Fabiani R, De Bartolomeo A, Rosignoli P, Morozzi G
Department of Biochemical Science and Molecular Biotechnology, University of Perugia, 06126 Perugia, Italy.
Nutr Cancer. 2001;39(2):284-91. doi: 10.1207/S15327914nc392_19.
Oxidative stress has been related to several degenerative diseases such as cancer and coronary heart disease. Reactive oxygen species can damage different cellular macromolecules, including DNA, which is directly responsible for mutation and carcinogenesis. In this study, monocytes that were activated by phorbol 12-myristate 13-acetate (PMA) were coincubated with lymphocytes, and the DNA damage was measured by single-cell gel electrophoresis (comet) assay. Stimulation of monocytes with PMA activates the "respiratory burst," which evokes DNA damage in lymphocytes. The extent of the damage is related to the concentration of monocytes and the exposure time. Exogenous addition of superoxide dismutase did not prevent the DNA damage, which suggests that superoxide ions are not directly responsible for the damage. Partial protection was observed when catalase was included (60% protection), which indicates that other reactive species, in addition to H2O2, are responsible for the damage. In this system, the protective activity of natural antioxidants at different concentrations was also investigated. After coincubation of PMA-activated monocytes with lymphocytes in the presence of each antioxidant for one hour at 37 degrees C, the lymphocyte DNA damage was determined. All the compounds protected the lymphocytes to a certain degree, with a maximum effect at different concentrations: 41% protection with 1 microM ascorbic acid, 55% protection with 40 microM alpha-tocopherol, 50% protection with 3 microM beta-carotene, and 56% protection with 5 microM quercetin. On the basis of these results, we maintain that this "ex vivo model," more closely related to physiological conditions, could be used to test the antioxidant activity of different compounds.
氧化应激与多种退行性疾病有关，如癌症和冠心病。活性氧物种可损伤不同的细胞大分子，包括DNA，而DNA直接与突变和致癌作用相关。在本研究中，用佛波酯12 -肉豆蔻酸酯13 -乙酸酯（PMA）激活的单核细胞与淋巴细胞共同孵育，并通过单细胞凝胶电泳（彗星）试验测量DNA损伤。用PMA刺激单核细胞可激活“呼吸爆发”，这会引起淋巴细胞中的DNA损伤。损伤程度与单核细胞浓度和暴露时间有关。外源添加超氧化物歧化酶并不能防止DNA损伤，这表明超氧离子并非直接导致损伤。当加入过氧化氢酶时观察到部分保护作用（60%的保护率），这表明除H2O2外，其他活性物种也与损伤有关。在该系统中，还研究了不同浓度天然抗氧化剂的保护活性。在37℃下，将每种抗氧化剂存在时PMA激活的单核细胞与淋巴细胞共同孵育1小时后，测定淋巴细胞DNA损伤。所有化合物都在一定程度上保护了淋巴细胞，在不同浓度下有最大保护效果：1μM抗坏血酸提供41%的保护，40μMα-生育酚提供55%的保护，3μMβ-胡萝卜素提供50%的保护，5μM槲皮素提供56%的保护。基于这些结果，我们认为这种与生理条件更密切相关的“体外模型”可用于测试不同化合物的抗氧化活性。
