Cytotoxic effect of spermine on retinal pigment epithelial cells
Cytotoxic effect of spermine on retinal pigment epithelial cells
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DOI：
10.1167/iovs.06-0379
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发表时间：
2007-01-01
影响因子：
4.4
通讯作者：
Ito, Seiji
中科院分区：
文献类型：
--
作者：
Kaneko, Shiho;Ueda-Yamada, Mami;Ito, Seiji
目的。先前的一项研究表明，在脉络膜和视网膜回旋萎缩（GA）体外模型中，特定的不可逆抑制剂（5-氟甲基鸟氨酸；5-FMO）可使鸟氨酸-δ-转氨酶（OAT）缺陷的人视网膜色素上皮（RPE）细胞失活，导致细胞死亡。本研究旨在探讨鸟氨酸代谢产物尤其是精胺对RPE细胞的细胞毒性，以阐明鸟氨酸对RPE细胞的细胞毒性作用机制。 RPE 细胞与鸟氨酸或参与鸟氨酸代谢途径的化合物一起孵育。使用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑 (MTT) 比色法和 [H-3] 胸苷掺入测定法评估对 RPE 细胞活力和增殖活性的影响。使用[C-14]精胺和丹酰精胺检查精胺掺入RPE细胞。为了评估精胺诱导的 RPE 细胞死亡，用膜联蛋白 V 和碘化丙啶对细胞进行双重染色，并进行流式细胞术。结果。鸟氨酸、精氨酸、谷氨酸、脯氨酸、肌酸、甘氨酸和腐胺对 RPE 细胞的活力和增殖活性没有影响，而亚精胺和精胺 (10 mM) 分别抑制 [H-3] 胸苷掺入 13% 和 89%。精胺对[H-3]胸苷掺入的抑制是剂量依赖性的，并且早在添加后4小时就观察到了。此外，精胺被掺入并积聚在RPE细胞的核周区域。精胺以剂量依赖性方式诱导 RPE 细胞凋亡。结论。目前的结果表明，过量的精胺对 RPE 细胞具有细胞毒性，并表明鸟氨酸的代谢物，特别是精胺，可能参与 GA 中 RPE 变性的机制。
PURPOSE. A prior study showed inactivation of ornithine-delta-aminotransferase (OAT)-deficient human retinal pigment epithelial (RPE) cells by a specific irreversible inhibitor (5-fluoromethylornithine; 5-FMO) leading to cell death, in an in vitro model of gyrate atrophy (GA) of the choroid and retina. In the present study, the cytotoxicity of metabolites of ornithine, especially spermine, in RPE cells was investigated, to clarify the mechanism of ornithine cytotoxicity in RPE cells.METHODS. RPE cells were incubated with ornithine or compounds involved in ornithine metabolic pathways. The effects on RPE cell viability and proliferative activity were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric and [H-3] thymidine incorporation assays. Incorporation of spermine into RPE cells was examined by using [C-14] spermine and dansyl-spermine. To assess spermine-induced RPE cell death, cells were double stained with annexin V and propidium iodide and subjected to flow cytometry.RESULTS. Ornithine, arginine, glutamate, proline, creatine, glycine, and putrescine exhibited no effects on the viability and proliferative activities of RPE cells, whereas spermidine and spermine (10 mM) inhibited [H-3] thymidine incorporation by 13% and 89%, respectively. The inhibition of [H-3] thymidine incorporation by spermine was dose dependent and was observed as early as 4 hours after addition. Further, spermine was incorporated and accumulated in the perinuclear region of RPE cells. Apoptotic RPE cell death was induced by spermine in a dose-dependent manner.CONCLUSIONS. The present results demonstrated that excessive spermine is cytotoxic to RPE cells and suggest that metabolites of ornithine, especially spermine, may be involved in the mechanism of RPE degeneration in GA.