The Zinc-Metallothionein Redox System Reduces Oxidative Stress in Retinal Pigment Epithelial Cells

Abstract

Oxidative stress affects all the structures of the human eye, particularly the retina and its retinal pigment epithelium (RPE). The RPE limits oxidative damage by several protective mechanisms, including the non-enzymatic antioxidant system zinc-metallothionein (Zn-MT). This work aimed to investigate the role of Zn-MT in the protection of RPE from the oxidative damage of reactive oxygen intermediates by analytical and biochemical-based techniques. The Zn-MT system was induced in an in vitro model of RPE cells and determined by elemental mass spectrometry with enriched isotopes and mathematical calculations. Induced-oxidative stress was quantified using fluorescent probes. We observed that 25, 50 or 100 μM of zinc induced Zn-MT synthesis (1.6-, 3.6- and 11.9-fold, respectively), while pre-treated cells with zinc (25, 50, and 100 μM) and subsequent 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) treatment increased Zn-MT levels in a lesser extent (0.8-, 2.1-, 6.1-fold, respectively), exerting a stoichiometric transition in the Zn-MT complex. Moreover, AAPH treatment decreased MT levels (0.4-fold), while the stoichiometry remained constant or slightly higher when compared to non-treated cells. Convincingly, induction of Zn-MT significantly attenuated oxidative stress produced by free radicals’ generators. We conclude that the stoichiometry of Zn-MT plays an important role in oxidative stress response, related with cellular metal homeostasis.

A Multi-Omics Approach Identifies Key Regulatory Pathways Induced by Long-Term Zinc Supplementation in Human Primary Retinal Pigment Epithelium

“…Both analyses revealed five main biological functions shared between apical and basal zinc supplementation: oxidative stress response, protein processing, protein export/import, adhesion to polarity and extracellular matrix organization (summarized with grouping of overlapping canonical pathways in Supplementary Figure S2 ). Zinc is known to affect oxidative processes by modulating the expression of metallothioneins [ 65 , 66 ], glutathione [ 67 , 68 , 69 ], and catalase [ 12 , 42 ], by stabilizing sulfhydryl groups on proteins [ 70 ] and by replacing redox-active metals, such as copper and iron [ 71 , 72 ]. With ageing in general and in AMD in particular, protein, lipid and mineral-rich deposits accumulate in the sub-RPE space.…”

Isotopically Enriched Tracers and Inductively Coupled Plasma Mass Spectrometry Methodologies to Study Zinc Supplementation in Single-Cells of Retinal Pigment Epithelium in Vitro

“…According to the microscopy images obtained in previous studies, the nucleus of HRPEsv cells is higher than 10 μM (ranging from 15-20 μm), while the size of HRPEsv cell (elongated single cells) is higher than 50 μm wide (ranging from 50-100 μm). 29,30…”

Transcriptomic Profiling of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Over Time

“…Metallothioneins are metal-binding proteins that are protective against oxidative stress.Compared to the 12-month-old RPE, the 1-month-old RPE cells express less transcripts for the metallothioneins MT1E, MT1F, MT1G, MT2A, MT1X and higher levels of the Dopachrome Tautomerase DCT. This data suggests a variation in the handling of metals and in the antioxidant abilities of RPE cells, which could be reflective of either a necessity to handle more oxidative stress in an aging in vitro environment or a maturation of RPE cells towards a more mature and protective phenotype[64]. The assessment of variations in other gene expressions between the two points in time indicates a maturation profile of cells rather than an increased stress.…”