基于血清促进的鸡肝微粒体离体代谢氯化石蜡方法及其清除规律

A method and clearance pattern for in vitro metabolism of chlorinated paraffin in chicken liver microsomes based on serum promotion

研究氯化石蜡(CPs)在动物体内代谢清除规律是探讨其生物富集机制的重要依据.肝微粒体离体代谢目标化合物是模拟其在动物体内代谢的重要手段.通过优化鸡肝微粒体离体代谢CPs实验方法,采用血清作为添加剂,建立一种高效的鸡肝微粒体离体代谢CPs方法.该方法使得溶解底物的有机溶剂体积高达反应总体积的3%,血清加入量为底物溶液体积的1—10倍;反应终止后,无需提取步骤,方法简洁,实现样品的快速处理.方法成功应用于鸡肝微粒体离体代谢CPs清除规律的研究.研究显示,在无血清条件下,鸡肝微粒体孵育底物(0.2μg·mL^(−1))90 min时,短链氯化石蜡(SCCPs)分子式同系物的代谢清除率均低于20%;然而,加入少量胎牛血清作为底物的载体后,相同孵育时间内,SCCPs分子式同系物的代谢清除率得到显著提高,C_(11)Cl_(6-8)的代谢清除率甚至超过80%;鸡肝微粒体对CPs的代谢清除率,随着底物浓度、CPs碳链长度以及氯原子数的增加而降低.本研究为动物肝微粒体离体代谢CPs研究提供一种高效的方法,同时也为其他疏水性毒害有机物的肝微粒体离体代谢研究提供一种新的方法参考.

It is an important basis for exploring their bioaccumulation mechanisms to study the metabolic clearance patterns of chlorinated paraffins(CPs)in animals.In vitro metabolism of target compounds by liver microsomes is an important means to simulate its metabolism in animals.By optimizing the method of CPs metabolism by chicken liver microsome in vitro,using serum as an additive agent,a high efficient method of CPs metabolism by chicken liver microsome in vitro was established.In this method,the volume of organic solvent dissolving the substrate could be as high as 3%of the total reaction volume,and the amount of serum added was one to ten times of the volume of substrate solution.After the termination of the reaction,there was no need to extract steps,and the method was simple,so that the rapid processing of samples was realized.The method was successfully applied to the study of CPs clearance by chicken liver microsomes in vitro.The results showed that under serum-free conditions,the metabolic clearance rates of all short chain chlorinated paraffins(SCCPs)with molecular formula homologues were lower than 20%when chicken liver microsomes were incubated with substrates for 90 min.However,after adding a small amount of serum as the carrier of substrate,the metabolic clearance ratio of SCCPs molecular formula homologues was significantly improved within the same incubation time,and the metabolic clearance ratio of C_(11)Cl_(6-8) was even more than 80%.This is the first time to reveal the metabolic clearance of CPs by liver microsomes in vitro:The metabolic clearance of CPs by chicken liver microsomes decreased with the increase of substrate concentration,CPs carbon chain length and their number of chlorine atoms.This study will provide an efficient method for the study of CPs metabolism by animal liver microsomes in vitro.Meanwhile,it will also provides a new method reference for the study of liver microsome metabolism of other hydrophobic compounds in vitro.