Glutathione dependence of neocarzinostatin cytotoxicity and mutagenicity in Chinese hamster V-79 cells.
Neocarzinostatin (NCS) is mutagenic in bacteria, yeast, fungi, and mammalian cells. In cell-free systems, DNA strand breakage induced by NCS requires a reducing agent like 2-mercaptoethanol, unless very high (greater than 100 micrograms/ml) concentrations of NCS are used. In this study, we have investigated the role of the sulfhydryl compound glutathione (GSH), which is usually the most common intracellular thiol, in the bioactivation of NCS to a toxic and mutagenic species. Chinese hamster V79 cells were pretreated with one of two GSH depleting agents, buthionine sulfoximine or diethyl maleate. These agents deplete GSH via different mechanisms, but both will lower GSH levels within the cell to less than 5% of control (untreated) values. GSH-depleted cells and control cells were then exposed to NCS concentrations of 0.5-2.5 micrograms/ml for 1 h, assayed for survival, and plated for expression of hypoxanthine-guanine phosphoribosyltransferase-negative (HGPRT-) mutants. After an expression period of 7 days, during which the cultures were subcultured twice, HGPRT- mutants were selected by plating in hypoxanthine-free medium containing 5 micrograms of 6-thioguanine per ml, at a density of 2 X 10(5) cells per 100 mm dish. NCS alone decreased the surviving fraction to about 1% at 2.5 micrograms/ml and produced dose-related increases in HGPRT-mutants that reached greater than 10 times the spontaneous mutation frequency at 2.5 micrograms NCS per ml. In GSH-depleted cells, however, NCS was only mildly cytotoxic (60-80% surviving fraction) and did not produce dose-related increases in HGPRT- mutants over cells treated only with diethyl maleate or buthionine sulfoximine. Thus, GSH appears to be the main reducing agent for the bioactivation of NCS to a toxic and mutagenic species in Chinese hamster V79 cells.
DeGraff
W G
WG
Mitchell
J B
JB
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000633:toxicity*; D001711:Biotransformation; D019328:Buthionine Sulfoximine; D006224:Cricetinae; D003412:Cricetulus; D005978:Glutathione / Q000502:physiology*; D008298:Maleates / Q000494:pharmacology; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000494:pharmacology; D009153:Mutagens; D009353:Zinostatin / Q000378:metabolism / Q000633:toxicity*
D000903:Antibiotics, Antineoplastic; D008298:Maleates; D009153:Mutagens; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; D009353:Zinostatin; C014476:diethyl maleate; D005978:Glutathione