OXIDANT INJURY OF CELLS - DNA STRAND-BREAKS ACTIVATE POLYADENOSINE DIPHOSPHATE-RIBOSE POLYMERASE AND LEAD TO DEPLETION OF NICOTINAMIDE ADENINE-DINUCLEOTIDE
OXIDANT INJURY OF CELLS - DNA STRAND-BREAKS ACTIVATE POLYADENOSINE DIPHOSPHATE-RIBOSE POLYMERASE AND LEAD TO DEPLETION OF NICOTINAMIDE ADENINE-DINUCLEOTIDE
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DOI：
10.1172/jci112436
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发表时间：
1986-04-01
影响因子：
15.9
通讯作者：
COCHRANE, CG
中科院分区：
文献类型：
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作者：
SCHRAUFSTATTER, IU;HINSHAW, DB;COCHRANE, CG
为了确定氧化剂诱导细胞损伤的生化基础，我们研究了P388D1暴露于过氧化氢后小鼠巨噬细胞的早期变化。当H_2O_2浓度为40微米或更高时，P388D_1细胞内细胞间总NAD+水平降低。0.1mM至2.5mM剂量的双氧水可使NAD在20min内耗尽80%。当H_2O_2的剂量为250微米或更低时，NAD和ATP的下降是可逆的。更高剂量的过氧化氢会导致细胞的最终溶解，导致NAD和ATP不可逆转的枯竭。Poly-ADP-核糖聚合酶是一种与DNA损伤和修复相关的核酶，它催化NAD转化为烟酰胺和蛋白质结合的多ADP-核糖，通过将细胞暴露在40µm或更高的H_2O_2中而激活。在佛波酯刺激的中性粒细胞孵育的外周淋巴细胞中也观察到多聚腺苷二磷酸核糖聚合酶的激活。通过测定胸腺嘧啶核苷掺入和细胞DNA单链断裂(SSB)来检测DNA改变的可能性。40微米或更高浓度的H_2O_2抑制DNA合成，并在不到30 S的时间内诱发SSB。这些结果表明，加入氧化剂后几秒钟内引起的DNA损伤可能导致多聚腺苷二磷酸-核糖聚合酶的刺激，从而导致NAD的下降。过度刺激多聚ADP-核糖聚合酶会导致NAD下降，足以干扰ATP的合成。
To determine the biochemical basis of the oxidant-induced injury of cells, we have studied early changes after exposure to P388D1 murine macrophages to hydrogen peroxide. Total interacelllar NAD+ levels in P388D1 cells decreased with H2O2 concentrations of 40 .mu.M or higher. Doses of H2O2 between 0.1 and 2.5 mM led to an 80% depletion of NAD within 20 min. With doses of H2O2 of 250 .mu.M or lower, the fall in NAD and, as shown previously, ATP, was reversible. Higher doses of H2O2 that cause ultimate lysis of the cells, induced an irreversible depletion of NAD and ATP. Poly-ADP-ribose polymerase, a nuclear enzyme associated with DNA damage and repair, which catalyzes conversion of NAD to nicotinamide and protein-bound poly-ADP-ribose, was activated by exposure of the cells to concentrations of 40 .mu.M H2O2 or higher. Activation of poly-ADP-ribose polymerase was also observed in peripheral lymphocytes incubated in the presence of phorbol myristate acetate-stimulated polymorphonuclear neutrophils. Examination of the possibility that DNA alteration was involved was performed by measurement of thymidine incorporation and determination of DNA single-strand breaks (SSB) in cells exposed to H2O2. H2O2 at 40 .mu.M or higher inhibited DNA synthesis, and induced SSB within less than 30 s. These results suggest that DNA damage induced within seconds after addition of oxidant may lead to stimulation of poly-ADP-ribose polymerase and a consequent fall in NAD. Excessive stimulation of poly-ADP-ribose polymerase leads to a fall in NAD sufficient to interfere with ATP synthesis.