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Alternatives to Laboratory Animals - ATLA

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The Coumarin 7-Hydroxylation Microassay in Living Hepatic Cells in Culture


M. Teresa Donato, José V. Castell and Maria José Gómez-Lechón

Coumarin 7-hydroxylation was evaluated in hepatic cells from various species, cultured in 96-well plates. This microassay involved incubating living cultured cells with the substrate, followed by fluorimetric quantification of the product released into the culture supernatant, after hydrolysis of the conjugates of 7-hydroxycoumarin that were formed. Fluorescence was measured directly in the wells by using a microplate fluorescence reader. The major advantages of this technique are its simplicity and automation, the small number of cells required, the reduction in sample handling and assay time, and the possibility of performing repeated assays with the same cell monolayer, since no injury to cells is detectable during the assay. By using this microassay, it was shown that human hepatocytes hydroxylated coumarin at higher rates than did rabbit, dog or rat hepatocytes, and that no appreciable metabolic activity was observed in hepatoma cells (Hep G2 and FaO). In addition, methoxsalen was found to be a potent inhibitor of cytochrome P4502A6 activity in living human hepatocytes.