Isolation of glutathione S-transferase P-positive hepatocytes from carcinogen treated rats by use of ethacrynic acid as selecting agent.
Rat liver responds to carcinogen treatment with growth of glutathione S-transferase P (GST-P)-positive enzyme-altered foci. In this paper a method is described where GST-P-positive hepatocytes are isolated from carcinogen-treated rats. The method utilizes ethacrynic acid, which is a good substrate for GST-P, and which induces toxicity mainly in GST-P-negative cells. The toxicity results in a loss of attachment to collagen. The method gives a 70% pure population of GST-P-positive cells attached to collagen-coated plates. Use of additional markers supports the conclusion that the GST-P-positive cells were derived from foci. Isolated GST-P-positive hepatocytes spread out and formed primary cultures of normal appearance. It was also shown that they synthesized DNA and did not respond to transforming growth factor beta 1. It is concluded that isolated GST-P-positive hepatocytes can be used for studies on alterations in enzyme-altered foci that cannot be done with in situ immunohistochemistry or in situ hybridization.
Stenius
U
U
Department of Toxicology, National Institute of Occupational Health, Solna, Sweden.
Warholm
M
M
Martens
U
U
Högberg
J
J
D000818:Animals; D002469:Cell Separation; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D004976:Ethacrynic Acid / Q000494:pharmacology*; D005260:Female; D005982:Glutathione Transferase / Q000032:analysis*; D007527:Isoenzymes / Q000032:analysis*; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D005723:gamma-Glutamyltransferase / Q000032:analysis
D007527:Isoenzymes; D016212:Transforming Growth Factor beta; D004247:DNA; D005723:gamma-Glutamyltransferase; D005982:Glutathione Transferase; D004976:Ethacrynic Acid