Growth Factors and Inflammatory Mediators in Arsenic-Induced Toxicity

砷已经被归类为已知的人类皮肤、肺和膀胱的致癌物，然而，由于缺乏合适的动物模型，导致砷致癌的机制尚不清楚。我们实验室正在进行的研究表明，砷通过刺激细胞因子和生长因子(包括粒细胞巨噬细胞集落刺激因子、转化生长因子α和肿瘤坏死因子α)的慢性产生，在皮肤中起到肿瘤增强剂或促进剂的作用。我们目前正在研究个体间对砷反应的差异，以解决为什么一些个人或人群似乎对砷诱导的毒性不那么敏感的问题。我们研究了几种不同的角质形成细胞供体对砷治疗的反应，发现尽管细胞因子反应在性质上是相似的，因为产生了相似的细胞因子，但对剂量和定量细胞因子分泌的敏感性因供体而异。与北卡罗来纳大学的米罗斯拉夫·斯蒂布洛博士合作，我们正在研究不同捐赠者的不同敏感性是否反映了他们甲基化砷的能力。到目前为止，这些研究表明，与膀胱和肝脏等其他组织的细胞相比，角质形成细胞对砷化合物的甲基化能力很低。然而，对无机砷的摄取以及单甲基砷的产量和分布的差异表明，在活体人体皮肤中可能存在个体间的差异。我们评估了砷暴露对胆碱缺乏和胆碱充足饮食改变其甲基化(和解毒)能力的p53小鼠细胞因子谱的影响。与相应的对照组相比，喂饲胆碱缺乏饲料的P53小鼠皮肤中的肿瘤坏死因子α、肿瘤生长因子α和GM-CSF的mRNAs水平更高。这些研究表明，甲基化缺陷可能会影响个体组织对砷的反应产生的细胞因子。我们还检测了砷III和砷V及其单甲基和二甲基代谢物在角质形成细胞培养中的毒性。正如预期的那样，无机三价砷对所有捐赠者的毒性最大。令人惊讶的是，单甲基砷对角质形成细胞也有很高的毒性。我们描述了毒性的剂量反应曲线，并检测了暴露于三价和五价代谢物后的细胞因子分泌模式和增殖活性。由于过敏性反应高度依赖于细胞因子的分泌和调节，而且我们以前已经证明生长和炎性细胞因子受砷暴露的调节，我们研究了低水平的砷暴露如何调节超敏反应。在砷暴露动物的引流淋巴结中，中等和强致敏剂诱导的抗原特异性增殖反应显著减少，尤其是在低剂量时。相比之下，引流淋巴结中丝裂原刺激的增殖反应不受砷暴露的影响。砷还减少了先前致敏小鼠的挑战诱导反应。为了探讨这些改变的可能机制，我们研究了砷对朗格汉斯细胞迁移和抗原提呈的影响。通过饮用水接触砷的小鼠耳部朗格汉斯细胞迁移增加。据报道，肿瘤坏死因子α是朗格汉斯细胞迁移的主要刺激因子，这与我们之前的发现一致，即砷处理的小鼠皮肤中的肿瘤坏死因子α水平升高。目前正在对这些动物皮肤中调节性细胞因子的表达进行定量研究。-砷，皮肤，角质形成细胞，癌症，细胞增殖，细胞因子，生长因子，Tg.AC转基因小鼠，PCR

Arsenic has been classified as a known human carcinogen of the skin, lung and urinary bladder, however, the mechanisms involved in carcinogenesis by arsenic are not known, due in part to a lack of appropriate animal models. Ongoing studies in our laboratory have suggested that arsenic serves as a tumor enhancer or progressor in the skin via stimulation of the chronic production of cytokines and growth factors, including granulocyte macrophage colony stimulating factor, transforming growth factor alpha and tumor necrosis factor alpha. We are currently investigating inter-individual variation in responses to arsenic, to address the question of why some individuals or populations appear to be less sensitive to arsenic-induced toxicities. We have examined the cytokine profiles produced by several different keratinocyte donors in response to arsenic treatment and have shown that while cytokine responses are qualitatively similar, in that similar cytokines are produced, sensitivity to dose and quantitative cytokine secretion vary from donor to donor. In collaboration with Dr. Miroslav Styblo at UNC we are examining whether the differential sensitivity seen with various donors may be a reflection of their ability to methylate arsenic. To date these studies have indicated that keratinocytes have very little ability to methylate arsenicals, as compared with cells from other tissues such as the bladder and liver. However, differences in the uptake of inorganic arsenic and in the yield and distribution of monomethylarsenic suggest that interindividual differences could occur in human skin in vivo. We have evaluated the effects of arsenic exposure on alterations in cytokine profiles in p53 mice fed choline deficient and choline sufficient diets to alter their ability to methylate (and detoxify) arsenals. p53 mice fed a choline deficient diet exhibit higher levels of TNFalpha, TGFalpha and GM-CSF mRNAs in their skin than corresponding controls. These studies suggest that deficiencies in methylation may influence the cytokines produced in individual tissues in response to arsenic. We have also examined the toxicity of both ArsenicIII and ArsenicV and their monomethyl and dimethyl metabolites in keratinocyte cultures. As expected, inorganic trivalent arsenic is the most toxic to all donors. Surprisingly, monomethyl arsenicals are also highly toxic to keratinocytes. We have characterized the dose response curves for toxicity and examined cytokine secretion patterns and proliferative activity after exposure to the trivalent and pentavalent metabolites. As hypersensitivity responses are highly dependent on cytokine secretion and regulation, and as we have previously demonstrated that growth and inflammatory cytokines are modulated by arsenic exposure, we have investigated how low level arsenic exposure modulates hypersensitivity responses. Antigen-specific proliferative responses induced by moderate and strong sensitizing agents are significantly reduced in the draining lymph nodes of arsenic-exposed animals, particularly at low doses. In contrast, mitogen-stimulated proliferative responses in the draining lymph nodes were not affected by arsenic exposure. Arsenic also reduced challenged-induced responses in previously sensitized mice. To investigate the potential mechanisms of these alterations we examined the effects of arsenic on Langerhans cell migration and antigen presentation. Langerhans cell migration out of the ear was enhanced in mice exposed to arsenic via the drinking water. As TNFalpha is reported to be a primary stimulator of Langerhans cell migration, this is consistent with our previous findings that TNFalpha levels are elevated in the skin of arsenic treated mice. Quantitation of the expression of regulatory cytokines in the skin of these animals is currently under investigation. - arsenic, skin, keratinocytes, cancer, cell proliferation, cytokines, growth factors, Tg.AC transgenic mice, PCR