Potts R J, Bespalov I A, Wallace S S, Melamede R J, Hart B A
Department of Biochemistry, C-440 Given Medical Building, University of Vermont College of Medicine, Burlington, VT 05405-0068, USA.
Toxicology. 2001 Mar 21;161(1-2):25-38. doi: 10.1016/s0300-483x(00)00419-4.
This study evaluated the effects of cadmium (Cd) adaptation in cultured alveolar epithelial cells on oxidant-induced DNA damage and its subsequent repair. Using the comet assay, we determined that lower levels of DNA damage occurred in Cd-adapted cells compared with non-adapted cells following treatment of cells with hydrogen peroxide (H(2)O(2)). This may be a consequence of increased thiol-containing antioxidants that were observed in adapted cells, including metallothionein and glutathione. Cd-adapted cells were, however, less efficient at repairing total oxidative DNA damage compared with non-adapted cells. Subsequently, we investigated the effect of Cd adaptation on the repair of particular oxidized DNA lesions by employing lesion-specific enzymes in the comet assay, namely formamidopyrimidine DNA glycosylase (Fpg), an enzyme that predominantly repairs 8-oxoguanine (8-oxoG), and endonuclease III, that is capable of repairing oxidized pyrimidines. The data demonstrated that adaptation to Cd results in significantly impaired repair of both Fpg- and endonuclease III-sensitive lesions. In addition, in situ detection of 8-oxoG using a recombinant monoclonal antibody showed that Cd-adaptation reduces the repair of this oxidative lesion after exposure of cells to H(2)O(2). Activities of 8-oxoG-DNA glycosylase and endonuclease III were determined in whole cell extracts using 32P-labeled synthetic oligonucleotides containing 8-oxoG and dihydrouracil sites, respectively. Cd adaptation was associated with an inhibition of 8-oxoG-DNA glycosylase and endonuclease III enzyme activity compared with non-adapted cells. In summary, this study has shown that Cd adaptation: (1) reduces oxidant-induced DNA damage; (2) increases the levels of key intracellular antioxidants; (3) inhibits the repair of oxidative DNA damage.
本研究评估了培养的肺泡上皮细胞中镉（Cd）适应对氧化剂诱导的DNA损伤及其后续修复的影响。使用彗星试验，我们确定在用过氧化氢（H₂O₂）处理细胞后，与未适应细胞相比，适应Cd的细胞中发生的DNA损伤水平较低。这可能是适应细胞中观察到的含硫醇抗氧化剂增加的结果，包括金属硫蛋白和谷胱甘肽。然而，与未适应细胞相比，适应Cd的细胞在修复总氧化DNA损伤方面效率较低。随后，我们通过在彗星试验中使用损伤特异性酶，即主要修复8-氧代鸟嘌呤（8-oxoG）的甲酰胺嘧啶DNA糖基化酶（Fpg）和能够修复氧化嘧啶的核酸内切酶III，研究了Cd适应对特定氧化DNA损伤修复的影响。数据表明，对Cd的适应导致Fpg和核酸内切酶III敏感损伤的修复显著受损。此外，使用重组单克隆抗体对8-oxoG进行原位检测表明，Cd适应降低了细胞暴露于H₂O₂后这种氧化损伤的修复。分别使用含有8-oxoG和二氢尿嘧啶位点的³²P标记合成寡核苷酸在全细胞提取物中测定8-oxoG-DNA糖基化酶和核酸内切酶III的活性。与未适应细胞相比，Cd适应与8-oxoG-DNA糖基化酶和核酸内切酶III酶活性的抑制有关。总之，本研究表明Cd适应：（1）减少氧化剂诱导的DNA损伤；（2）增加关键细胞内抗氧化剂的水平；（3）抑制氧化DNA损伤的修复。
