Investigating Markers of DNA Oxidation, Lipid Peroxidation and Stress Response in Human Lens Epithelial Cell Lines

Abstract

Abstract: : Purpose: Damage resulting from oxidative stress includes DNA oxidation and mutations, protein destruction or alterations, and lipid peroxidation. Oxidative stress and damage have been implicated in cataract formation. Compounds that induce oxidative stress in the lens therefore may be cataractogenic. The ability to identify drug candidates with a potential to result in cataract formation early using in vitro assays is desirable. The purpose of this study was to examine changes in several markers of oxidative stress and damage in human lens epithelial cells treated with known oxidants and stressors. The markers included 8–hydroxyguanine [8–OHG], a marker of DNA oxidation, 4–hydroxy–2–nonenal [4–HNE], a marker of lipid peroxidation, and translocation of nuclear factor kappa B [NFΚB], a transcription factor involved in early cellular stress response. Methods: The human lens epithelial cell lines SRA 01/ 04 and/or HLE B3 were treated with TNF–α [1 – 10 ng/ml], IL–1α [2.5 – 250 ng/ml], H₂O₂ [0.5 – 1mM], diamide [50 – 300 µM] or U18666A [50 – 100 µM] in chamber slides or 96–well plates for 0.5 – 4 hr. Cells were fixed and labeled with antibodies for 8–OHG, 4–HNE, or NFΚB. Fluorescent detection of labeling was quantified using a Laser Scanning Cytometer for 8–OHG and 4–HNE and a Cellomics ArrayScan for NFΚB. Cytotoxicity was determined by the WST–1 assay. Results: Time and concentration–dependent changes in fluorescent labeling of 8–OHG, 4–HNE, or NFΚB were observed with all treatments. Increases in 4–HNE labeling were greatest at 0.5 hr with H₂O₂ or diamide treatment. Increases in 8–OHG labeling were greatest at 0.5 hr with diamide treatment and at 1.5 hr with H₂O₂ treatment. Peak NFΚB translocation occurred at 0.5 hr with TNF–α, IL–1α, or U18666A treatment. H₂O₂ or diamide treatment resulted in only a small increase in NFΚB translocation at 1 hr. Conclusions: Changes in 8–OHG, 4–HNE, or NFΚB may be useful in vitro markers of drug–induced oxidative stress or damage. However, the relatively high concentrations of H₂O₂ or diamide needed to cause significant changes in lens epithelial cells may reflect strong antioxidant defense mechanisms in these cells. Future studies will assess the effects of additional known oxidants and cataractogens on these markers as well as investigating changes in 8–OHG, 4–HNE, or NFΚB in other ocular [ex. RPE] and non–ocular cell types to determine if the resistance observed is unique to lens cells.

Keywords: oxidation/oxidative or free radical damage • stress response • transcription factors