THE COUMARIN 7-HYDROXYLATION MICROASSAY IN LIVING HEPATIC CELLS IN CULTURE

Coumarin 7-hydroxylation was evaluated in hepatic cells from various s pecies, cultured in 96-well plates. This microassay involved incubatin g living cultured cells with the substrate, followed by fluorimetric q uantification of the product released! into the culture supernatant, a fter hydrolysis of the conjugates of 7-hydroxycoumarin that were forme d. Fluorescence was measured directly in the wells by using a micropla te fluorescence reader. The major advantages of this technique are its simplicity and automation. the small number of cells required, the re duction in sample handling and assay time, and the possibility of perf orming repeated assays with the same cell monolayer, since no injury t o cells is detectable during the assay. By using this microassay, it w as shown that human hepatocytes hydroxylated coumarin at higher rates than did rabbit, dog or rat hepatocytes, and that no appreciable metab olic activity was observed in hepatoma cells (Hep G2 and FaO). In addi tion, methoxsalen was found to be a potent inhibitor of cytochrome P45 02A6 activity in living human hepatocytes.