Thies R L, Autor A P
Department of Pathology, University of British Columbia, Vancouver, Canada.
Arch Biochem Biophys. 1991 May 1;286(2):353-63. doi: 10.1016/0003-9861(91)90051-j.
The vascular endothelium is a significant site for tissue injury following exposure to reactive oxygen species derived from a number of sources. In order to develop a better understanding of the mechanism(s) of oxidative damage, monolayer cultures of endothelial cells obtained from bovine pulmonary arteries were exposed to reactive oxygen species generated from the oxidation of dihydroxyfumarate (DHF) to diketosuccinate. Exposure to oxidizing DHF caused a loss of cell membrane integrity that was delayed in onset; that is, it did not begin until 2 h after the addition of DHF although reactive oxygen species are produced immediately by DHF in solution. Endothelial cell lysis by DHF was prevented by the simultaneous addition of superoxide dismutase (SOD), catalase (CAT), or deferoximine (DFX). This oxidant-induced lysis was unaffected by N,N,-diphenyl-p-phenylenediamine (DPPD), a potent inhibitor of lipid peroxidation. However, simultaneous addition of 3-aminobenzamide (3AB) and nicotinamide (NA), inhibitors of poly(ADP-ribose) polymerase, prevented cell lysis. Oxidant-induced loss of membrane integrity was preceded by the early appearance of DNA strand breaks, by increased levels of poly(ADP-ribose), the product of polymerase activity, and by depletion of NAD+ and ATP, followed by a decline in the energy charge ratio of the cells. None of these intracellular changes occurred when either SOD, CAT, or DFX were added at the same time as DHF, suggesting that O2-., H2O2, and HO. mediated these changes. The O2-. appears to be important in the autoxidation reaction of DHF. The latter two reactive oxygen species may be part of cellular-catalyzed Fenton chemistry. The increase in poly(ADP-ribose), depletion of NAD+, and the decline in ATP were also prevented by the addition of 3AB. The oxidant-induced DNA strand breakage was, however, unaffected by either 3AB or NA. Addition of 3AB immediately prior to the onset of cell lysis (2 h after the addition of DHF), prevented cell lysis, i.e., "rescued" the cells when neither SOD, CAT, nor DFX addition were effective. Concurrent with the "rescue" from lysis by 3AB, there was an increase in NAD+ content and a return of the energy charge ratio to control levels. The data presented in this study suggests that in endothelial cells, DNA is a very sensitive target for reactive oxygen species and HO. is the likely proximal damaging species.(ABSTRACT TRUNCATED AT 400 WORDS)
血管内皮是暴露于多种来源产生的活性氧后发生组织损伤的重要部位。为了更好地理解氧化损伤的机制，将从牛肺动脉获得的内皮细胞单层培养物暴露于二羟基富马酸（DHF）氧化为二酮琥珀酸产生的活性氧中。暴露于氧化型DHF会导致细胞膜完整性丧失，且这种丧失在开始时会延迟；也就是说，虽然DHF在溶液中会立即产生活性氧，但直到添加DHF后2小时才开始出现细胞膜完整性丧失。同时添加超氧化物歧化酶（SOD）、过氧化氢酶（CAT）或去铁胺（DFX）可防止DHF引起的内皮细胞裂解。这种氧化剂诱导的裂解不受脂质过氧化的有效抑制剂N，N-二苯基对苯二胺（DPPD）的影响。然而，同时添加聚（ADP-核糖）聚合酶的抑制剂3-氨基苯甲酰胺（3AB）和烟酰胺（NA）可防止细胞裂解。在氧化剂诱导的膜完整性丧失之前，会先出现DNA链断裂、聚合酶活性产物聚（ADP-核糖）水平升高、NAD +和ATP消耗，随后细胞的能量电荷比下降。当与DHF同时添加SOD、CAT或DFX时，这些细胞内变化均未发生，这表明超氧阴离子（O2-.）、过氧化氢（H2O2）和羟基自由基（HO.）介导了这些变化。O2-.似乎在DHF的自氧化反应中很重要。后两种活性氧可能是细胞催化的芬顿化学反应的一部分。添加3AB也可防止聚（ADP-核糖）增加、NAD +消耗和ATP下降。然而，氧化剂诱导的DNA链断裂不受3AB或NA的影响。在细胞裂解开始前（添加DHF后2小时）立即添加3AB可防止细胞裂解，即当添加SOD、CAT或DFX均无效时“挽救”细胞。与3AB对细胞裂解的“挽救”作用同时发生的是，NAD +含量增加，能量电荷比恢复到对照水平。本研究中的数据表明，在内皮细胞中，DNA是活性氧的非常敏感的靶点，HO.可能是直接造成损伤的活性物种。（摘要截短为400字）
