives（Sunset Yellow FCF,Tartrazine,Green complex and Sodium Nitrite） to make the joint toxicity test.That’s because the Sunset Yellow FCF, Tartrazine and Green complex are coloring matters,and the Sodium Nitrite is a kind of antiseptic,actually it is common that the coloring matters and antiseptic are used together. So it is better to do such a joint toxicity test using the Sodium Nitrite and these three coloring matters react each other.And the joint toxicity between them are tested by the SCGE experiment and cell apoptosis experiment.Results1.MTF exam the cells toxicity of additives:In the study,the inhibitory actions on CHL cell growth were potential obviously with the dose increase of Amarante,Sunset Yellow FCF,Green complex and Sodium Nitrite,which showed considerable cell toxicity. However,various doses of Brillant Blue FCF,Purple complexand and Tartrazine showed little difference among the inhibitory actions on CHL cell growth.Considering IC₅₀ of CHL cell growth,the toxicity of Green complex（0.37 g/L）,Tartrazine（0.5 g/L） and Sunset Yellow FCF（0.7 g/L） are higher.2.Use SCGE to exam damage of additives to CHL cell DNA:With the dose increase, all the chemicals brought a raise result of cell comet ratio and indicated a clear dose dependant relation.Sunset Yellow FCF（0.037 g/L）,Tartarazina（0.1 g/L）,Amarante （10 g/L）,Brillant Blue FCF（10 g/L）,Green complex（0.1 g/L）,Purple complex（0.1 g/L）and Sodium Nitrite（0.1 g/L）showed significant higher cell DNA comet ratio than the blank control group with the doses listed（P＜0.05）,and the comet ratio of Sunset Yellow FCF,Green complex,Tartrazine,Sodium Nitrite is most obvious.3.Chromosome aberration experiment exam DNA injury:After analysis on the chromosome aberration of seven additives,we found that 4 additives（they are Sunset Yellow,Green complex,Tartrazine,and Sodium Nitrite） cause the cells chromosome aberration obviously,there is the statistical significance comparing to the blank group （P＜0.05）,for these 4 additives,in the experiment,the chromosome aberration ratio can increase obviously when the dosage is in the middle（the chromosome aberration ratio is above 10%）,but the chromosome aberration action of the other three coloring matters （Purple complex,Brilliant Blue FCF and Amarante） is not obvious.4.Cell apoptosis and Cycle experiment of additives:In the seven additives,we found that there are only three additives can accelerate the cell apoptosis obviously （Green complex-0.1 g/L,Tartrazine-0.04 g/L and Sodium Nitrite-1.5 g/L）,there is the statistical significance comparing to the blank group（P＜0.05）.The accelerating cell apoptosis action is not very obvious for the other.additives.In the cells cycle experiment, compare the additives to the blank group（except the Green complex）,the proportion of the S phrase for the cells period is enhance,it shows the S phrase retardarce and hint that the food additives have the genotoxicity.For the Green complex,the proportion enhances when it is in the G1 phrase and G2 phrase,but the proportion is decline when it is in S phrase.5.Joint action analysis:Through the SECG experiment,the result shows that there is joint action between the Sodium Nitrite and Tartrazine（P =0.015）.The comet ratio will increase as the additives dosage increase,and it behaves the synergistic joint action.The joint action is not obvious between the Sodium Nitrite and Green Complex（P =0.387）,it seems that it is just a simple addition action.There is also joint action between the Sodium Nitrite and Sunset Yellow FCF（P=0.010） and that behaves the synergistic joint action,too.Conclusions1.All the additives have cells toxicity in a way,and the dosage-react relationship is obvious,the toxicity of the Sunset Yellow FCF,Green complex,Tartrazine is highest.2.Sodium nitrite perform for certain genotoxicity（the comet ratio the chromosome aberration ratio can increase obviously when the concentration is 0.1 g/L,it causes cell apoptosis rate increase obviously when the concentration is 0.15 g/L）,Green complex, Sunset Yellow and Tartrazine all perform for obviously genotoxicity.3.There is joint action between the Sodium Nitrite and Tartrazine,as well as between Sodium Nitrite and Sunset Yellow.Both of them behave synergistic joint action.更多还原