A MICROASSAY FOR MEASURING CYTOCHROME-P450IA1 AND CYTOCHROME-P450IIB1 ACTIVITIES IN INTACT HUMAN AND RAT HEPATOCYTES CULTURED ON 96-WELL PLATES
A MICROASSAY FOR MEASURING CYTOCHROME-P450IA1 AND CYTOCHROME-P450IIB1 ACTIVITIES IN INTACT HUMAN AND RAT HEPATOCYTES CULTURED ON 96-WELL PLATES
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DOI：
10.1006/abio.1993.1381
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发表时间：
1993-08-15
影响因子：
2.9
通讯作者：
CASTELL, JV
中科院分区：
文献类型：
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作者：
DONATO, MT;GOMEZLECHON, MJ;CASTELL, JV
本研究涉及开发一种灵敏且简单的微测定法，用于测量完整人和大鼠肝细胞中细胞色素 P4501A1 和 P45011B1 的活性。该方法基于在无细胞毒性浓度（8 μM 7-乙氧基-试卤灵和 15 μM 7-戊氧基试卤灵）的适当底物的微孔中培养的细胞的代谢。将探针与细胞孵育后，对形成并释放到培养基中的脱烷基化试卤灵进行定量。为了确保最终形成试卤灵缀合物的水解，将培养物上清液在微孔中与β-葡萄糖醛酸酶和芳基硫酸酯酶一起孵育。然后使用微孔板荧光读数器读取荧光。发现在相同细胞的微粒体部分中通过该方法测量的单加氧酶活性与通过常规方法测量的单加氧酶活性之间存在高度相关性。该方法的主要优点是：（1）所需细胞数量少； (2) 测定时间大幅缩短； (3) 该测定是在完整的细胞中进行的； (4) 由于在测定过程中未检测到细胞损伤，因此可以在几天内对同一细胞单层进行重复测定。该方法被证明对于研究人肝细胞原代培养物中异生素诱导细胞色素 P450 非常方便。
This study deals with the development of a sensitive and simple microassay for measuring cytochrome P4501A1 and P45011B1 activities in intact human and rat hepatocytes. The method is based on the metabolism by cells cultured in microwells of appropriate substrates at noncytotoxic concentrations (8 μM 7-ethoxy-resorufin and 15 μM 7-pentoxyresorufin). After incubation of the probes with the cells, the dealkylated resorufin formed and released into culture medium was quantified. To ensure the hydrolysis of the resorufin conjugates eventually formed, culture supernatants were incubated in the microwells with β-glucuronidase and arylsulfatase. The fluorescence was then read using a microplate fluorescence reader. A high correlation between the monooxygenase activity measured by this procedure and that measured by conventional procedures in the microsomal fraction of the same cells was found. The major advantages of this method are: (1) the small number of cells required; (2) a drastic reduction in assay time; (3) that the assay is performed in intact cells; and (4) the possibility of performing repeated assays with the same cell monolayer over a period of several days since no injury to cells is detectable during the assay. This method proved to be very convenient for studying cytochrome P450 induction by xenobiotics in primary cultures of human hepatocytes.