Diamide is a commonly used thiol oxidant, also recognized as a highly cell-permeable reagent that can oxidize reduced glutathione (GSH) to oxidized glutathione (GSSG) without generating reactive oxygen species (ROS)[1-2]. Diamide has been widely applied in cell-free systems, in vitro cellular experiments, and in vivo studies to rapidly oxidize GSH and/or protein thiols[3-4].
In vitro, Diamide (0–400μM) treatment of LoVo cells significantly reversed the increase in intracellular lactate levels induced by DDR1a knockdown and counteracted the regulatory effect of DDR1a on pyruvate kinase activity[5]. Diamide (0.1–0.5mM) treatment of rat aortic smooth muscle cells concentration-dependently increased protein glutathionylation, induced mitochondrial proton leak, and reduced ATP-linked oxygen consumption; higher concentrations (≥0.25mM) led to bioenergetic failure and cell death[6].
In vivo, stereotaxic injection of Diamide (1.45μmol) into the substantia nigra of C57BL/6 mice (single injection), Diamide significantly reduced glutathione levels in brain tissue, increased oxidative stress markers, and induced degeneration of dopaminergic neurons in the substantia nigra, which can be utilized to establish a mouse model of Parkinson's disease[7].
References:
[1] Kosower NS, Kosower EM, Koppel RL. Sensitivity of hemoglobin thiol groups within red blood cells of rat during oxidation of glutathione. Eur J Biochem. 1977 Aug 1;77(3):529-34.
[2] Kosower NS, Kosower EM, Wertheim B, et al. Diamide, a new reagent for the intracellular oxidation of glutathione to the disulfide. Biochem Biophys Res Commun. 1969 Nov 6;37(4):593-6.
[3] Liu Y, Min W. Thioredoxin promotes ASK1 ubiquitination and degradation to inhibit ASK1-mediated apoptosis in a redox activity-independent manner. Circ Res. 2002 Jun 28;90(12):1259-66.
[4] Marey-Semper I, Gelman M, Lévi-Strauss M. A selective toxicity toward cultured mesencephalic dopaminergic neurons is induced by the synergistic effects of energetic metabolism impairment and NMDA receptor activation. J Neurosci. 1995 Sep;15(9):5912-8.
[5] Xiong B, Xie Z, Song F, et al. DDR1 promotes LoVo cell proliferation by regulating energy metabolism. Acta Biochim Biophys Sin (Shanghai). 2022 May 25;54(5):615-624.
[6] Hill BG, Higdon AN, Dranka BP, et al. Regulation of vascular smooth muscle cell bioenergetic function by protein glutathiolation. Biochim Biophys Acta. 2010 Feb;1797(2):285-95.
[7] Ray A, Kambali M, Ravindranath V. Thiol Oxidation by Diamide Leads to Dopaminergic Degeneration and Parkinsonism Phenotype in Mice: A Model for Parkinson's Disease. Antioxid Redox Signal. 2016 Aug 10;25(5):252-67.