EFFECTS OF CHRONIC ETHACRYNIC-ACID EXPOSURE ON GLUTATHIONE CONJUGATION AND MRP EXPRESSION IN HUMAN COLON-TUMOR CELLS

Chronic exposure to ethacrynic acid of a subcloned HT29 human cion can cer cell line produces a 3- to 4-fold increase in the level of resista nce to this agent. The resistant cells (HT6-8) have an enhanced capaci ty to metabolize the parent drug and efflux it from the cell. This is reflected in a 5-fold enhanced decompositioning rate constant for etha crynic acid in HT6-8 (3.47 x 10(-3) min(-1)) versus wild type cells (1 .58 x 10(-2) min(-1)). We observed that the glutathione conjugate of e thacrynic acid is an effective competitive inhibitor for binding to th e multidrug resistance-associated protein by [S-35]azidophenacyl-gluta thione, a photoaffinity analog of glutathione. In addition, the HT6-8 cells overexpressed multidrug resistance-associated transcript 2- to 3 -fold. These results suggest that resistance to ethacrynic acid result s from a concerted, coordinate increase in defense mechanisms which de toxify the drug and remove its conjugate via plasma membrane efflux. ( C) 1996 Academic Press, Inc.