THE ANTIOXIDANT TROLOX ENHANCES THE OXIDATION OF 2',7'-DICHLOROFLUORESCIN TO 2',7'-DICHLOROFLUORESCEIN

The use of antioxidants to prevent intracellular free radical damage i s an area currently attracting considerable research interest. The com pound 2',7'-dichlorofluorescin diacetate (DCFH-DA) is a probe for intr acellular peroxide formation commonly used in such studies. During our studies we unexpectedly found that incubation of Trolox, a water solu ble vitamin E analog, with DCFH-DA in cell-free physiological buffers resulted in the deacetylation and oxidation of DCFH-DA to form the flu orescent compound, 2',7'-dichlorofluororescein (DCF). The reaction was time-, temperature-, and pH-dependent. Fluorescence intensity increas ed with an increase in either Trolox or DCFH-DA concentration. These r esults indicate that even at physiological pH, DCFH-DA can be deacetyl ated to form 2',7'-dichlorofluorescin (DCFH). DCFH can then be oxidize d to DCF by abstraction of a hydrogen atom by the phenoxyl radical of Trolox. Exposure of the reaction mixture to 10 Gy of Co-60 gamma radia tion greatly increased production of DCF. Antioxidant compounds report ed to ''repair'' the Trolox phenoxyl radical (e.g., ascorbic acid, sal icylate) can also prevent the Trolox-induced DCFH-DA fluorescence. How ever, compounds that cannot repair the Trolox phenoxyl radical (e.g., catechin) or can themselves form a radical (e.g., uric acid, TEMPOL) e ither have no effect or can increase levels of DCF. These results demo nstrate that experimental design must be carefully considered when usi ng DCFH-DA to measure peroxide formation in combination with certain a ntioxidants.