Abstract
Abstract: :
Purpose: Metallothioneins (MTs) play important roles in defending cells against metals and oxidative stress. It is documented that Cd++ levels increase in cataract relative to clear human lenses, and we have previously detected increased expression of MTIIA in age–related cataract relative to clear lenses. These results collectively suggest a role for MTIIA in the maintenance of lens transparency. Here, we sought to further explore the role of MTs in lens transparency by examining the ability of MTIIA to directly protect lens epithelial cells from damage by either Cd++ or the oxidant tertiary butyl hydroperoxide (TBHP). Methods: Human lens epithelial cells (SRA01/04) that stably over–express MTIIA were created using retroviral–mediated gene transfer. Control and experimental cells were treated with increasing amounts of CdCl2 and assayed for mitochondrial function using MTS colorimetric assays. In another set of experiments, control and MTIIA–overexpressed (MT) cells were challenged with 0.5 mM TBHP and monitored by light microscopy after 3, 6 and 15hr of culture. For cells exposed to TBHP for 3hr plus 3–6hr of normal culture, changes in gene expression for three antioxidant enzymes were evaluated with the use of Real–Time PCR. Results: Analysis of the over–expressing lens cell lines demonstrated an approximately 2–3 fold level of MTIIA over–expression relative to control cells. Overexpression of MTIIA resulted in as much as 20% protection against Cd++–induced cell damage relative to control cells. MT cells were significantly more resistant to TBHP compared to controls. Control cells treated with TBHP for 3 to 6hr showed substantial shrinking and rounding–up, compared to TBHP–treated MT cells, which appeared normal during this time course. At 15hr, control cells exposed to TBHP appeared damaged and suffered nearly 50% cell death, compared to 15–20% cell death in the TBHP–treated MT cells. TBHP induced an upregulation of message for heme oxygenase, thioredoxin reductase and MnSOD that was 2–3 fold higher for control cells, compared to MT cells. Conclusions: Overexpression of MTIIA in human lens epithelial cells provided the cells with increased protection against Cd++ and TBHP. The up–regulation of other antioxidant transcripts in response to TBHP–treatment is lower in MT–over–expressing cells compared to controls. The results establish a role for MT protection of human lens epithelium against Cd++–induced toxicity and oxidative agents.
Keywords: oxidation/oxidative or free radical damage • cataract • antioxidants