Reddan J R, Giblin F J, Dziedzic D C, McCready J P, Schrimscher L, Reddy V N
Department of Biological Sciences, Oakland University, Rochester, MI 48309-4401.
Exp Eye Res. 1988 Feb;46(2):209-21. doi: 10.1016/s0014-4835(88)80078-2.
Our previous studies on cultured rabbit lens epithelial cells from 4-day-old rabbits showed that the glutathione redox cycle plays an important role in detoxifying H2O2, a potentially damaging oxidant present in the aqueous humor. Here we report the effect of donor age and cell density on the ability of cultured rabbit lens epithelial cells to detoxify H2O2. Lens epithelial cells (8 x 10(5] from a 4-day-old and an 8-year-old rabbit were cultured for 3 hr in minimal essential medium (MEM) or in MEM containing 0.01-0.1 mM H2O2 maintained with glucose oxidase. We determined the effect of H2O2 on the level of reduced glutathione (GSH), hexose monophosphate shunt activity, cell growth, and morphology. For growth studies, cells were exposed to the desired concentration of H2O2 for 3 hr and then cultured in MEM plus 10% rabbit serum for 7 days and counted. Young and old untreated cells contained high levels (30-40 nmol/8 x 10(5) cells) of GSH. Cells from 4-day-old rabbits tolerated 0.03 mM H2O2 with no effect on GSH and a minimal decrease in subsequent cell growth. However, in the older cells, GSH and growth were substantially diminished following treatment with 0.03 mM H2O2. Cells plated out at high density (8 x 10(5] were more tolerant of 0.03 mM H2O2 than cells plated out at low density (5 x 10(4]. Maximum shunt activity in the younger cells exposed to H2O2 was twice that of the older cells and occurred at a higher level of H2O2 (0.04 compared with 0.03 mM). Enzyme activities in untreated young and old cells were comparable for hexokinase, glucose-6-phosphate dehydrogenase, and glutathione peroxidase. However, glutathione reductase activity was 50% lower in the cells from the 8-year-old rabbit. The toxicity of H2O2 to cultured lens epithelial cells was directly related to donor age and inversely related to cell density. The damage in the older lens epithelial cells at 0.03 mM H2O2 was apparently due, in part, to a diminished response of the glutathione redox cycle to oxidative challenge.
我们之前对4日龄兔培养的晶状体上皮细胞进行的研究表明，谷胱甘肽氧化还原循环在清除过氧化氢（房水中存在的一种具有潜在损伤性的氧化剂）方面发挥着重要作用。在此，我们报告供体年龄和细胞密度对培养的兔晶状体上皮细胞清除过氧化氢能力的影响。将来自4日龄和8岁兔的晶状体上皮细胞（8×10⁵个）在最低限度基本培养基（MEM）中或在含有0.01 - 0.1 mM过氧化氢且用葡萄糖氧化酶维持的MEM中培养3小时。我们测定了过氧化氢对还原型谷胱甘肽（GSH）水平、磷酸己糖旁路活性、细胞生长和形态的影响。对于生长研究，将细胞暴露于所需浓度的过氧化氢3小时，然后在添加10%兔血清的MEM中培养7天并计数。未处理的年轻和年老细胞含有高水平（30 - 40 nmol/8×10⁵个细胞）的GSH。4日龄兔的细胞能耐受0.03 mM过氧化氢，对GSH无影响，且后续细胞生长仅有轻微下降。然而，在年老细胞中，用0.03 mM过氧化氢处理后，GSH和细胞生长显著减少。高密度接种（8×10⁵个）的细胞比低密度接种（5×10⁴个）的细胞更能耐受0.03 mM过氧化氢。暴露于过氧化氢的年轻细胞中的最大旁路活性是年老细胞的两倍，且出现在更高水平的过氧化氢（0.04 mM，而年老细胞为0.03 mM）时。未处理的年轻和年老细胞中己糖激酶、葡萄糖 - 6 - 磷酸脱氢酶和谷胱甘肽过氧化物酶的酶活性相当。然而，8岁兔的细胞中谷胱甘肽还原酶活性低50%。过氧化氢对培养的晶状体上皮细胞的毒性与供体年龄直接相关，与细胞密度呈负相关。0.03 mM过氧化氢对年老晶状体上皮细胞的损伤显然部分归因于谷胱甘肽氧化还原循环对氧化应激的反应减弱。
