Protection from Oxidative Insult in Glutathione Depleted Lens Epithelial Cells

doi:10.1006/exer.1998.0606

Experimental Eye Research

Volume 68, Issue 1, January 1999, Pages 117-127

https://doi.org/10.1006/exer.1998.0606 Get rights and content

Abstract

It has previously been shown that TEMPOL, n-propyl gallate and deferoxamine, compounds that limit the availability of Fe⁺²and prevent the generation of hydroxyl radicals, protect cultured rabbit lens epithelial cells from H₂O₂-induced damage. In view of the importance of glutathione as an antioxidant and the decrease in GSH that is known to accompany most forms of cataract, we investigated whether these compounds protected cultured lens epithelial cells from H₂O₂when the cells were artificially depleted of glutathione. Treatment of lens epithelial cells with 1-chloro-2,4-dinitrobenzene (CDNB), a compound that irreversibly binds to glutathione, or buthionine sulfoximine (BSO), an inhibitor of glutathione biosynthesis, reduced the glutathione content to an average of 15–20% of the control values without a concomitant increase in oxidized glutathione. Morphological changes were assessed by phase contrast and electron microscopy. In order to assess growth, cells in 5 ml serum-free MEM were exposed to an initial concentration of 0.05 mmH₂O₂(for 50,000 cells) or 2 doses of 0.5 mmH₂O₂(for 800,000 cells). After exposure to H₂O₂, medium was replaced with MEM plus 8% rabbit serum; cells were fed on days 3 and 6 and counted on day 7.

When 50,000 or 800,000 cells with decreased glutathione were exposed to 0.05 or 0.5 mmH₂O₂the H₂O₂was cytotoxic, whereas cells treated with H₂O₂alone remained viable but showed inhibited proliferation. An unexpected finding was that cells continued to remove H₂O₂from the medium at normal rates even when the GSH level was reduced. Cells treated with CDNB or BSO alone exhibited morphological and growth properties comparable to untreated cells. Cells treated with CDNB or BSO and then with H₂O₂exhibited decreased cell-to-cell contact, nuclear shrinkage, and arborization when viewed with phase-contrast microscopy and showed extensive nuclear and cytoplasmic degeneration at the EM level. Cell death was determined by dye exclusion and confirmed by video microscopy. When cells were treated with CDNB or BSO and subsequently treated with TEMPOL, n-propyl gallate or deferoxamine and then challenged with H₂O₂cytotoxicity was prevented and the cells were capable of growth. The data show that H₂O₂was not lethal to glutathione-depleted lens epithelial cells when they were treated with compounds that prevented the generation of reactive oxygen species. In addition, the results indicate that GSH has an important protective role independent of its ability to decompose H₂O₂via glutathione peroxidase.

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Cited by (40)

