Orhan H, Gurer-Orhan H, Vriese E, Vermeulen N P E, Meerman J H N
Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, Amsterdam, The Netherlands.
Toxicol In Vitro. 2006 Sep;20(6):1005-13. doi: 10.1016/j.tiv.2005.12.012. Epub 2006 Feb 20.
We recently developed two biomarker sets for oxidative damage: one for determination of lipid peroxidation (LPO) degradation products; acetaldehyde, propanal, butanal, pentanal, hexanal, heptanal, octanal, nonanal, malondialdehyde and acetone, by a gas chromatography-electron capture detection method, and the other for protein oxidation products such as o,o'-dityrosine, by an isotope dilution high performance liquid chromatography-tandem mass spectrometry method. In the present study, we explored the possibility to utilize these biomarkers for determining the oxidative damage in liver mammalian cells in vitro. Two different treatments were chosen for inducing oxidative stress in Chinese Hamster ovary cells: menadione and copper plus hydrogen peroxide (Cu2+/H2O2). Cells were incubated with the model compounds in the presence or absence of vitamin E and C, and cytotoxicity was evaluated by a nuclear-dye method. Results were compared to two fluorescent probes, H2DCF-DA and C11 -BODIPY581/591, which have been used for determining the formation of free radicals in the cells. From ten LPO degradation products, eight were increased significantly following incubation with menadione in cell lysate or incubation media. Menadione-induced oxidative stress was also confirmed by oxidation of fluorescent probes. However, no increased formation of protein oxidation products was observed. Vitamin E and C did not diminish the formation of LPO degradation products that were increased by menadione. Although Cu2+/H2O2 did not induce oxidation of fluorescent probes, it induced formation of six out of ten LPO degradation products. Vitamin E and C did not diminish the formation of LPO degradation products; vitamin C even substantially increased the formation of acetaldehyde and propanal, which is in line with its reported prooxidant action under certain conditions. Vitamin C also caused two-fold increase in Cu2+/H2O2-induced o,o'-dityrosine formation when applied simultaneously. In conclusion, our present results show that the LPO biomarker set can be used for evaluation of oxidant capacity and the toxic potential of various chemicals in an in vitro cell model. These biomarkers might even be more sensitive than measuring protein oxidation products or oxidation of fluorescent probes.
一组通过气相色谱 - 电子捕获检测法测定脂质过氧化（LPO）降解产物，包括乙醛、丙醛、丁醛、戊醛、己醛、庚醛、辛醛、壬醛、丙二醛和丙酮；另一组通过同位素稀释高效液相色谱 - 串联质谱法测定蛋白质氧化产物，如邻，邻'-二酪氨酸。在本研究中，我们探讨了利用这些生物标志物来测定体外培养的哺乳动物肝细胞氧化损伤的可能性。我们选择了两种不同的处理方法来诱导中国仓鼠卵巢细胞的氧化应激：甲萘醌和铜加过氧化氢（Cu2+/H2O2）。细胞在有或没有维生素E和C的情况下与模型化合物一起孵育，并通过核染料法评估细胞毒性。将结果与两种荧光探针H2DCF - DA和C11 - BODIPY581/591进行比较，这两种探针已用于测定细胞内自由基的形成。在细胞裂解物或培养液中与甲萘醌孵育后，十种LPO降解产物中有八种显著增加。甲萘醌诱导的氧化应激也通过荧光探针的氧化得到证实。然而，未观察到蛋白质氧化产物的形成增加。维生素E和C并未减少由甲萘醌增加的LPO降解产物的形成。虽然Cu2+/H2O2未诱导荧光探针的氧化，但它诱导了十种LPO降解产物中的六种的形成。维生素E和C并未减少LPO降解产物的形成；维生素C甚至大幅增加了乙醛和丙醛的形成，这与其在某些条件下所报道的促氧化作用一致。当同时应用时，维生素C还使Cu2+/H2O2诱导的邻，邻'-二酪氨酸形成增加了两倍。总之，我们目前的结果表明，LPO生物标志物组可用于评估体外细胞模型中各种化学物质的氧化能力和毒性潜力。这些生物标志物甚至可能比测量蛋白质氧化产物或荧光探针的氧化更敏感。
