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.
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