EFFECTS OF GLUTATHIONE AND PH ON THE OXIDATION OF BIOMARKERS OF CELLULAR OXIDATIVE STRESS

Cellular oxidative stress is associated with such pathological conditi ons as arteriosclerosis, inflammatory diseases and cancer. The oxidati on of the biomarkers 2',7'-dichlorofluorescin (DCFH), 2-deoxyribose, a nd lipid peroxidation are often used to assess the status of oxidative stress in cells and tissues. Since high levels of reduced glutathione (GSH) and acidic conditions have been associated with diminished chem ical lethality, we evaluated the influence of these parameters on the cellular response to oxidative stress. We used a cultured hepatocyte l ine (ch/ch cells) that is susceptible to oxidative toxicity. A hydroxy l radical-generating system consisting of H2O2, ascorbate and iron pro duced a pH-dependent lethality, with complete cell killing at pH 7.4 a nd none at pH 6.8. Lethality correlated with the depletion of intracel lular GSH, and with an increase in DNA fragmentation. The influence of GSH and pH was assessed for DCFH and 2-deoxyribose oxidation, and for lipid peroxidation. The oxidation of DCFH and 2-deoxyribose was inhib ited by GSH, with about 4-fold greater inhibition efficacy at pH 6.8 t han at pH 7.4 [IC50 values (mu M GSH) for pH 6.8 and 7.4, respectively : DCFH = 7 and 30; 2-deoxyribose = 125 and 490]. GSH did not affect li pid peroxidation at either pH, even at a high intracellular concentrat ion of 10 mM. We conclude: 1) GSH is not inhibiting DCFH and 2-deoxyri bose oxidation by simply quenching reactive oxygen (hydroxyl radical o r perferryl oxygen), since GSH did not inhibit lipid peroxidation; 2) the protonated form GSH is more likely to be the inhibitory species ra ther than GS(-), since even in the simple cell-free systems lower pH i nhibited biomarker oxidation; and; 3) hydroxyl radical may not be the primary intracellular oxidant of DCFH, since intracellular GSH concent rations are typically 10- to 100-fold higher than the IC50 values for GSH inhibiting reactive oxygen-mediated DCFH oxidation.