Hallmarks of lens aging and cataractogenesis
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Models of GSH depletion have provided a useful tool for studying the mechanisms of impaired GSH homeostasis observed in aging lenses. GSH-depleted lens epithelial cells were particularly susceptible to H2O2 (Reddan et al., 1999). To study the functional implications of reducing GSH levels in lens epithelial cells, applying inhibitors of GSH synthesis (e.g. buthionine sulfoximine [BSO]), GSH inhibitors (e.g. 1-chloro-2,4-dinitrobenzene, [CDNB]), leads to GSH pool depletion, decreased resistance to oxidative stress-induced damage, and cataract formation, both in vivo (Liang and Pelletier, 1988; Mårtensson et al., 1989; Calvin et al. 1992, 1996; Elanchezhian et al., 2009; Carey et al., 2011) and in vitro (Reddan et al., 1999; Shang et al., 2003).
Lens homeostasis and transparency are dependent on the function and intercellular communication of its epithelia. While the lens epithelium is uniquely equipped with functional repair systems to withstand reactive oxygen species (ROS)-mediated oxidative insult, ROS are not necessarily detrimental to lens cells. Lens aging, and the onset of pathogenesis leading to cataract share an underlying theme; a progressive breakdown of oxidative stress repair systems driving a pro-oxidant shift in the intracellular environment, with cumulative ROS-induced damage to lens cell biomolecules leading to cellular dysfunction and pathology. Here we provide an overview of our current understanding of the sources and essential functions of lens ROS, antioxidative defenses, and changes in the major regulatory systems that serve to maintain the finely tuned balance of oxidative signaling vs. oxidative stress in lens cells. Age-related breakdown of these redox homeostasis systems in the lens leads to the onset of cataractogenesis. We propose eight candidate hallmarks that represent common denominators of aging and cataractogenesis in the mammalian lens: oxidative stress, altered cell signaling, loss of proteostasis, mitochondrial dysfunction, dysregulated ion homeostasis, cell senescence, genomic instability and intrinsic apoptotic cell death.
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Previous measurements [35] demonstrated that the quantum yields of photodecomposition for KN-RNO conjugates are 2–3 orders of magnitude higher than that for KN itself. Besides, in opposite to stable RNO radicals, which demonstrate antioxidative properties [51,52], the short-lived radicals are expected to cause oxidative damages to biological tissues. In the present work, we tried to reduce the interaction between the photoexcited KN moiety and nitroxide group, and to improve the photostability of KN-RNO conjugates by either elongation of the linker connecting KN and RNO moieties (KN-R1NO), or by synthesis of a conjugate in which the radical center is shielded by bulky substituents (KN-R2NO).
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Tempol is a redox-cycling nitroxide that promotes the metabolism of many reactive oxygen species (ROS) and improves nitric oxide bioavailability. It has been studied extensively in animal models of oxidative stress. Tempol has been shown to preserve mitochondria against oxidative damage and improve tissue oxygenation. Tempol improved insulin responsiveness in models of diabetes mellitus and improved the dyslipidemia, reduced the weight gain and prevented diastolic dysfunction and heart failure in fat-fed models of the metabolic syndrome. Tempol protected many organs, including the heart and brain, from ischemia/reperfusion damage. Tempol prevented podocyte damage, glomerulosclerosis, proteinuria and progressive loss of renal function in models of salt and mineralocorticosteroid excess. It reduced brain or spinal cord damage after ischemia or trauma and exerted a spinal analgesic action. Tempol improved survival in several models of shock. It protected normal cells from radiation while maintaining radiation sensitivity of tumor cells. Its paradoxical pro-oxidant action in tumor cells accounted for a reduction in spontaneous tumor formation. Tempol was effective in some models of neurodegeneration. Thus, tempol has been effective in preventing several of the adverse consequences of oxidative stress and inflammation that underlie radiation damage and many of the diseases associated with aging. Indeed, tempol given from birth prolonged the life span of normal mice. However, presently tempol has been used only in human subjects as a topical agent to prevent radiation-induced alopecia.
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Citation Excerpt :
In this case, the diagnosis of pellagra was based on the patient's history and the presence of the 3 D syndrome: dermatitis, diarrhea, and dementia. Vitamin B3 plays a role in glutathione synthesis, the most important antioxidant of the crystalline lens.3,4 Our patient also suffered from beta thalassemia.
Soon after the diagnosis of pellagra in a 20-year-old patient with beta thalassemia, bilateral intumescent cataracts rapidly developed. We believe the patient's crystalline lenses were at an increased oxidative state due to iron overload from the thalassemia. Depletion of the lens epithelial cells of an important antioxidative agent (glutathione) as a result of niacin (vitamin B3) deficiency due to pellagra reduced the antioxidative capacity of the lenses. The oxidative damage led to rapid development of cataracts.

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^f1: Address correspondence to: John R. Reddan, Department of Biological Sciences, Oakland University, Rochester, M1 48309-4476, U.S.A.

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Regular articleProtection from Oxidative Insult in Glutathione Depleted Lens Epithelial Cells

Abstract

Regular article
Protection from Oxidative Insult in Glutathione Depleted Lens Epithelial Cells