基于化学计量学的合成食品添加剂体外DNA靶向作用检测技术及其作用机制研究

The abuse of the chemical synthesis food additives is one of the important risk factors to food safety. Studies show that the excessive use of the synthetic food additives can cause acute or chronic toxicity and even induce cancer, and the mutagenicity of the food additives is closely related to their DNA targeted interaction. However, not only the studies on the interaction mechanism between the synthetic food additives and DNA are very lack, but also the detection methods of their interaction are also serious lag. This project intends to construct a new technique, which uses calf thymus DNA as a target and adopts the strategy combined the multivariate curve resolution-alternating least squares chemometrics algorithm with fluorescence and UV-vis absorption spectroscopic methods. This technique can be applied to determine the interacton of the chemical synthesis food additives with DNA. Predict,confirm and elucidate the binding modes and binding sites of the synthetic food additives with DNA as well as the molecule mechanism of damage DNA with the use of various methods including circular dichroism, Fourier transform infrared spectroscopy, molecular modeling and so on. Clarify the structure characteristics of the stabilized DNA-food additive complexes, and determine the binding parameters between the synthetic food additives and DNA. Moreover,the correlation and regularity between the mutagenicity of the synthetic food additives and the binding sites on DNA and the structural damage of DNA will be explored. The implementation of this project has important significance for further understanding toxicity mechanism of the synthetic food additives with potential mutagenicity along with exploring innovative and effective detection technique for the interaction of DNA with the synthetic food additives.

滥用化学合成食品添加剂是危害食品安全的重要因素之一。研究表明，过量使用合成食品添加剂会引起急性或慢性毒性甚至诱发癌变，且此诱变性与食品添加剂作用于DNA密切相关。然而目前针对二者相互作用机制的研究还非常缺乏，相互作用的检测技术也严重滞后。本项目拟以小牛胸腺DNA为靶点，构建化学计量学模型多元曲线分辨-交替最小二乘法与荧光和紫外光谱法联用新技术，应用于检测BHA等合成食品添加剂与DNA的相互作用；运用圆二色谱、红外光谱和分子模拟等手段，预测、验证并阐明合成食品添加剂的体外DNA靶向作用模式、作用位点及其损伤DNA的分子机制，探明稳定复合物的结构特征，测定相互作用的结合参数；探索合成食品添加剂的诱变性与其作用DNA的位点和引起DNA损伤之间的相互关系，揭示其可能的规律性。本项目的实施对于深入认识潜在诱变性合成食品添加剂的毒性机理和探寻新颖、有效的DNA靶向作用检测技术具有重要的理论和实践意义。

食品安全问题已成为我国突出的社会问题之一，在诸多的食品安全隐患中，食品加工过程中滥用化学合成食品添加剂是重要的因素之一。研究表明，过量使用合成食品添加剂会引起急性或慢性毒性甚至诱发癌变，且此诱变性与食品添加剂结合DNA密切相关，然而目前针对二者相互作用机制的研究还非常缺乏，检测技术也严重滞后。为此，本项目以体外小牛胸腺DNA为靶分子，通过构建化学计量学模型多元曲线分辨-交替最小二乘法与荧光和紫外光谱法联用新技术，并结合圆二色谱、红外光谱和分子模拟等手段，研究了食品抗氧化剂丁基羟基茴香醚和2-叔丁基对甲苯酚、食品着色剂赤藓红、甜味剂糖精钠、食品增香剂香兰素和乙基香兰素、面粉增白剂过氧化苯甲酰及食品保鲜剂2, 4–二氯苯氧乙酸等合成食品添加剂与DNA的相互作用机制。获得了丁基羟基茴香醚等食品添加剂与DNA反应过程中各组分的浓度变化及其光谱曲线，从而确证了食品添加剂与DNA的相互作用并对其反应进程进行定量监测。研究发现，丁基羟基茴香醚、2-叔丁基对甲苯酚、特丁基对苯二酚、赤藓红、过氧化苯甲酰等主要通过嵌插模式与DNA结合，而糖精钠、香兰素、乙基香兰素、麦芽酚、2,4–二氯苯氧乙酸等则以沟槽结合方式与DNA发生作用，其结合常数在103 ~ 104数量级。同时测定了上述食品添加剂与DNA作用的热力学参数、主要作用力、作用位点以及DNA结构的变化。结果表明，这些食品添加剂能够与ctDNA的不同碱基发生作用并在一定程度上影响DNA的结构，推测这可能是其中一些合成食品添加剂具有潜在诱变性的原因之一。本项目的研究结果为从分子水平上认识合成食品添加剂与DNA的相互作用机制及其毒性机理提供了理论依据，本研究建立的方法新颖、普适，为合成食品添加剂仍至其他小分子化合物与DNA相互作用研究提供了新思路和新策略。