Growth Factors /Inflammatory Mediators /Target-organ Tox

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。阵列研究进一步表明，在皮肤癌中起重要作用的环氧合酶-2(COX-2)基因的表达在砷暴露后以剂量依赖的方式高度诱导。后续研究表明，砷还可提高NHEK中COX-2蛋白的水平。这些事件似乎依赖于通过有丝分裂原和压力相关的激酶的信号；其活性受砷的调节。砷对COX-2的诱导也与培养液中COX-2活性的最终产物前列腺素水平升高和DNA合成增加有关。我们已经确定，砷暴露后COX-2的诱导在一定程度上依赖于特定的MAPK信号通路的激活，特别是ERK或p42/44MAPK。我们的研究表明，COX-2的诱导不依赖于p38MAPK的磷酸化。这种模式类似于角质形成细胞特有的生长因子，如成纤维细胞生长因子-7，但与同时激活p38和p42/44的表皮生长因子相反。有趣的是，抑制p38和p42/44通路的化合物可以抑制COX-2的升高。COX-2抑制剂抑制角质形成细胞的生物学过程，包括细胞增殖和转化生长因子α的分泌。我们目前正在研究皮肤成纤维细胞暴露砷后生长和炎症介质的变化。与NHEK相似，这些细胞已被证明对低剂量砷暴露敏感。然而，它们显示了许多在NHEK中没有观察到的细胞特异性反应，并将使我们能够更准确地模拟人类皮肤中两种细胞类型之间的相互作用。此外，我们正在与Miroslv Styblo博士和Luz Maria Del Razo博士合作，评估转化生长因子α作为砷暴露生物标记物的实用性。 我们目前正在与Michael Waalkes博士的实验室合作，对使用Tg.AC小鼠进行的活体研究中的组织进行微阵列研究，以确定与氧化应激、新陈代谢、热休克/应激反应、细胞增殖和DNA损伤相关的基因是否发生类似的变化。这些研究的组织也被提供给一些合作者，并被用来开发砷的组织分布、对大脑信号通路的影响以及对与氧化应激相关的酶的影响的模型。其他体内研究正在检验环氧合酶-2抑制剂塞来昔布对暴露于砷后的Tg.AC小鼠乳头状瘤的调节效果。在此期间，完成了砷暴露对Balb/c小鼠皮肤致敏作用的研究。在免疫反应的诱导和激发阶段，通过饮用水暴露于砷可减轻化学物质引起的接触性皮炎。机制研究表明，这种抑制可能是由于细胞因子诱导的朗格汉斯细胞从皮肤迁移到引流淋巴结的改变，以及随后对抗原提呈和细胞增殖的影响。 在本报告所述期间，我们还完成了肿瘤坏死因子α在TCDD诱导细胞凋亡中的作用的研究。作为这些研究的一个分支，我们与Nigel Walker博士合作，正在比较TCDD暴露后啮齿动物特定物种和性别的基因表达，以阐明可能导致TCDD诱导的肝癌发生的不同易感性的遗传机制。TSC-22是一种可以诱导细胞凋亡的转化生长因子β可诱导转录因子，在大鼠中受到抑制，但在小鼠中不会因慢性或急性TCDD暴露而改变。

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). Array 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. We have determined that the induction of COX-2 following arsenic exposure is, in part, dependent on activation of specific MAP kinase (MAPK) signaling pathways, in particular the ERK or p42/44 MAPK. Our studies indicate that the induction of COX-2 is independent of p38 MAPK phosphorylation. This pattern is similar to that observed with growth factors specific for keratinocytes, such as Fibroblast Growth Factor-7, but contrasts with that of Epidermal Growth Factor, which activates both p38 and p42/44. Interestingly, compounds that inhibit either the p38 and p42/44 pathway can attenuate elevation of COX-2. COX-2 inhibitors suppress biological processes in keratinocytes that we associate with the neoplastic process, including cell proliferation and TGF alpha secretion. We are currently investigating alterations in growth and inflammatory mediators following arsenic exposure in dermal fibroblasts. Similar to NHEK, these cells have been shown to be sensitive to low-dose arsenic exposure. However, they exhibit a number of cell-specific responses not observed in NHEK and will allow us to more accurately model the interactions between the two cell types that occur in human skin. In addition, we are collaborating with Drs. Miroslav Styblo and Luz Maria Del Razo to evaluate the utility of Transforming Growth Factor alpha as a biomarker for arsenic exposure. In collaboration with Dr. Michael Waalkes laboratory, we are currently conducting microarray studies in tissues from in vivo studies conducted using the Tg.AC mouse to determine if similar alterations in genes associated with oxidative stress, metabolism, heat shock/stress response, cell proliferation and DNA damage occur. Tissues from these studies have also been provided to a number of collaborators and are being used to develop models of tissue distribution of arsenic, effects on signaling pathways in the brain and the effects on enzymes associated with oxidative stress. Additional in vivo studies are examining the efficacy of Celecoxib, a COX-2 inhibitor, in modulating papillomagenesis in Tg.AC mice following arsenic exposure. Studies on the effects of arsenic exposure on dermal sensitization in Balb/c mice were completed during this period. Exposure to arsenic via the drinking water attenuated chemical-induced contact dermatitis during both the induction and elicitation phases of the immune response. Mechanistic studies suggest that this suppression is likely due to cytokine-induced alterations in the migration of Langerhans cells from the skin to the draining lymph node and subsequent effects on antigen presentation and cell proliferation. During this reporting period we have also completed studies on the role of Tumor Necrosis Factor alpha in TCDD-induced induction of apoptosis. As an offshoot of these studies, in collaboration with Dr. Nigel Walker, we are comparing species- and gender-specific gene expression in rodents following TCDD exposure to elucidate the genetic mechanisms that may underlie differential susceptibility to TCDD-induced hepatocarcinogenesis. TSC-22, a Transforming Growth Factor beta inducible transcription factor that can induce apoptosis was suppressed in the rat, but was not altered by either chronic or acute TCDD exposure in the mouse.