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Glutathione and Related Enzymes in Rat Brain Tumor Cell Resistance to 1,3-Bis(2-chloroethyl)-1-nitrosourea and Nitrogen Mustard¹

Department of Biomedical and Environmental Health Sciences, School of Public Health, University of California, Berkeley, California 94720 [C. G. E., P. D-S., M. T. S.]; and Brain Tumor Research Center, University of California, San Francisco, California 94143 [W. J. B., K. T.]

Reduced glutathione (GSH) and activities of several glutathione-related enzymes were measured in two 9L rat brain tumor cell lines with differing sensitivities to both 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and nitrogen mustard. GSH, measured by a specific high-performance liquid chromatographic method, was found to be approximately twice as high in 9L cells sensitive to BCNU but resistant to nitrogen mustard. The nitrogen mustard resistant cell line was also found to have 2.5-fold more bulk glutathione transferase activity and approximately 3-fold more -glutamyl transpeptidase activity. Glutathione reductase activity, protein thiol, and total protein content were similar in the two cell lines.

Pretreatment of 9L cells with 50 µM buthionine sulfoximine for 24 h to deplete GSH only slightly potentiated BCNU cytotoxicity in a clonogenic assay whereas that of nitrogen mustard was markedly potentiated in both cell lines. Similarly, buthionine sulfoximine pretreatment had little effect on the induction of sister chromatid exchanges by BCNU, but significantly increased the number of sister chromatid exchanges induced by nitrogen mustard in both cell lines. Depleting GSH also had no significant effect on the cytotoxicity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea to 9L cells.

Pretreatment of 9L cells with 1 mM GSH significantly protected against nitrogen mustard cytotoxicity. Moreover, nitrogen mustard incubated with GSH and glutathione transferase was 4-fold less cytotoxic than nitrogen mustard incubated with GSH alone. Incubation of BCNU with GSH alone or with glutathione transferase had no effect on BCNU cytotoxicity. These results indicate that elevated GSH and glutathione transferase activity is one mechanism of cellular resistance to nitrogen mustard in the 9L cell line, but it does not correlate with resistance to BCNU or other clinically important nitrosoureas.

¹ This research was supported by Grant CA 13525 from the National Cancer Institute.

² To whom requests for reprints should be addressed, at the University of California, School of Public Health, 322 Warren Hall, Berkeley, CA 94720.

Received 10/15/86. Revised 1/27/87. Accepted 2/16/87.

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