Thiol antioxidants protect human lens epithelial (HLE B-3) cells against tert-butyl hydroperoxide-induced oxidative damage and cytotoxicity

Abstract

Oxidative damage to lens epithelial cells plays an important role in the development of age-related cataract, and the health of the lens has important implications for overall ocular health. As a result, there is a need for effective therapeutic agents that prevent oxidative damage to the lens. Thiol antioxidants such as tiopronin or N-(2-mercaptopropionyl)glycine (MPG), N-acetylcysteine amide (NACA), N-acetylcysteine (NAC), and exogenous glutathione (GSH) may be promising candidates for this purpose, but their ability to protect lens epithelial cells is not well understood. The effectiveness of these compounds was compared by exposing human lens epithelial cells (HLE B-3) to the chemical oxidant tert-butyl hydroperoxide (tBHP) and treating the cells with each of the antioxidant compounds. MTT cell viability, apoptosis, reactive oxygen species (ROS), and levels of intracellular GSH, the most important antioxidant in the lens, were measured after treatment. All four compounds provided some degree of protection against tBHP-induced oxidative stress and cytotoxicity. Cells treated with NACA exhibited the highest viability after exposure to tBHP, as well as decreased ROS and increased intracellular GSH. Exogenous GSH also preserved viability and increased intracellular GSH levels. MPG scavenged significant amounts of ROS, and NAC increased intracellular GSH levels. Our results suggest that both scavenging ROS and increasing GSH may be necessary for effective protection of lens epithelial cells. Further, the compounds tested may be useful for the development of therapeutic strategies that aim to prevent oxidative damage to the lens.

Keywords: 7-AAD, 7-aminoactinomycin D; ATCC, American Type Culture Collection; Antioxidant; Carboxy-H2DCFDA, 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate; Cataract; EMEM, Eagle's minimum essential medium; FBS, fetal bovine serum; FDA, United States Food and Drug Administration; GSH, glutathione; GSSG, glutathione disulfide; Glutathione; H2O2, hydrogen peroxide; HLE B-3, human (eye) lens epithelial cell line B-3; Lens; MPG, N-(2-mercaptopropionyl)glycine; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); NAC, N-acetylcysteine; NACA, N-acetylcysteine amide; OH•, hydroxyl radical; Oxidative stress; PBS, phosphate-buffered saline; ROS, reactive oxygen species; Thiol; tBHP, tert-butyl hydroperoxide.