Branco Vasco, Coppo Lucia, Solá Susana, Lu Jun, Rodrigues Cecília M P, Holmgren Arne, Carvalho Cristina
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Redox Biol. 2017 Oct;13:278-287. doi: 10.1016/j.redox.2017.05.024. Epub 2017 Jun 1.
Mercury (Hg) compounds target both cysteine (Cys) and selenocysteine (Sec) residues in peptides and proteins. Thus, the components of the two major cellular antioxidant systems - glutathione (GSH) and thioredoxin (Trx) systems - are likely targets for mercurials. Hg exposure results in GSH depletion and Trx and thioredoxin reductase (TrxR) are prime targets for mercury. These systems have a wide-range of common functions and interaction between their components has been reported. However, toxic effects over both systems are normally treated as isolated events. To study how the interaction between the glutathione and thioredoxin systems is affected by Hg, human neuroblastoma (SH-SY5Y) cells were exposed to 1 and 5μM of inorganic mercury (Hg), methylmercury (MeHg) or ethylmercury (EtHg) and examined for TrxR, GSH and Grx levels and activities, as well as for Trx redox state. Phosphorylation of apoptosis signalling kinase 1 (ASK1), caspase-3 activity and the number of apoptotic cells were evaluated to investigate the induction of Trx-mediated apoptotic cell death. Additionally, primary cerebellar neurons from mice depleted of mitochondrial Grx2 (mGrx2D) were used to examine the link between Grx activity and Trx function. Results showed that Trx was affected at higher exposure levels than TrxR, especially for EtHg. GSH levels were only significantly affected by exposure to a high concentration of EtHg. Depletion of GSH with buthionine sulfoximine (BSO) severely increased Trx oxidation by Hg. Notably, EtHg-induced oxidation of Trx was significantly enhanced in primary neurons of mGrx2D mice. Our results suggest that GSH/Grx acts as backups for TrxR in neuronal cells to maintain Trx turnover during Hg exposure, thus linking different mechanisms of molecular and cellular toxicity. Finally, Trx oxidation by Hg compounds was associated to apoptotic hallmarks, including increased ASK-1 phosphorylation, caspase-3 activation and increased number of apoptotic cells.
汞（Hg）化合物可作用于肽和蛋白质中的半胱氨酸（Cys）和硒代半胱氨酸（Sec）残基。因此，细胞内两大主要抗氧化系统——谷胱甘肽（GSH）和硫氧还蛋白（Trx）系统的组成成分可能都是汞类化合物的作用靶点。汞暴露会导致谷胱甘肽耗竭，硫氧还蛋白和硫氧还蛋白还原酶（TrxR）是汞的主要作用靶点。这些系统具有广泛的共同功能，且其组成成分之间已被报道存在相互作用。然而，对这两个系统的毒性作用通常被视为孤立事件。为了研究谷胱甘肽系统和硫氧还蛋白系统之间的相互作用如何受到汞的影响，将人神经母细胞瘤（SH-SY5Y）细胞暴露于1μM和5μM的无机汞（Hg）、甲基汞（MeHg）或乙基汞（EtHg）中，并检测硫氧还蛋白还原酶、谷胱甘肽和谷氧还蛋白的水平及活性，以及硫氧还蛋白的氧化还原状态。评估凋亡信号激酶1（ASK1）的磷酸化、半胱天冬酶-3的活性和凋亡细胞数量，以研究硫氧还蛋白介导的凋亡性细胞死亡的诱导情况。此外，使用来自线粒体谷氧还蛋白2（mGrx2）缺失小鼠的原代小脑神经元来研究谷氧还蛋白活性与硫氧还蛋白功能之间的联系。结果表明，硫氧还蛋白比硫氧还蛋白还原酶在更高的暴露水平下受到影响，尤其是对于乙基汞。谷胱甘肽水平仅在暴露于高浓度乙基汞时受到显著影响。用丁硫氨酸亚砜胺（BSO）消耗谷胱甘肽会严重增加汞对硫氧还蛋白的氧化作用。值得注意的是，在mGrx2缺失小鼠的原代神经元中，乙基汞诱导的硫氧还蛋白氧化显著增强。我们的结果表明，在神经元细胞中，谷胱甘肽/谷氧还蛋白作为硫氧还蛋白还原酶的后备物质，在汞暴露期间维持硫氧还蛋白的周转，从而将分子和细胞毒性的不同机制联系起来。最后，汞化合物对硫氧还蛋白的氧化作用与凋亡特征相关，包括ASK-1磷酸化增加、半胱天冬酶-3激活和凋亡细胞数量增加。
