Abstract
Abstract: :
Purpose: Thyroid hormone homeostasis is important in the regulation of the resistance of cell membranes to oxidative insult. Furthermore, hypothyroidism can contribute to cataractogenesis. We quantified thyroxin ( T4) induced changes in the phospholipid composition of cultured human lens epithelial cells and measured their resistance to oxidative stress. Methods: Immortalized human lens epithelial cells were a gift from Dr. Usha Andley. Cells were treated with and without 0.335nM free T4 in culture medium containing 5% FBS and the medium was changed every 3 days for a period of 10 days. The concentration of free T4 in culture medium was measured using electrochemiluminescent immunoassay. In addition, control and T4 treated cells were challenged with 80% oxygen for 32 hours. Lipids were extracted and phospholipid composition quantified by 31P NMR and matrix–assisted laser desorption/ionization time–of–flight mass spectrometry. Reactive oxygen species were detected using the fluorescent probe DCFH–DA. Cell confluence and growth were monitored by phase–contrast microscopy and phase hemacytometers. Results: T4 treatment significantly increased lens epithelial cell sphingomyelins, cardiolipins, phosphatidylethanolamine plasmalogens and lipid hydrocarbon chain saturation and decreased phosphatidylcholines. Hyperoxia caused a similar increase in the reactive oxygen species in control and T4 treated cells. Within normaxia groups, proliferation and cell survival were similar. Under hyperoxia conditions, cell survival was greater for T4 treated cells than for untreated cells. Cell survival was greater for both untreated and T4 treated cells in normaxia conditions compared with hyperoxia conditions. Conclusions: T4 treatment caused phospholipid changes in the epithelial cells that resulted in membranes more resistant to oxidative insult. This supports in vitro studies which showed that membranes with elevated sphingolipids, plasmalogens or saturation are more resistant to oxidative insult. Our results provide a basis for future studies involving treatment with T4 or its analogs to render membranes more resistant to oxidation that may delay cataract formation.
Keywords: cataract • cell membrane/membrane specializations • stress response