THE MEASUREMENT OF BIOREDUCTIVE CAPACITY OF TUMOR-CELLS USING METHYLENE-BLUE

Purpose: Methylene blue (MB) can be used as an intracellular electron acceptor. The purpose of this study was to demonstrate the usefulness of MB for the determination of total bioreductive capacity of cell sus pensions. Methods and Materials: We measured oxygen consumption by Cla rk electrode and pentose cycle activity by release of (CO2)-C-14 from 1-C-14-glucose. Results: Methylene blue catalyzes the reaction of intr acellular reductants NADPH, NADH, and reduced glutathione (GSH) with o xygen, causing the production of hydrogen peroxide. The reaction rate correlates with the negative charge of molecule (NADPH(-4) > NADH(-2) > GSH(-1)), suggesting that reaction with positively charged oxidized MB is the limiting step of the reaction. In a cellular system MB cause s the electron flow from cellular endogenous substrates to oxygen. It is activated by the disruption of the NADP(+)/NADPH ratio due to sever al processes. These are direct oxidation of NADPH and GSH, the GSH per oxidase catalyzed reaction of GSH with H2O2, followed by NADPH oxidati on by oxidized glutathione (GSSG). This results in increased cellular oxygen consumption and stimulation of the oxidative limb of pentose cy cle (PC) in the presence of MB. The cellular effect of MB differs from other electron accepting drugs. Diamide and tert-butylhydroperoxide a ct as direct oxidants, while MB is an electron carrier to oxygen. Acco rdingly, MB shows the highest effect on PC activation and oxygen consu mption. Conclusions: Our results indicate that MB may be used for the determination of the total bioreductive capacity of the cells, measure d by oxygen consumption and PC activation. (C) 1998 Elsevier Science I nc.