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Influence of Glutathione S-Transferases on Cellular Glutathione Determination by Flow Cytometry Using Monochlorobimane¹

Departments of Human Oncology [G. A. U., R. T. M.] and Pediatrics and Physiological Chemistry [J. A. J., F. L. S.], University of Wisconsin-Madison, Madison, Wisconsin 53792

Intracellular glutathione (GSH) levels for seven mammalian cell lines (four human tumors, two rodent, one monkey) were determined by flow cytometry following staining with monochlorobimane (MBCl), and the results were compared with GSH levels measured by the Tietze assay. The mean fluorescence intensity for all but the two rodent lines did not correlate with GSH levels determined biochemically. Good agreement between the two assays was observed for the rodent lines following depletion of GSH by buthionine sulfoximine, but the level of GSH depletion achieved in the human and monkey lines was always underestimated by MBCl/flow cytometry. These discrepancies were not resolved by increasing stain concentration or staining time. Total glutathione S-transferase (GST) activity and GST isozyme profiles were determined for each of the cell lines. Western analysis with antibodies raised against rat Y_a, Y_b1, and Y_c and human isozymes revealed that the rodent cell lines expressed abundant (Y_a, Y_c subunits) and µ (Y_b1 subunits) class isozymes. In contrast, GST- was the predominant isozyme detected in the human tumor cell lines and Cos-7 monkey cells. Michaelis-Menten analysis with purified GSTs from rat liver as well as purified human placental () GST revealed that the conjugation of MBCl and GSH catalyzed by the (1-1 and 2-2) and µ (3-3 and 3–4) class GST isozymes was approximately 10 and 80 times more efficient than was conjugation by the GST form, respectively. These data indicate that the GST-catalyzed conjugation of GSH and MBCl is isozyme dependent and that MBCl is a relatively poor substrate for the isozyme. As a consequence of this isozyme rate differential, the MBCl/flow cytometry technique for GSH quantitation must be applied cautiously, particularly with human tumor cells, many of which have been shown to have high GST- activity. Application to other cell types should also be made after careful characterization of GSH levels and GST isozyme composition and only after comparison with other independent assays of GSH concentration.

¹ Supported by NIH Grant CA42325 and Grants NS24969 and GM38497.

² To whom requests for reprints should be addressed, at Department of Human Oncology, K4/338, 600 Highland Avenue, Madison, WI 53792.

Received 10/24/90. Accepted 1/23/91.

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