Reactive oxygen species in Ni(II) carcinogenesis

描述（由申请人提供）：含镍化合物是人类致癌物。其致癌作用的机制仍有待研究。近年来的研究表明，活性氧（ROS）可能起着重要的作用。我们假设镍诱导活性氧的产生，活性氧激活核转录因子，导致细胞转化和肿瘤发生。特异性Aim 1将检测和鉴定暴露于镍化合物的人支气管上皮细胞（BEAS-2B）和小鼠胚胎成纤维细胞产生的ROS，并研究其机制。我们假设镍（Ni3S2和NiCl2）可以通过cdc42和p47phox刺激细胞激活NADPH氧化酶，产生超氧自由基，然后转化为过氧化氢和羟基自由基。特异性目标2将验证细胞和体内对镍化合物的反应中NFAT和NFkappaB的激活需要ROS的假设。通过共转染NFkappaB-荧光素酶或NFAT-荧光素酶报告质粒和特异性ROS清除酶来评估ROS在细胞中镍诱导的NFAT和NFkappaB活化中的作用。在体内研究中，将使用BALB/c转基因小鼠，交替使用抗氧化酶或NADPH氧化酶（ROS生成酶）。特异性目的3将研究ROS、NFAT和NFkappaB在镍诱导的细胞转化和肿瘤发生中的作用。我们假设ROS激活转录因子并导致细胞转化和肿瘤发生。我们将利用DN-NFAT、DN- ikappabalpha和DN- IKKbeta的过表达来研究NFAT和NFkappaB在镍细胞转化和诱导肿瘤发生中的作用。ROS的作用将通过特异性抗氧化酶表达和NADPH氧化酶的变化来研究。我们预计镍通过ROS反应激活NFAT和NFkappaB，导致细胞转化和肿瘤发生。我们试图将细胞转化和肿瘤发生与特定的转录因子和特定的活性氧联系起来。本研究结果将阐明ROS和NFAT/NFkappaB信号在Ni化合物诱导的癌变中的作用。长期目标是对Ni的致癌作用机制有一个基本的认识；填补对暴露致癌风险评估机制信息的需求；提出早期发现的方法；并制定干预和预防策略。含镍化合物是人类致癌物。本项目将通过验证镍诱导ROS的产生，激活核转录因子，导致细胞转化和肿瘤发生的假说，探讨镍诱导癌变的机制。长期目标是了解镍致癌的机制；提出早期发现的方法；并制定干预和预防策略。

DESCRIPTION (provided by applicant): Nickel-containing compounds are human carcinogens. The mechanisms of their carcinogenic actions remain to be investigated. Recent studies have indicated that reactive oxygen species (ROS) may play an important role. We hypothesize that nickel induces generation of ROS, which activate nuclear transcription factors, leading to cell transformation and tumorigenesis. Specific Aim 1 will detect and identify ROS generated in human bronchial epithelial cells (BEAS-2B) and mouse embryo fibroblast cells exposed to nickel compounds and investigate the mechanism involved. We hypothesize that nickel (Ni3S2 and NiCl2) can stimulate the cells to activate NADPH oxidase via cdc42 and p47phox to produce superoxide radical, which is then converted to hydrogen peroxide and hydroxyl radical. Specific Aim 2 will test the hypothesis that ROS are required for activation of NFAT and NFkappaB in cells and in vivo in response to nickel compounds. The role of ROS in nickel-induced activation of NFAT and NFkappaB in cells will be evaluated by co-transfection of NFkappaB-luciferase or NFAT-luciferase reporter plasmids and specific ROS scavenger enzymes. For in vivo study, BALB/c transgenic mice with alternation of antioxidant enzymes or NADPH oxidase (ROS generating enzyme) will be employed. Specific Aim 3 will investigate the role of ROS, NFAT and NFkappaB in nickel-induced cell transformation and tumorigenesis. We hypothesize that ROS activate transcription factors and cause cell transformation and tumorigenesis. We will use overexpression of DN-NFAT, DN-IkappaBalpha and DN- IKKbeta to investigate the involvement of NFAT and NFkappaB in nickel-cell transformation and induced tumorigenesis. The role of ROS will be investigated using specific antioxidant enzyme expressions and NADPH oxidase alternation. We anticipate that nickel causes activation of NFAT and NFkappaB through ROS reactions, leading to cell transformation and tumorigenesis. We attempt to link the cell transformation and tumorigenesis with specific transcription factors and specific reactive oxygen species. The results obtained from this proposal will elucidate the role of ROS and NFAT/NFkappaB signaling in Ni compounds-induced carcinogenesis. The long term goals are to provide a fundamental understanding concerning the mechanism of carcinogenic actions of Ni; to fill a need for the mechanistic information of cancer risk assessment for exposure; to propose methods for early detection; and to develop intervention and prevention strategies. Nickel-containing compounds are human carcinogens. This project will investigate the mechanism of Ni-induced carcinogenesis by testing the hypothesis that nickel induces generation of ROS, which activate nuclear transcription factors, leading to cell transformation and tumorigenesis. The long term goals are to understand the mechanism of Ni-carcinogenesis; to propose methods for early detection; and to develop intervention and prevention strategies.