环境化学与生态毒理学国家重点实验室知识库

二恶英类化合物是首批列入斯德哥尔摩公约受控的持久性有机污染物 
(Persistent organic pollutants, POPs)，其 排放监管与环境风险评估不仅关系公共卫生健康，也关系国家的履约任务。快速有效的检测方法可以为二恶英源头控 制、污染筛查以及高暴露风险样品的评估提供有效的技术支撑。基于芳香烃受体(Aryl Hydrocarbon Receptor AhR)荧光素酶 报告基因 的 生物检测技术 能够实现 快速 、有效的二恶英定量检测并且具有健康风险评估的潜力 目前 已被多个国家和地区列为 二恶英 标准 筛查 方法 。由 于我国缺乏具有自主知识产权的该类技术，因此在推行与使用二恶英生物检测时受到阻力，为此实验室自主构建了新型荧光素酶报告基因检测质粒并获得了稳定转染该质粒的小鼠肝癌检测细胞株 CBG2.8D。本研究基于该细胞株，建立了二恶英新型生物检测系统，制定 标准化操作流程，稳定系统各项参数，并在此基础上开展该生物 系统在飞灰和大气两种环境样品以及牛奶样品中的适用性研究，针对不同样品提出实际检测策略及方案，为报告基因生物检测法的实际应用提供可靠保证。同时 提出了 总 AhR激活效应 (Total AhR Activation, TAA)的概念 并 使用 TAA评估大气样品粗提物对 AhR激活的效率，在大气样品中粗提物的 TAA显著高于二恶英类化合物对芳香烃受体的激活效率，而 TAA与样品中多环芳烃的毒性当量之间具有相关性。因此使用该系统不仅能够实现二恶英的定量检测，还能从 一定程度上 实现对 环境样品 的毒性效应 评估 。

本研究所获得的结果总结如下：

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
CBG2.8D细胞 对系列浓度 (1.5×10-13-1×10-9 mol/L)标准物质 响应 的稳定性， 多 次独立检测数据显示不同 时期 的细胞对标准物质 响应的 变异系数均在 30%以内，其中浓度范围在 4.5×10-13-3.7×10-11mol/L的标 准曲 线，稳定性更好，各浓度点的变异系数均在 20%以内 。
二、基于标准化检测流程，该系统体现了良好的适用性，在二恶英含量普遍较高的飞灰样品中，其生物检测结果与仪器分析结果相关性R2=0.99。 通过对比
两种检测方法得到的数据 生物检测结果相比于仪器 分析 结果数据偏差基本在
0.5-2倍 的动态变化区间之内 。 根据该动态检测区间，我们比较了浓度在飞灰填
埋限量值 0.5-2倍之间 的 临界样品 生物检测法 与仪器分析法 之间的相关性，结果显示两种检测方法相关性 R2=0.97，且 生物 检测结果 无漏检 现象 。随机抽检飞灰样品 进行重复性检验， 18个样品 3次独立 检测结果的 变异系数均在 20%以内。此外， 我们根据飞灰样品的特点在实际操作过程中增加了样品稀释的标准操作，并开发了操作简便的配套计算软件。

三、 基于系统良好的稳定性与重复性，该系统在二恶英含量较低的大气样品
中同样展示出 与仪器分析 结果之间良好的 相关 性 R2=0.97）。 在 AhR激活效率探索应用中，针对 大气 雾霾 样品 ，分别进行了样本 粗提 与二恶英组分净化提取，然后进行 TAA的检测 。 结果显示 粗提物 的 TAA显著高于二恶英组分对 AhR的激活效应， 结合仪器分析结果，我们将 TAA与颗粒物中含量最高的多环 芳烃类化合物毒性当量进行 比较，发现 TAA与 多环芳烃毒性当量 总和之间具有 相关 性R2=0.90）。
四、在食品样品（奶制品）的生物检测应用研究方面，通过制作标准质控奶样，检验系统稳定性。质控奶样浓度接近欧盟限量值，质控奶样共进行 24次独立 检测 变异系数为 17.53%，系统呈现出良好的稳定性 说明 该生物检测系统具备 应用于 奶制品二恶英 检测 的潜力。
      综上，本研究基于高灵敏检测细胞株 CBG2.8D，建立了二恶英生物检测系统，从标准化操作出发，探索该检测系统在不同介质样品中的应用能力，结 合不
同样品的特点，提供相 应的检测策略；首次提出 TAA概念，并通过 TAA检测发现大气样品粗提物中 TAA与多环芳烃类化合物毒性当量间具有相关性。

      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%.
2. Based on the standardized detection process, the system shows a good application, in fly ash samples with high dioxin content, the correlation coefficient between bioassay and chemical analysis R2 was 0.99. Samples with the concentration in the range of 0.5-2 times of the landfill limit showed a superb correlation between bioassay and chemical analysis, R2=0.97, no false positive detection. 18 random selected samples with triplicated tests to show the repeatability, the variation coefficients of samples were all within 20%. According to the characteristics of fly ash samples, the dilution strategy was formulated and the corresponding calculation software was developed.

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.
4. In the application of food samples (milk samples), The stability of bioassay system was tested by making standard quality milk samples. The concentration of quality-controlled milk samples was close to the EU limit, 24 independent tests were carried out on quality-controlled milk samples with a CV of 17.53% and the results within 23 experiments showed the coefficient variation were within 30%, which suggested the repeatability of the detection system and the potential application value for dioxin screening in milk samples.
      Above all, this study was focus on the development and application of a rapid and highly sensitive bioassay detection system based on the CBG2.8D cell line, the application of this bioassay system from high concentration environmental samples to low concentration food samples was explored, and the corresponding detection strategies were provided according to the characteristics of different samples. TAA was put forward in this study and the correlation between TAA and toxicity equivalent of PAHs in crude extracts of atmospheric samples was found by TAA detection.