Altman S A, Zastawny T H, Randers L, Lin Z, Lumpkin J A, Remacle J, Dizdaroglu M, Rao G
Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County 21228.
Mutat Res. 1994 Apr 1;306(1):35-44. doi: 10.1016/0027-5107(94)90165-1.
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.
叔丁基过氧化氢已被用于研究氧化应激对活细胞的影响；然而，其对细胞中DNA碱基的影响尚未得到表征。在本研究中，我们调查了暴露于这种有机过氧化氢的哺乳动物细胞中的DNA碱基损伤。用4种浓度的叔丁基过氧化氢处理SP2/0衍生的鼠杂交瘤细胞不同时间。从处理过的细胞和对照细胞中分离出染色质，随后通过气相色谱-质谱联用仪进行选择离子监测，以分析DNA碱基损伤。通过同位素稀释质谱法对受损DNA碱基进行定量。在0.01-0.1 mM浓度范围内，用叔丁基过氧化氢处理的细胞中8种产物的量显著高于对照水平。在1.0至10 mM的浓度下，产物形成受到抑制，产物量与对照细胞中的相似。剂量反应的双峰性质在定性上可能类似于先前关于过氧化氢对大肠杆菌的双峰杀伤的报道。所鉴定的DNA碱基损伤的性质表明羟基自由基参与了它们的形成。已知叔丁基过氧化氢在与金属离子的反应中会产生叔丁氧基自由基。然而，叔丁氧基自由基不太可能产生这些DNA损伤。有人认为，DNA碱基损伤源于细胞中叔丁基过氧化氢介导的氧化应激，导致在DNA附近形成羟基自由基。高浓度叔丁基过氧化氢对产物形成的抑制作用可能是由于叔丁基过氧化氢对叔丁氧基自由基的清除，从而抑制了氧化应激。通过对含有过氧化氢的溶液中DNA损伤剂量反应的数学模拟，评估了清除机制的合理性。模拟模型预测了双峰剂量反应，这在定性上与本研究结果以及文献中报道的其他体内和体外研究结果一致。
