|Alternative Title||Application of a novel bioassay system for dioxin determination and total toxicity assessment|
|Place of Conferral||北京|
|Keyword||二恶英，生物检测，芳香烃受体，荧光素酶报告基因，标准化 Dioxin, Bioassay, Aryl Hydrocarbon Receptor, Luciferase Reporter Gene, Standardization|
一、基于CBG2.8D细胞株，在优化多项细胞检测操作参数的基础上，建立二恶英检测系统，形成标准化生物检测操作流程。通过背景值， EC50值 ，激活倍数等参数 质控 细胞状态，形成了 稳定检测系统 的管理方案 。同时， 我们分析了
Dioxin is a group of typical persistent organic pollutants (POPs) which were in the first list of the Stockholm convention. The emission regulation on dioxins is more than a duty of our country, but also an important issue to the public health. Rapid and effective detection methods can provide effective technical support for dioxin source control, screening and assessment of high exposure risk samples. Aryl hydrocarbon receptor (AhR) based luciferase reporter gene bioassay technology can be used as a rapid and effective method for dioxin determination and this method has been listed as one of the standard detection methods in many countries. Due to the lack of such bioassay detection system with proprietary intellectual property rights, there are many limitations on the implement and application of bioassay detection system in China. Our team developed a highly sensitive mouse hepatic cell line, CBG2.8D which was stable transfected with luciferase reporter gene in previous work. In this study, a bioassay detection system based on the CBG2.8D has been designed and optimized, together with the standardized operation procedures and stabilized the various parameters of the system has been established. Furthermore, application of this system to detect dioxins in a different kind of samples, including fly ash samples, ambient air samples and food samples was studied. Practical detection strategies and schemes for the different samples providing reliable guarantee for the practical application of the bioassay detection system has been put forward. Nevertheless, the term of total activation of aryl hydrocarbon receptor (TAA) was proposed in this study to assess the potential AhR activation effects of atmospheric samples. Polycyclic aromatic hydrocarbons (PAHs) have been found as a dominant contribution of total AhR activation in air samples and the content of PAHs is correlated with TAA. Therefore, the system can be used to not only detect dioxins, but also evaluate the toxicity of environmental samples.
The major findings in this study are as follows:
1. Based on CBG2.8D cell line, a dioxin bioassay system was established by optimizing a series of cell detection parameters and a standardized operation process was output. Through the background value, EC50 value, activation factor and other parameters to control the cell state, a stable detection system management program was formed. By accumulating the standard curves and analyzing the stability of the standard curves, the coefficient variation of each point of the standard curves were all within 30%, the CV of the points in the range from 4.5×10-13 to 3.7×10-11 mol/L of 2,3,7,8-TCDD were all less than 20%.
3. Based on the stability and repeatability，the detection values obtained from bioassay system show a good correlation with instrumental analysis in ambient-air samples which R2 = 0.97. In this study, haze ambient-air samples were collected, and the AhR activation efficiency was detected after crude extraction and dioxin purified extraction separately. According to the results of the bioassay system, the TAA in the crude extracts was considerably higher than the BEQ of dioxin-rich fractions. Notably, the haze samples contained abundant amounts of PAHs whose relative toxicity equivalent was correlated with the TAA (R2=0.9); this finding suggests that PAHs critically contribute to the AhR-related biological impacts of haze ambient-air samples.
|张嵩岩. 二恶英新型生物检测系统的应用及总毒性评估的拓展研究[D]. 北京. 中国科学院生态环境研究中心,2018.|
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