SENSITIVE ENZYMATIC CYCLING ASSAY FOR GLUTATHIONE - MEASUREMENTS OF GLUTATHIONE CONTENT AND ITS MODULATION BY BUTHIONINE SULFOXIMINE IN-VIVO AND IN-VITRO IN HUMAN COLON-CANCER

We have measured glutathione content in small tissue samples derived f rom biopsies of primary and metastatic human colon tumors and from col on cancer cell lines in tissue culture and xenografts in athymic mice. Measurements were performed using an enzymatic cycling assay designed to quantitate extremely low levels of glutathione (GSH) (down to 10(- 14) mol) from perchlorate extracts of tissue samples weighing less tha n 1 mg wet weight. Glutathione was stable in these acid extracts for a t least 6 months when stored at -80 degrees C. A survey of normal tiss ues in mice, rats, and some human tissues showed considerable variatio n in GSH content of different tissues but generally similar levels wer e identifiable for the same tissues from different species. The highes t GSH level was 56.9 nmol/mg protein in rat liver and the lowest was 1 .8 nmol/mg protein in rat skeletal muscle. High GSH levels were also d etermined in mouse and human liver, while low GSH levels were detected in mouse muscle. Human colon cancer cell lines showed slightly higher GSH levels than did colon cancer tumor samples obtained from biopsies . These studies revealed a marked interindividual difference in tumor GSH content, as well as a difference in GSH content between tumor depo sits at different metastatic sites in the same individual. These resul ts indicate the importance of direct tumor measurements of GSH content in clinical trials designed to modulate tumor glutathione content to try to increase sensitivity to chemotherapy or radiation therapy. Buth ionine sulfoximine, an inhibitor of gamma-glutamyl cysteine synthetase , was shown to produce almost complete depletion of GSH in four differ ent human colon cancer cell lines in 24 h. Buthionine sulfoximine was also shown to be capable of producing drastic depletion of GSH in huma n colon cancer grown as xenografts in athymic animals.