The Role Of Growth Factors And Inflammatory Mediators In

本项目的主要重点是研究参与砷皮肤毒性的复杂细胞因子调控网络。利用正常人表皮角质形成细胞(NHEK)进行的路径图谱研究表明，短期、无毒的砷暴露导致几类基因(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)的调控。我们目前正在使用多重RT-PCR技术分析暴露于饮用水中的砷化合物的Tg.AC小鼠皮肤中的基因表达。将小鼠暴露于饮用水中的亚砷酸钠(AsIII)24周。在第7周和第8周，一组小鼠接受了TPA以促进乳头状瘤的发展；另一组仍然没有得到促进。检测细胞因子的表达，以评估AsIII在促进和非促进、受损和非受损皮肤中调节基因表达的潜力。砷可促进TPA促进的Tg.AC皮肤中IL-6、转化生长因子-β、IL-1α和IL-1β的表达，而非促进皮肤中GM-CSF的表达。大多数环境砷污染是以无机三价亚砷酸盐和五价砷酸盐的形式存在的，它们在体内和体外经历了一系列的还原和甲基化反应，形成甲基化的代谢物。虽然曾经被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.AC小鼠暴露于四种砷制剂中的一种--AsIII、饮用水中的亚砷酸钠(ASV)、单甲基砷酸(MMA)或二甲基砷酸(DMA)--的皮肤基因表达；TPA促进了所有小鼠的皮肤基因表达。甲基化的代谢产物比AsIII或ASV的毒性更大。MMA和DMA下调IL-6、IL-1α和转化生长因子-β，上调HMOX。此外，MMA上调c-myc和转化生长因子-α的表达，而DMA上调EGR1的表达和下调GM-CSF的表达。ASV上调IL-6的表达。我们的初步结果支持细胞因子和生长因子调节是皮肤毒性和致癌的潜在机制的概念。我们将继续研究炎症和氧化应激相关基因的表达，并将与Michael Waalkes博士和Jie Liu博士合作，测量这些组织中的DNA甲基化，试图将基因表达谱与在这些小鼠中评估的其他毒性参数相关联。我们还在研究抗氧化剂在调节砷诱导的信号转导和细胞因子产生变化中的作用，因为活性氧物种(ROS)被认为与砷的致癌作用有关，而在皮肤中，ROS激活了调节细胞生长的丝裂原活化蛋白激酶(MAPKs)。绿茶多酚是一种抗癌化学防腐剂，具有清除ROS和调节MAPK活性的作用。由于ROS和MAPKs是由多酚调节的，我们研究了单独用AsIII和绿茶多酚表没食子儿茶素没食子酸酯(EGCG)处理人真皮成纤维细胞(HDF)后p42/44和p38MAPKs的激活(磷酸化)。在这些条件下，细胞抗氧化酶和应激感受器血红素加氧酶-1(HO-1)的表达被检测为潜在的ROS作用的衡量标准。Western印迹分析表明，非细胞毒性AsIII或过氧化氢可刺激HDF中p42/44和p38的磷酸化，并上调HO-1的表达。40微摩尔的EGCG短期(3小时)和长期(24小时)处理降低了p42/44的磷酸化，而与AsIII无关。此外，EGCG还阻止了AsIII后p42/44的最大磷酸化。EGCG对AsIII刺激的p38MAPK磷酸化无影响。与EGCG同时处理对AsIII刺激的HO-1表达无影响。这些数据表明，EGCG可阻止任何一种AsIII刺激的p42/44MAPK的最大磷酸化。我们继续与Miroslv Styblo博士和Luz Maria Del Razo博士合作，评估转化生长因子-α作为生物标志物在墨西哥饮用被砷污染的饮用水的个人暴露于砷中的有效性。我们还与A·杰伊·甘道菲博士进行了一项新的合作，评估了来自更多墨西哥人口的样本中的尿液转化生长因子α水平。

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.