Loss of Glutathione, Ascorbate Recycling, and Free Radical Scavenging in Human Erythrocytes Exposed to Filtered Cigarette Smoke

Abstract

Exposure of human erythrocytes to filtered cigarette smokein vitroinhibited their capacity to reduce dehydroascorbic acid (ascorbate recycling activity). Glucose uptake was not affected, implying that dehydroascorbic acid transport was not inhibited by the smoke treatment. The intracellular reduction of cationic nitroxide free radicals, which provides a measure of ascorbate recycling, was also inhibited by cigarette smoke. A major factor in the inhibition of free radical reduction was glutathione depletion. However, glutathione depletion alone could not account for the inhibition of free radical reduction because a restoration of the glutathione pool in hemolyzed cells only partially restored free radical reduction activity. Another factor inhibiting free radical reduction was a lowering of pH, which was attributed mainly to the uptake of CO2and was reversible by restoring the physiological pH. Exogenous glutathione spared both intracellular glutathione and free radical reduction activity. The rate of depletion of intracellular glutathione was similar to that of extracellular glutathione, indicating that the erythrocyte membrane did not significantly attenuate thiol-reactive species in smoke. Protein thiols were also depleted by cigarette smoke, but to a much lesser extent than was glutathione. Ascorbate was relatively unaffected by cigarette smoke; significant intracellular ascorbate levels remained after glutathione was barely detectable. Autooxidizable reducing agents, capable of reducing both reduced piperidinyl (Tempo) and pyrrolidinyl (Proxyl) nitroxides partitioned from filtered cigarette smoke into aqueous solutions. Attempts to detect cigarette smoke-derived oxidants in buffer solutions or in cell suspensions with a prereduced Tempo nitroxide, whose oxidation properties resemble those of ascorbate, were unsuccessful. The results of this study suggest that chemical modification of glutathione is a major damage mechanism of filtered cigarette smoke, whereas free radical oxidations are relatively insignificant.