Reddan J R, Giblin F J, Kadry R, Leverenz V R, Pena J T, Dziedzic D C
Department of Biological Sciences, Oakland University, Rochester, MI, 48309-4476, USA.
Exp Eye Res. 1999 Jan;68(1):117-27. doi: 10.1006/exer.1998.0606.
It has previously been shown that TEMPOL, n-propyl gallate and deferoxamine, compounds that limit the availability of Fe+2 and 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 H2O2 when 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 mm H2O2 (for 50,000 cells) or 2 doses of 0.5 mm H2O2 (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 mm H2O2 the H2O2 was cytotoxic, whereas cells treated with H2O2 alone remained viable but showed inhibited proliferation. An unexpected finding was that cells continued to remove H2O2 from 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 H2O2 exhibited 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 H2O2 cytotoxicity was prevented and the cells were capable of growth. The data show that H2O2 was 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 H2O2 via glutathione peroxidase.
先前的研究表明，TEMPOL、没食子酸正丙酯和去铁胺等化合物能够限制Fe+2的可用性并防止羟基自由基的产生，可保护培养的兔晶状体上皮细胞免受H2O2诱导的损伤。鉴于谷胱甘肽作为抗氧化剂的重要性以及已知在大多数形式的白内障中伴随出现的谷胱甘肽减少情况，我们研究了在人工耗尽谷胱甘肽的情况下，这些化合物是否能保护培养的晶状体上皮细胞免受H2O2的损伤。用1-氯-2,4-二硝基苯（CDNB，一种与谷胱甘肽不可逆结合的化合物）或丁硫氨酸亚砜胺（BSO，谷胱甘肽生物合成的抑制剂）处理晶状体上皮细胞，可将谷胱甘肽含量降低至对照值的平均15 - 20%，而氧化型谷胱甘肽没有相应增加。通过相差显微镜和电子显微镜评估形态学变化。为了评估生长情况，将5 ml无血清MEM中的细胞暴露于初始浓度为0.05 mM H2O2（针对50,000个细胞）或2剂0.5 mM H2O2（针对800,000个细胞）。暴露于H2O2后，将培养基换成含8%兔血清的MEM；在第3天和第6天给细胞喂食，并在第7天计数。当谷胱甘肽减少的50,000个或800,000个细胞暴露于0.05或0.5 mM H2O2时，H2O2具有细胞毒性，而仅用H2O2处理的细胞仍存活但增殖受到抑制。一个意外的发现是，即使谷胱甘肽水平降低，细胞仍以正常速率继续从培养基中清除H2O2。单独用CDNB或BSO处理的细胞表现出与未处理细胞相当的形态学和生长特性。用CDNB或BSO处理后再用H2O2处理的细胞，在相差显微镜下观察显示细胞间接触减少、核收缩和分支，在电子显微镜水平显示广泛的核和细胞质变性。通过染料排斥法确定细胞死亡，并通过视频显微镜进行确认。当细胞先用CDNB或BSO处理，随后用TEMPOL、没食子酸正丙酯或去铁胺处理，然后再用H2O2攻击时，细胞毒性得到预防且细胞能够生长。数据表明，当用防止活性氧产生的化合物处理时，H2O2对谷胱甘肽耗尽的晶状体上皮细胞并不致命。此外，结果表明谷胱甘肽具有重要的保护作用，这与其通过谷胱甘肽过氧化物酶分解H2O2的能力无关。
