tert.-butyl hydroperoxide-mediated DNA base damage in cultured mammalian cells

tert.-Butyl hydroperoxide has been utilized to study the effect of oxidative stress on living cells; however, its effect on DNA bases in cells has not been characterized. In the present work, we have investigated DNA base damage in mammalian cells exposed to this organic hydroperoxide. SP2/0 derived murine hybridoma cells were treated with 4 concentrations of tert.-butyl hydroperoxide for varying periods of time. Chromatin was isolated from treated and control cells and subsequently analyzed by gas chromatography-mass spectrometry with selected-ion monitoring for DNA base damage. Quantification of damaged DNA bases was achieved by isotope-dilution mass spectrometry. The amounts of 8 products were significantly higher than control levels in cells treated with tert.-butyl hydroperoxide at a concentration range of 0.01-0.1 mM. At concentrations from 1.0 to 10 mM, product formation was inhibited and the amounts of products were similar to those in control cells. The bimodal nature of the dose-response may be qualitatively analogous to previous reports of bimodal killing of E. coli bacteria by hydrogen peroxide. The nature of the identified DNA base lesions suggests the involvement of the hydroxyl radical in their formation. tert.-Butyl hydroperoxide is known to produce the tert.-butoxyl radical in reactions with metal ions. However, it is unlikely that the tert.-butoxyl radical produces these DNA lesions. It is suggested that DNA base damage arises from tert.-butyl hydroperoxide-mediated oxidative stress in cells, resulting in formation of hydroxyl radicals in close proximity to DNA. The inhibition of product formation at high concentrations of tert.-butyl hydroperoxide may be explained by the scavenging of tert.-butoxyl radical by tert.-butyl hydroperoxide resulting in inhibition of oxidative stress. The plausibility of the scavenging mechanism was evaluated with a mathematical simulation of the dose-response for DNA damage in solutions containing hydrogen peroxide. The simulation model predicted a bimodal dose-response which agreed qualitatively with the results in this study and with other in vivo and in vitro studies reported in the literature.