Terephthalic acid: a dosimeter for the detection of hydroxyl radicals in vitro

Hydroxylation reactions of aromatic compounds have been used to detect hydroxyl radicals produced by gamma irradiation and ultrasound. The present study investigated the suitability of terephthalic acid (THA) as a hydroxyl radical dosimeter for general use in biologically relevant reactions. Hydroxyl radicals were generated by: (1) irradiating THA with a 254 nm ultraviolet light; (2) irradiating with gamma rays from a cesium source; and (3) generating hydroxyl radicals with 1 mM H2O2 and 10 microM Cu+2. In each of the three experiments, a fluorescent product was generated which exhibited identical fluorescent excitation and emission spectra. THA is non-fluorescent, eliminating the problem of a high initial background. Because THA has four identical ring hydrogens, only one mono-hydroxylated isomer was formed. The hydrogen peroxide reaction was dependent on the presence of a metal and cupric ions were effective in enhancing the reaction. With a Cu+2 concentration of 10 microM, the reaction was linear between 0-30 mM H2O2. Catalase abolished the reaction at a concentration of 100 micrograms/ml and the effects could still be observed at 10 ng/ml, consistent with the very high rate at which catalase destroys hydrogen peroxide. Tertbutyl- hydroperoxide did not generate any fluorescence in this system which makes THA a very specific detector of hydroxyl radicals.