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Glutathione Dependence of Neocarzinostatin Cytotoxicity and Mutagenicity in Chinese Hamster V-79 Cells

Radiation Biology Section, Radiation Oncology Branch, Clinical Oncology Program, Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, Maryland 20205

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 (>100 µg/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 <5% of control (untreated) values. GSH-depleted cells and control cells were then exposed to NCS concentrations of 0.5–2.5 µg/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 µg of 6-thioguanine per ml, at a density of 2 x 10⁵ cells per 100 mm dish. NCS alone decreased the surviving fraction to about 1% at 2.5 µg/ml and produced dose-related increases in HGPRT^- mutants that reached >10 times the spontaneous mutation frequency at 2.5 µg 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.

¹ To whom requests for reprints should be addressed.

Received 4/24/85. Revised 7/ 3/85. Accepted 7/ 8/85.

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