The Role Of Growth Factors And Inflammatory Mediators In

本项目的主要重点是研究砷诱导的皮肤毒性中复杂的细胞因子调控网络。我们已经完成了体外研究，评估了砷III和砷V的毒性，以及它们在正常人角质形成细胞培养物中的单甲基和二甲基代谢产物。我们正在将这些结果与正在进行的研究进行比较，以检查砷III和砷V及其单甲基和二甲基代谢产物在Tg.AC转基因小鼠模型中的相对毒性。使用NHEK的微阵列研究表明，短期、无毒的砷暴露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）。我们的微阵列研究进一步揭示，环氧合酶-2（考克斯-2），一个在皮肤癌中起重要作用的基因，在砷暴露后以剂量依赖的方式被高度诱导。随后的研究表明，砷还提高了NHEK中考克斯-2蛋白的水平。这些事件似乎是依赖于信号通过有丝分裂原和应力相关的激酶，其活动是由砷调制。砷对考克斯-2的诱导作用还与培养基中前列腺素水平的增加和DNA合成的增加有关，前列腺素是考克斯-2活性的终产物。正在进行的其他微阵列研究旨在研究砷暴露后NHEK和HaCaT角质形成细胞（常用于砷，氧化应激和表皮研究的永生化细胞系）中相似/不相似的基因表达谱。还进行了途径作图、FACs分析和活力研究，并将用于将基因表达改变与生理效应（例如，细胞周期停滞、DNA损伤、升高的细胞增殖反应等）。谷胱甘肽在砷诱导的毒性中的作用也正在研究中，我们的研究表明，谷胱甘肽可能发挥作用，不仅在减弱砷的毒性作用，但也在砷诱导的增殖的积极调制。砷介导的影响，真皮成纤维细胞的活力和长期生长进行了评估。数据表明，活力的影响程度相同，在人类角质形成细胞，这表明砷耐受性在两种细胞类型之间的密切关系。真皮成纤维细胞不能耐受砷浓度> 5 mM的长期生长，与角质形成细胞相似。已从时间和剂量反应实验中获得了真皮成纤维细胞的RNA样品，并正在通过RT-PCR和北方印迹法进行分析。假设砷暴露改变了真皮成纤维细胞中有丝分裂原如KGF和TGF的表达/产生/诱导，并且预测这些变化会改变角质形成细胞的生理学/功能（例如，增殖、分化和细胞因子表达）。该项目的第二部分使用内毒素超敏反应的体内模型来检查TNF信号传导和细胞凋亡之间的关系。我们的特点是TCDD诱导的肝损伤的动力学在肝脏中，通过评估血清酶水平，定量凋亡细胞，并在关键时间点在B6 C3 F1小鼠暴露于TCDD在存在或不存在内毒素的情况下，在基因表达，半胱天冬酶活性和NF κ B活性的测量变化。联合TCDD/内毒素治疗改变了TCDD诱导的肝毒性的动力学，血清酶水平峰值比单独TCDD提前3-4天。与对照组相比，TCDD处理小鼠的肝细胞百分比增加，显示经典的凋亡表型。与诱导细胞凋亡一致，TCDD刺激LPS和盐水处理的小鼠在第10天和第14天TNF α的表达。与此相反，Fas mRNA的表达迅速调制TCDD曝光后。在40 mg LPS下，在TCDD暴露的小鼠中，caspase 3活性在第3天和第7天被刺激，然后在第10天和第14天被抑制。TNFR 1、TNFR 2和NF κ B基因表达、IkappaB α和IkappaB β蛋白表达以及NF κ B DNA结合活性在研究条件下似乎不受TCDD调节。先前的研究表明，当啮齿动物在内毒素暴露之前用TCDD处理时，毒性显著增加，并且在该模型中用放线菌酮抑制蛋白质合成阻断了TCDD诱导的对TNF的敏感性。放线菌酮的保护作用，TNF α基因表达的改变，以及缺乏参与NF κ B表明，TCDD诱导的细胞凋亡可能是由TNF通过TNF α/TNFR 2/TRAF途径介导的，但它不是抑制NF κ B的结果。

The main focus of this project is to investigate the complex cytokine regulatory network involved in arsenic-induced dermatotoxicity. We have completed in vitro studies that evaluate the toxicity of both ArsenicIII and ArsenicV, and their monomethyl and dimethyl metabolites in normal human keratinocyte cultures. We are comparing these results with ongoing studies to examine the relative toxicity of ArsenicIII and ArsenicV, and their monomethyl and dimethyl metabolites in the Tg.AC transgenic mouse model. Microarray studies using NHEK indicate 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). Our microarray studies further revealed that the expression of cyclooxygenase-2 (COX-2), a gene that plays a prominent role in skin cancer, is highly induced in a dose-dependent manner following arsenic exposure. Subsequent studies indicate that arsenic also elevates the level of COX-2 protein in NHEK. These events appear to be dependent on signaling via mitogen- and stress-related kinases; the activities of which are modulated by arsenic. The induction of COX-2 by arsenic also correlated with increased prostaglandin levels, an end product of COX-2 activty, in culture media and increased DNA synthesis. Additional microarray studies in progress are designed to examine similar/dissimilar gene expression profiles in NHEK and HaCaT keratinocytes (an immortalized cell line commonly used in arsenic, oxidative stress, and epidermal research) following arsenic exposure. Pathway mapping, FACs analysis and viability studies also have been performed