Zhang H, Zdolsek J M, Brunk U T
Department of Pathology II, Faculty of Health Sciences, Linköping University, Sweden.
APMIS. 1991 Nov;99(11):1038-48. doi: 10.1111/j.1699-0463.1991.tb01297.x.
The diabetogenic effect of the quinonoid compound alloxan is not understood in detail although it supposedly involves reactions mediated by alloxan and oxygen radicals. These reactive species may form extra- or intracellularly and cause cell damage through a variety of complex interactions with several macromolecules. The purpose of this study was to elucidate early (less than or equal to 60 min) effects of alloxan and reducing agents (cysteine and ascorbic acid) on cultured macrophages, as assayed by the trypan blue dye exclusion test and the sensitive fluorescein diacetate and propidium iodide (FDA/PI) double staining technique. During the reactions between alloxan and reducing agents, oxygen was consumed as a sign of superoxide anion radical formation. When alloxan alone was added to two different culture media without serum, oxygen was still consumed, indicating formation of oxygen radicals due to the occurrence of reducing substances in cell culture media. This finding demonstrated the necessity of performing further studies in solutions without reducing capacity, e.g. in phosphate-buffered saline. The experiments showed that exposure of normal and malignant macrophages to alloxan and reducing substances resulted in rapidly occurring plasma membrane damage and ensuing cell death. Separate addition of catalase, desferrioxamine or superoxide dismutase resulted in evident, slight and no protection, respectively. The combinations of (i) catalase and desferrioxamine, and (ii) catalase, desferrioxamine and superoxide dismutase, however, inhibited cell damage in a pronounced and complete way, respectively. The results are interpreted as indicating cell damage due to the extracellular formation of hydrogen peroxide and hydroxyl radicals. The latter in close proximity to the cells and acting on the plasma membrane, while the former, after diffusing into the cell, may have several intracellular targets. The FDA/PI technique proved its value as a quantifiable method for the evaluation not only of cell death but also of cell damage with computer-based fluorometry.
尽管据推测醌类化合物四氧嘧啶的致糖尿病作用涉及由四氧嘧啶和氧自由基介导的反应，但其具体机制尚不清楚。这些活性物质可能在细胞外或细胞内形成，并通过与多种大分子的各种复杂相互作用导致细胞损伤。本研究的目的是通过台盼蓝染料排除试验以及灵敏的荧光素二乙酸酯和碘化丙啶（FDA/PI）双重染色技术，阐明四氧嘧啶和还原剂（半胱氨酸和抗坏血酸）对培养巨噬细胞的早期（小于或等于60分钟）影响。在四氧嘧啶与还原剂的反应过程中，氧气被消耗，这是超氧阴离子自由基形成的标志。当仅将四氧嘧啶添加到两种不同的无血清培养基中时，氧气仍被消耗，这表明由于细胞培养基中存在还原物质而形成了氧自由基。这一发现表明有必要在没有还原能力的溶液中，例如在磷酸盐缓冲盐水中进行进一步研究。实验表明，正常和恶性巨噬细胞暴露于四氧嘧啶和还原物质会导致质膜迅速受损并随后导致细胞死亡。分别添加过氧化氢酶、去铁胺或超氧化物歧化酶，结果分别导致明显、轻微和无保护作用。然而，（i）过氧化氢酶和去铁胺的组合，以及（ii）过氧化氢酶、去铁胺和超氧化物歧化酶的组合，分别以显著和完全的方式抑制了细胞损伤。这些结果被解释为表明细胞损伤是由于细胞外形成过氧化氢和羟基自由基所致。后者紧邻细胞并作用于质膜，而前者扩散到细胞内后可能有多个细胞内靶点。FDA/PI技术证明了其作为一种可量化方法的价值，不仅可用于评估细胞死亡，还可用于通过基于计算机的荧光测定法评估细胞损伤。
