TERT-BUTYL HYDROPEROXIDE-MEDIATED DNA-BASE DAMAGE IN CULTURED-MAMMALIAN-CELLS
TERT-BUTYL HYDROPEROXIDE-MEDIATED DNA-BASE DAMAGE IN CULTURED-MAMMALIAN-CELLS
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DOI：
10.1016/0027-5107(94)90165-1
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发表时间：
1994-04-01
期刊：
影响因子：
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通讯作者：
RAO, G
中科院分区：
文献类型：
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作者：
ALTMAN, SA;ZASTAWNY, TH;RAO, G
第三，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碱基的影响尚未被表征。在目前的工作中，我们已经调查了哺乳动物细胞暴露于这种有机氢过氧化物的DNA碱基损伤。将SP2/0衍生的鼠杂交瘤细胞用4种浓度的叔丁基二甲基甲酰胺处理。丁基过氧化氢反应不同的时间。染色质从处理和对照细胞中分离，随后通过气相色谱-质谱分析，选择离子监测DNA碱基损伤。通过同位素稀释质谱法实现对受损DNA碱基的定量。在用tert.-处理的细胞中，8种产物的量显著高于对照水平。浓度范围为0.01- 0.1mM的丁基过氧化氢。在1.0 - 10 mM的浓度下，产物形成受到抑制，产物的量与对照细胞中的那些相似。剂量反应的双峰性质可能与以前报道的双峰杀灭大肠杆菌的性质类似。过氧化氢对大肠杆菌的作用。所确定的DNA碱基损伤的性质表明羟基自由基参与其形成。第三个已知丁基过氧化氢产生叔丁基氢。丁氧基自由基与金属离子反应。然而，这是不可能的，第三。丁氧基自由基产生这些DNA损伤。这表明DNA碱基损伤是由tert.-丁基过氧化氢介导的细胞氧化应激，导致在DNA附近形成羟基自由基。在高浓度的叔丁基对产物形成的抑制丁基过氧化氢可以通过清除叔丁基过氧化氢来解释。叔丁氧基丁基过氧化氢导致氧化应激的抑制。通过对过氧化氢溶液中DNA损伤的剂量-效应关系的数学模拟，评价了其清除机制的合理性。模拟模型预测的双峰剂量反应，同意定性与本研究中的结果，并与其他在体内和体外研究中的文献报道。
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.