THE PRODUCTION OF DNA STRAND BREAKS IN HUMAN-LEUKOCYTES BY SUPEROXIDE ANION MAY INVOLVE A METABOLIC PROCESS
THE PRODUCTION OF DNA STRAND BREAKS IN HUMAN-LEUKOCYTES BY SUPEROXIDE ANION MAY INVOLVE A METABOLIC PROCESS
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DOI：
10.1073/pnas.82.20.6820
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发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
KANABUSKAMINSKA, M
中科院分区：
文献类型：
--
作者：
BIRNBOIM, HC;KANABUSKAMINSKA, M
已知H2 O2能够诱导细胞内DNA的链断裂损伤，但在没有H2 O2的情况下O2-是否也能这样做尚不确定。区分两者的影响的困难在于，在生理条件下，O2-歧化为H2 O2很容易发生。当人白细胞被佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）刺激时，它们释放O2-，并且在几分钟内可以观察到细胞内DNA的断裂。我们试图确定是否O2-产生的是本身能够引起DNA损伤或是否H12 O2单独，或与O2-结合，是负责观察到的损伤。添加过氧化氢酶（高达250 μ g/ml）以除去H2 O2防止不超过约50%的DNA损伤。剩余的大部分损伤可以被阻断，以剂量依赖性的方式，由超氧化物歧化酶（SOD）或SOD模拟铜络合物，确定依赖于细胞外O2-的细胞内DNA损伤的分数。我们通过使用三种代谢毒物（氟化物、2-脱氧葡萄糖和A23187）研究了这一O2特异性组分。这些药物在很大程度上阻断了DNA损伤，而对细胞外O2-水平的影响很小。为了比较，H2 O2诱导的DNA损伤与葡萄糖氧化酶产生的H2 O2通量进行了研究。前两种代谢毒物几乎没有影响，而A23187确实抑制H2 O2诱导的DNA损伤。我们的结论是，O2-诱导的损伤发生的机制不同，至少在一定程度上，从H2 O2损伤途径，前者可能涉及一个或多个代谢步骤。
H2O2 is known to be capable of inducing strand-break damage in intracellular DNA, but whether O2- also can do so in the absence of H2O2 is uncertain. The difficulty in distinguishing the effects of the two is that, under physiological conditions, dismutation of O2- to H2O2 can readily occur. When human leukocytes are stimulated with phorbol 12-myrstate 13-acetate (PMA), they release O2- and within a few minutes strnd breakage in intracellular DNA can be observed. We have attempted to determine whether the O2- produced is itself capable of causing DNA damage or whether H12O2 alone, or in combination with O2-, is responsible for the observed damage. Addition of catalase (up to 250 .mu.g/ml) to remove H2O2 prevented no more than about 50% of the DNA damage. The majority of the remaining damage could be blocked, in a dose-dependent manner, by superoxide dismutase (SOD) or a SOD-mimetic copper complex, identifying a fraction of damage to intracellular DNA dependent upon extracellular O2-. We studied this O2--specific fraction through the use of three metabolic poisons (fluoride, 2-deoxyglucose, and A23187). These agents largely blocked DNA damage, while affecting extracellular O2- levels only slightly. For comparison, H2O2-induced DNA damage was studied with glucose oxidase to generate a flux of H2O2. The first two metabolic poisons had little effect, whereas A23187 did inhibit H2O2-induced DNA damage. We conclude that O2- induced damage occurs through a mechanism that differs, at least in part, from the H2O2 damage pathway and that the former may involve one or more metabolic steps.