Resistance against ethacrynic acid in glutathione transferase 7-7 (GST-P)-positive hepatocytes isolated from carcinogen-treated rats: the role of cytoskeletal changes and ATP depletion.
Ethacrynic acid (Ea) is a substrate for glutathione transferase 7-7 (GST-P) in rat. Toxic effects of Ea have been related to its metabolism and GSH depletion, but resistance conferred by GSTP1-1 (the human homologue) has also been reported. Hepatocytes from enzyme altered foci (EAF) express GST-P, and a model for selection of resistant EAF cells has been developed using Ea as a toxic agent. In the present study the effects of Ea in this model have been characterized. Hepatocytes from foci-bearing rats were isolated. Isolated cells were exposed to Ea for 1-4 hr in suspension. They were then allowed to attach to collagen-coated plates in a serum-containing medium. Preferentially GST-P-positive cells attached after Ea treatment, thus increasing the number of positive cells per attached cells (GST-P-%). Extracellular GSH, as well as alpha-tocopherol, did not influence the Ea effect. However, the effect of Ea was counteracted by inhibitors of glutathione transferase activity. Taxol, a microtubule stabilizing agent, also counteracted the effect of Ea on GST-P-%. 1,2-Dichloro-4-nitrobenzene (DCNB, 0.4 mM), which is a substrate for other glutathione transferase isoenzymes than GST-P, also increased the GST-P-%. However, the effect of DCNB was not inhibited by taxol. It was also found that Ea induced a drop in ATP levels, but this effect, as well as cell leakage, came later than the loss of attachment. The data suggest that the critical effect of Ea was cytoskeletal changes, and that GST-P conferred resistance by detoxification of Ea.
Warholm
M
M
Department of Toxicology, National Institute of Occupational Health, S-171 84 Solna, Sweden.
Stenius
U
U
Ståhl
A
A
Högberg
J
J