Zhang Z, Huang C, Li J, Leonard S S, Lanciotti R, Butterworth L, Shi X
Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505, USA.
Arch Biochem Biophys. 2001 Aug 15;392(2):311-20. doi: 10.1006/abbi.2001.2464.
While vanadium compounds are known as potent toxicants as well as carcinogens, the mechanisms of their toxic and carcinogenic actions remain to be investigated. It is believed that an improper cell growth regulation leads to cancer development. The present study examines the effects of vanadate on cell cycle control and involvement of reactive oxygen species (ROS) in these vanadate-mediated responses in a human lung epithelial cell line, A549. Under vanadate stimulation, A549 cells generated hydroxyl radical (OH), as determined by electron spin resonance (ESR), and hydrogen peroxide (H2O2) and superoxide anion (O2-), as detected by flow cytometry using specific dyes. The mechanism of ROS generation involved the reduction of molecular oxygen to O2*- by both a flavoenzyme-containing NADPH complex and the mitochondria electron transport chain. The O2*- in turn generated H2O2, which reacted with vanadium(IV) to generate *OH radical through a Fenton-type reaction (V(IV) + H2O2 --> V(V) +OH + OH-). The ROS generated by vanadate induced G2/M phase arrest in a time- and dose-dependent manner as determined by measuring DNA content. Vanadate also increased p21 and Chk1 levels and reduced Cdc25C expression, leading to phosphorylation of Cdc2 and a slight increase in cyclin B1 expression as analyzed by Western blot. Catalase, a specific antioxidant for H2O2, decreased vanadate-induced expression of p21 and Chk1, reduced phosphorylation of Cdc2Tyr15, and decreased cyclin B1 levels. Superoxide dismutase, a scavenger of O2-, or sodium formate, an inhibitor of *OH, had no significant effects. The results obtained from the present study demonstrate that among ROS, H2O2 is the species responsible for vanadate-induced G2/M phase arrest. Several regulatory pathways are involved: (1) activation of p21, (2) an increase of Chk1 expression and inhibition of Cdc25C, which results in phosphorylation of Cdc2 and possible inactivation of cyclin B1/Cdc2 complex.
虽然钒化合物被认为是强效毒物和致癌物，但其毒性和致癌作用机制仍有待研究。人们认为细胞生长调节不当会导致癌症发展。本研究考察了钒酸盐对细胞周期调控的影响，以及活性氧（ROS）在人肺上皮细胞系A549中这些钒酸盐介导反应中的作用。在钒酸盐刺激下，通过电子自旋共振（ESR）测定，A549细胞产生了羟基自由基（OH），并通过使用特定染料的流式细胞术检测到过氧化氢（H2O2）和超氧阴离子（O2-）。ROS产生的机制涉及含黄素酶的NADPH复合物和线粒体电子传递链将分子氧还原为O2*-。O2*-进而产生H2O2，H2O2与钒（IV）反应通过芬顿型反应（V（IV）+H2O2→V（V）+OH+OH-）生成OH自由基。通过测量DNA含量确定，钒酸盐产生的ROS以时间和剂量依赖性方式诱导G2/M期阻滞。通过蛋白质印迹分析，钒酸盐还增加了p21和Chk1水平，降低了Cdc25C表达，导致Cdc2磷酸化以及细胞周期蛋白B1表达略有增加。过氧化氢酶是H2O2的特异性抗氧化剂，它降低了钒酸盐诱导的p21和Chk1表达，减少了Cdc2Tyr15的磷酸化，并降低了细胞周期蛋白B1水平。超氧化物歧化酶是O2*-的清除剂，或甲酸是*OH的抑制剂，它们没有显著影响。本研究获得的结果表明，在ROS中，H2O2是导致钒酸盐诱导G2/M期阻滞的物质。涉及几种调节途径：（1）p21的激活，（2）Chk1表达的增加和Cdc25C的抑制，这导致Cdc2磷酸化以及细胞周期蛋白B1/Cdc2复合物可能失活。
