BIOREDUCTIVE METABOLISM OF SR-4233 (WIN-59075) BY WHOLE-CELL SUSPENSIONS UNDER AEROBIC AND HYPOXIC CONDITIONS - ROLE OF THE PENTOSE CYCLE AND IMPLICATIONS FOR THE MECHANISM OF CYTOTOXICITY OBSERVED IN AIR

Purpose: Measurement of pentose cycle (PC) activity is shown to be a noninvasive means for monitoring the reduction of SR-4233 in whole cells. Comparing these measurements to the actual measurements of drug loss under aerobic and hypoxic conditions helps to define the mechanism for the associated aerobic toxicity. Methods and Materials: SR-4233 is activated to a toxic species by bioreductive metabolism. NADPH is required for the activation of the drug by purified enzymes, cell homogenates and whole cells. In viva the NADPH:NADP+ ratio is maintained by the oxidation of glucose via the oxidative limb of the pentose cycle. By measuring radiolabeled (CO2)-C-14 released as a product of this oxidation one can get an accurate measurement of the rate of drug metabolism in whole cells. These results are compared to measurements of drug consumption under aerobic and hypoxic conditions using an HPLC assay. Results: SR-4233 stimulates pentose cycle activity to a greater extent in air then under hypoxia, however, in the presence of added catalase, pentose cycle activity is stimulated to a similar extent under both conditions. The higher levels of PC activity observed in air are due to the production of hydrogen peroxide by the nitroxide free radical undergoing futile redox cycling. The contribution of H2O2 to the observed aerobic cytotoxicity of SR-4233 is minimal however, since toxicity is only slightly reduced in the presence of exogenous catalase and antioxidants such as vitamin E. The level of PC stimulation by SR-4233 suggests that the rate of electron addition to the drug is independent of O-2 concentration. The loss of drug from the incubation medium, i.e., conversion to a stable intermediate species, occurs approximately five times faster under nitrogen than in air for A549 cells. It is the rate of drug loss from the cell and not the rate of reduction which best correlates with the observed aerobic and hypoxic toxicity. Conclusion: Toxicity in air and in nitrogen is directly related to the rate of drug reduction, i.e., at equivalent levels of drug loss we observe equal levels of cytotoxicity.