Lapshina Elena A, Zavodnik Ilya B, Labieniec Magdalena, Rekawiecka Katarzyna, Bryszewska Maria
Institute of Biochemistry, National Academy of Sciences of Belarus, BLK-50, 230017 Grodno, Belarus.
Mutat Res. 2005 Jun 6;583(2):189-97. doi: 10.1016/j.mrgentox.2005.03.005.
The organic hydroperoxide, tert-butyl hydroperoxide (t-BHP), is a useful model compound to study mechanisms of oxidative cell injury. In the present work, we examined the features of the interactions of this oxidant with Chinese hamster B14 cells. The aim of our study was to reveal a possible role of structural modifications in membranes and loss of DNA integrity in t-BHP-induced cell injury and death. The tert-butyl hydroperoxide treatment (100-1000 microM, 37 degrees C for 1h) did not decrease cell viability (as measured by cell-specific functional activity with an MTT test), but completely prevented cell growth. We observed intracellular reduced glutathione (GSH) oxidation and total glutathione (GSH+GSSG) depletion, a slight increase in the level of lipid-peroxidation products, an enhancement of membrane fluidity, intracellular potassium leakage and a significant decrease of membrane potential. At oxidant concentrations of 100-1500 microM, a significant damage to DNA integrity was observed as revealed by the Comet assay. The inhibition of cell proliferation (cell-growth arrest) may be explained by genotoxicity of t-BHP, by disturbance of the cellular redox-equilibrium (GSH oxidation) and by structural membrane modifications, which result in ion-non-selective pore formation. The disturbance in passive membrane permeability and the DNA damage may be the most dramatic cell impairments induced by t-BHP treatment. The presence of another oxidant, hypochlorous acid (HOCl), completely prevented t-BHP-induced DNA strand breaks, perhaps due to extracellular oxidation of t-BHP by HOCl.
有机氢过氧化物叔丁基过氧化氢（t-BHP）是研究氧化细胞损伤机制的一种有用的模型化合物。在本研究中，我们检测了这种氧化剂与中国仓鼠B14细胞相互作用的特征。我们研究的目的是揭示膜结构修饰和DNA完整性丧失在t-BHP诱导的细胞损伤和死亡中可能发挥的作用。叔丁基过氧化氢处理（100 - 1000微摩尔，37℃处理1小时）并未降低细胞活力（通过MTT试验以细胞特异性功能活性来衡量），但完全抑制了细胞生长。我们观察到细胞内还原型谷胱甘肽（GSH）氧化和总谷胱甘肽（GSH + GSSG）耗竭，脂质过氧化产物水平略有升高，膜流动性增强，细胞内钾离子泄漏以及膜电位显著降低。通过彗星试验发现，在氧化剂浓度为100 - 1500微摩尔时，DNA完整性受到显著破坏。细胞增殖的抑制（细胞生长停滞）可能是由于t-BHP的遗传毒性、细胞氧化还原平衡的紊乱（GSH氧化）以及膜结构修饰导致离子非选择性孔形成所引起的。被动膜通透性的紊乱和DNA损伤可能是t-BHP处理诱导的最显著的细胞损伤。另一种氧化剂次氯酸（HOCl）的存在完全阻止了t-BHP诱导的DNA链断裂，这可能是由于HOCl对t-BHP进行了细胞外氧化。
