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The Role Of Growth Factors And Inflammatory Mediators In

生长因子和炎症介质在疾病中的作用

基本信息

批准号：
7168266
负责人：
Dori R Germolec
金额：
--
依托单位：
NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

The main focus of this project is to investigate the complex cytokine regulatory network involved in arsenic-induced dermatotoxicity. Pathway mapping studies using normal human epidermal keratinocytes (NHEK) indicated that short-term, non-toxic arsenic-exposure results in the modulation of multiple genes from several classes (e.g., oxidative stress, glutathione metabolism, heat shock/stress response, cell proliferation and DNA damage). We are currently profiling gene expression in the skin of Tg.AC mice exposed to arsenicals in the drinking water using multiplex RT-PCR. Mice were exposed to sodium arsenite (AsIII) in drinking water for 24 weeks. At weeks 7 and 8, one group of mice received TPA to promote papilloma development; another group remained unpromoted. Cytokine expression was measured to evaluate the potential for AsIII to regulate gene expression in promoted and non-promoted, lesioned and non-lesioned skin. Arsenic enhanced IL-6, TGF-beta, IL-1alpha, and IL-1beta expression in TPA-promoted Tg.AC skin, and GM-CSF expression in non-promoted skin. Most environmental arsenic contamination is in the form of inorganic trivalent arsenite and pentavalent arsenate, which undergo a series of reduction and methylation reactions in vivo and in vitro to form methylated metabolites. While it was once believed that inorganic arsenicals were detoxified by reduction and methylation, recent evidence suggests that the metabolites may be more toxic than the parent compounds. We are comparing gene expression in skin from Tg.AC mice exposed to one of four arsenicals, AsIII, sodium arsenate (AsV), monomethylarsenic acid (MMA) or dimethylarsenic acid (DMA) in the drinking water; all mice were promoted with TPA. The methylated metabolites were more toxic than either AsIII or AsV. MMA and DMA down-regulated IL-6, IL-1alpha, and TGF-beta, and they up-regulated HMOX. In addition, MMA up-regulated c-myc and TGF-alpha, while DMA up-regulated EGR1 and down-regulated GM-CSF. AsV up-regulated IL-6. Our preliminary results support the concept of cytokine and growth factor regulation as a potential mechanism for dermal toxicity and carcinogenesis. We will continue to examine the expression of genes involved in inflammation and oxidative stress, and in collaboration with Drs. Michael Waalkes and Jie Liu, will measure DNA methylation in these tissues in an attempt to correlate gene expression profiles with other parameters of toxicity evaluated in these mice. We are also examining the role of antioxidants in modulating arsenic-induced alterations in signal transduction and cytokine production, as reactive oxygen species (ROS) are proposed to contribute to arsenic carcinogenesis and in skin ROS activate mitogen-activated protein kinases (MAPKs) that regulate cell growth. Green tea polyphenols are purported cancer chemopreventive agents that exert their scavenging effects against ROS and also modulate MAPK activity. Because ROS and MAPKs are modulated by polyphenols, we have examined the activation (phosphorylation) of p42/44 and p38 MAPKs in human dermal fibroblasts (HDF) following treatment with AsIII alone and in the presence of the green tea polyphenol epigallocatechin 3-gallate (EGCG). Expression of the cellular antioxidant enzyme and stress sensor heme oxygenase-1 (HO-1) was examined under these conditions as a measure of potential ROS action. Western blot analyses indicated that noncytotoxic AsIII or hydrogen peroxide stimulated p42/44 and p38 phosphorylation and elevated HO-1 expression in HDF. Short-term (3 hr) and long-term (24 hr) treatment with 40 micromolar EGCG reduced p42/44 phosphorylation independent of AsIII. In addition, EGCG prevented maximal p42/44 phosphorylation post-AsIII. EGCG did not influence p38 MAPK phosphorylation stimulated by AsIII. No effect on HO-1 expression stimulated by AsIII was observed with concurrent EGCG treatment. These data indicate that EGCG prevents maximal p42/44 MAPK phosphorylation stimulated by either AsIII. We have continued an on-going collaboration with Drs. Miroslav Styblo and Luz Maria Del Razo to evaluate the utility of TGF-alpha as a biomarker for arsenic exposure in individuals consuming arsenic contaminated drinking water in Mexico. We are also evaluating urinary-TGF alpha levels in samples from additional Mexican populations in a new collaboration with Dr. A. Jay Gandolfi.

