Iron contributes to the formation of catechol isoquinolines and oxidative toxicity induced by overdose dopamine in dopaminergic SH-SY5Y cells

Abstract

Objective

The selective loss of dopaminergic neurons in Parkinson’s disease is suspected to correlate with the increase of cellular iron, which may be involved in the pathogenesis of PD by promotion of oxidative stress. This research investigated dopamine-induced oxidative stress toxicity contributed by iron and the production of dopamine-derived neurotoxins in dopaminergic SH-SY5Y cells.

Methods

After the SH-SY5Y cells were pre-incubated with dopamine and Fe²⁺ for 24 h, the cell viability, hydroxyl radical, melondialdehyde, cell apoptosis, and catechol isoquinolines were measured by lactate dehydrogenase assay, salicylic acid trapping method, thiobarbuteric acid assay, Hoechst 33258 staining and HPLC-electrochemical detection (HPLC-ECD), respectively.

Results

(1) Optimal dopamine (150 μmol/L) and Fe²⁺ (40 or 80 μmol/L) significantly increased the concentrations of hydroxy radicals and melondialdehyde in SH-SY5Y cells. (2) Induction with dopamine alone or dopamine and Fe²⁺ (dopamine/Fe²⁺) caused cell apoptosis. (3) Compared with untreated cells, the catechol isoquinolines, salsolinol and N-methyl-salsolinol in dopamine/Fe²⁺-induced cells were detected in increasing amounts.

Conclusion

Due to dopamine/Fe²⁺-induced oxidative stress similar to the state in the parkinsonian substantia nigra neurons, dopamine and Fe²⁺ impaired SH-SY5Y cells could be used as the cell oxidative stress model of Parkinson’s disease. The catechol isoquinolines detected in cells may be involved in the pathogenesis of Parkinson’s disease as potential neurotoxins.

摘要

目的

帕金森氏病(Pakinson’s disease, PD)中多巴胺能神经元选择性缺失与胞内铁水平升高有密切关系, 提示铁可能通过参与氧化应激在PD发病机制中起重要作用。本研究使用一定浓度的Fe²⁺和多巴胺诱导人多巴胺能成神经细胞瘤SH-SY5Y细胞产生氧化应激状态, 并且检测胞内是否有多巴胺衍生类的神经内毒素物质产生。

方法

多巴胺添加不同浓度的Fe²⁺诱导SH-SY5Y细胞, 24 h后用乳酸脱氢酶法、 水杨酸捕获法、 硫代巴比妥酸法、 Hoechst33258染色法和带有电化学检测器的高效液相色谱仪分别检测细胞存活率、 羟自由基生成量、 丙二醛含量、 细胞凋亡和儿茶酚异喹啉物质的生成情况。

结果

(1) 150 μmol/L 多巴胺添加40或80 μmol/L Fe²⁺后, 胞内羟自由基和丙二醛含量较对照组显著增加; (2) 单独多巴胺以及多巴胺加40或80 μmol/L Fe²⁺诱导后细胞发生凋亡; (3)在诱导后的胞内检测到Salsolinol和N-methylsalsolinol的含量高于对照组。

结论

一定浓度的Fe²⁺和多巴胺诱导SH-SY5Y细胞可模拟帕金森氏病人黑质区多巴胺能神经元所受到的氧化应激状态, 胞内检测到的儿茶酚异喹啉物质, 如去甲猪毛菜碱和N-methyl-salsolinol, 可能作为一类潜在的神经毒性物质与帕金森氏病的发病有关。