and will be used to correlate gene expression alterations with physiological effects (e.g., cell cycle arrest, DNA damage, elevated cell proliferative responses, etc.). The role of glutathione in arsenic-induced toxicity also is being examined and our studies suggest that glutathione may play a role not only in attenuating the toxic effects of arsenic but also in the positive modulation of arsenic-induced proliferation. Arsenic-mediated effects on viability and long-term growth in dermal fibroblasts have been evaluated. Data indicate that viability is affected to the same degree as that seen in human keratinocytes, suggesting a close relationship between arsenic tolerance in both cell types. Dermal fibroblasts cannot tolerate long-term growth in concentrations of arsenic > 5 mM, similar to that of keratinocytes. RNA samples from dermal fibroblasts have been obtained from time- and dose-response experiments and are in the process of being analyzed via RT-PCR and northern blotting. It is hypothesized that arsenic exposure alters the expression/production/inducibility of mitogens such as KGF and TGF in dermal fibroblasts, and these changes are predicted to alter keratinocyte physiology/function (e.g., proliferation, differentiation and cytokine expression). A second component of this project uses in vivo models of endotoxin hypersensitivity to examine the relationship between TNF signaling and apoptosis. We have characterized the kinetics of TCDD-induced hepatic damage in the liver by evaluating serum enzyme levels, quantifying apoptotic cells, and measuring alterations in gene expression, caspase activity, and NFkappaB activity at critical time points in B6C3F1 mice exposed to TCDD in the presence or absence of endotoxin. Combined TCDD/endotoxin treatment altered the kinetics of TCDD-induced hepatotoxicity, with peak serum enzyme levels occurring 3-4 days earlier than with TCDD alone. An increased percentage of hepatocytes from TCDD-treated mice displayed the classical apoptotic phenotype, compared to controls. Consistent with induction of apoptosis, TCDD stimulated expression of TNFalpha at days 10 and 14 in both LPS- and saline-treated mice. In contrast, Fas mRNA expression was modulated rapidly following TCDD exposure. At 40 mg LPS, caspase 3 activity was stimulated in TCDD exposed mice at 3 and 7 days, and then suppressed at 10 and 14 days. TNFR1, TNFR2 and NFkappaB gene expression, IkappaBalpha and IkappaBbeta protein expression, and NFkappaB DNA-binding activity did not appear to be modulated by TCDD under the conditions of the study. Previous studies had shown that when rodents are treated with TCDD prior to endotoxin exposure a significant increase in toxicity occurs, and that inhibition of protein synthesis with cycloheximide blocks the TCDD-induced sensitivity to TNF in this model. The protective effects of cycloheximide, alterations in TNFalpha gene expression, and lack of involvement of NFkappaB suggest that TCDD-induced apoptosis may be mediated by TNF via the TNFalpha/TNFR2/TRAF pathway, but that it is not the result of suppression of NFkappaB.