本项目的主要重点是研究涉及砷诱导皮肤毒性的复杂细胞因子调节网络。利用正常人类表皮角质形成细胞（NHEK）进行的途径作图研究表明，短期、无毒的砷暴露会导致来自几个类别的多个基因的调节（例如，氧化应激、谷胱甘肽代谢、热休克/应激反应、细胞增殖和DNA损伤）。我们目前正在分析Tg皮肤中的基因表达。使用多重RT-PCR检测暴露于饮用水中砷的交流小鼠。小鼠在饮用水中接触亚砷酸钠（AsIII） 24周。在第7周和第8周，一组小鼠接受TPA促进乳头瘤的发展；另一组人仍然没有晋升。通过测量细胞因子表达来评估AsIII在促进和非促进、损伤和非损伤皮肤中调节基因表达的潜力。砷增强了tpa促进的Tg中IL-6、tgf - β、il -1 α和il -1 β的表达。AC皮肤和GM-CSF在非促进皮肤中的表达。环境砷污染大多以无机三价亚砷酸盐和五价砷酸盐的形式存在，在体内和体外经过一系列的还原和甲基化反应，形成甲基化代谢物。虽然曾经认为无机砷是通过还原和甲基化来解毒的，但最近的证据表明，代谢物可能比母体化合物毒性更大。我们正在比较Tg在皮肤中的基因表达。小鼠暴露于四种砷，砷酸钠（AsV），单甲基拉森酸（MMA）或二甲基拉森酸（DMA）的饮用水中的一种；所有小鼠都被TPA促进。甲基化代谢物毒性大于asii或AsV。MMA和DMA下调IL-6、il -1 α和tgf - β，上调HMOX。此外，MMA上调c-myc和tgf - α，而DMA上调EGR1，下调GM-CSF。AsV上调IL-6。我们的初步结果支持细胞因子和生长因子调节作为皮肤毒性和致癌的潜在机制的概念。我们将继续研究与炎症和氧化应激有关的基因表达，并与dr。Michael Waalkes和Jie Liu将测量这些组织中的DNA甲基化，试图将基因表达谱与这些小鼠毒性评估的其他参数联系起来。我们还研究了抗氧化剂在砷诱导的信号转导和细胞因子产生改变中的作用，因为活性氧（ROS）被认为有助于砷致癌，并且在皮肤中ROS激活丝裂原活化蛋白激酶（MAPKs）调节细胞生长。绿茶多酚被认为是癌症化学预防剂，对活性氧发挥清除作用，并调节MAPK活性。由于ROS和mapk是由多酚调节的，我们研究了单独用AsIII治疗和绿茶多酚表没食子儿茶素3-没食子酸酯（EGCG）存在下，人真皮成纤维细胞（HDF）中p42/44和p38 mapk的激活（磷酸化）。在这些条件下检测细胞抗氧化酶和应激传感器血红素氧化酶-1 （HO-1）的表达，以衡量潜在的ROS作用。Western blot分析表明，非细胞毒性的AsIII或过氧化氢刺激了HDF中p42/44和p38的磷酸化和HO-1的表达升高。40微摩尔EGCG短期（3小时）和长期（24小时）治疗可降低p42/44磷酸化，不依赖于AsIII。此外，EGCG阻止了asiii后p42/44的最大磷酸化。EGCG不影响AsIII刺激的p38 MAPK磷酸化。同时处理EGCG对AsIII刺激HO-1表达无影响。这些数据表明，EGCG可以阻止任何一种AsIII刺激的p42/44 MAPK的最大磷酸化。我们继续与dr。Miroslav stybloo和Luz Maria Del Razo评估了tgf - α在墨西哥饮用砷污染饮用水的个体中作为砷暴露生物标志物的效用。在与a . Jay Gandolfi博士的新合作中，我们还评估了来自其他墨西哥人群样本中的尿液tgf - α水平。