Rapid and sensitive detection of hydroxyl radicals formed by activated neutrophils in the presence of chelated iron: Hydroxylation of deoxyguanosine to 8-hydroxydeoxyguanosine

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Abstract
Hydroxyl radicals (OH) can react with deoxyguanosine (dG) leading to the formation of 8-hydroxydeoxyguanosine (8OHdG). In this study, this has been used to detect the hydroxyl radicals formed when human polymorphonuclear leukocytes (PMNL) are stimulated with phorbol myristate acetate (PMA) in the presence of chelated iron. Reaction mixtures containing PMNL, PMA, dG and Fe-EDTA were incubated at 37°C, and the formation of 8OHdG was analysed with high-performance liquid chromatography and electrochemical detection. 8OHdG formation was detected at PMA concentrations of 2 nM or higher, and half-maximal 8OHdG formation was found at around 6 nM PMA. Stimulation of 500 000 cells with 10 nM PMA for 20 min resulted in a 500 to 1000-fold increase in 8OHdG formation as compared to unstimulated cells. The 8OHdG formation decreased after addition of hydroxyl radical scavengers (sodium benzoate, dimethylsulfoxide, and mannitol) and increased after addition of platelet-activating factor (PAF), an agent known to stimulate the generation of reactive oxygen metabolites in neutrophils. These results demonstrate that hydroxylation of dG to 8OHdG can be used to determine neutrophil-generated hydroxyl radicals in different experimental systems. Since the analysis of 8OHdG is rapid, sensitive and easy, this may have wide applications in inflammation and cancer research.

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