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.

Fig. 1

Cell viability of HLE B-3 cells after long-term treatment (4-h exposure to 0.5 mM tBHP + 1 mM antioxidant, followed by 20-h treatment with 1 mM antioxidant). Cell viability was determined by MTT assay and is expressed as a percentage of absorbance at 570 nm compared to that of the control group. The height of the columns represents the mean of at least fourteen replicates, and the error bars indicate the standard deviation. *p ≤ 0.05 and ****p ≤ 0.0001 compared to the tBHP-only group. Cell viability results from the antioxidant-only groups (groups 2–5 in Table 2) are not shown because they were very similar to that of the control group.

Fig. 2

Representative 2D plots from flow cytometry analysis of apoptotic cells after long-term treatment (4-h exposure to 0.5 mM tBHP + 1 mM antioxidant, followed by 20-h treatment with 1 mM antioxidant). The cells were gated excluding debris. The analysis was performed using 7-AAD (FL-3) and annexin V-Alexa Fluor 647 (FL-4) double staining.

Fig. 3

Quantitative results of flow cytometry on early apoptotic cells (A) and late apoptotic and necrotic cells (B). The measurements were taken after short-term (4-h exposure to 0.5 mM tBHP + 1 mM antioxidant, followed by 1-h treatment with 1 mM antioxidant) and long-term (4-h exposure to 0.5 mM tBHP + 1 mM antioxidant, followed by 20-h treatment with 1 mM antioxidant) treatments. The height of the columns indicates the mean of at least six replicates, and the error bars represent the standard deviation. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001 compared to the control group. a: p ≤ 0.0001, b: p ≤ 0.001, c: p ≤ 0.01 compared to the tBHP-only group. The antioxidant-only groups (groups 2–5 in Table 2) are not shown because the percentages of early and late apoptotic/necrotic cells in these groups were not significantly different from those in the control group (p > 0.05).

Fig. 4

Time-response for tBHP-induced generation of ROS during the short-term treatment (4-h exposure to 0.5 mM tBHP + 1 mM antioxidant, followed by 1-h treatment with 1 mM antioxidant). The ROS levels of the antioxidant-only groups (groups 2–5 in Table 2) are not shown because they were not significantly different from those of the control group (p > 0.05). A. Time-response for tBHP-induced generation of ROS during treatment with 0.5 mM tBHP and 1 mM antioxidants. The height of the columns represents the mean of at least three replicates, and the error bars indicate the standard deviation. *p ≤ 0.05, and ****p ≤ 0.0001 compared to the control group. B. ROS levels after the short-term treatment. The height of the columns represents the mean of at least seven replicates, and the error bars represent the standard deviation. **p ≤ 0.01 and ****p ≤ 0.0001 compared to the control group. ####p ≤ 0.0001 compared to tBHP-only group.

Fig. 5

Intracellular GSH levels in HLE B-3 cells after short-term treatment (4-h exposure to 0.5 mM tBHP + 1 mM antioxidant, followed by 1-h treatment with 1 mM antioxidant). The height of the columns represents the mean of three replicates, and the error bars indicate the standard deviation. ****p ≤ 0.0001 compared to the tBHP-only group. The GSH levels of the antioxidant-only groups (groups 2–5 in Table 2) are not shown because they were not significantly different from those of the control group (p > 0.05).

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