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). Gene expression changes are currently being validated via PCR and Northern analysis. 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 proliferatory 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 at critical time points in B6C3F1 mice exposed to TCDD in the presence or absence of endotoxin. TCDD modulated endotoxin-mediated early expression of Fas, and upregulated expression of caspase 3 (day 10), NFkappaB (day 14), and TNFalpha (days 10 and 14). In addition, combined TCDD/endotoxin altered the kinetics of TCDD-induced hepatoxicity and caspase activity, with peak levels occurring 3-4 days earlier. TNFR1 and TNFR2 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. Overexpression of caspases has been shown to induce apoptosis, and activation of the caspases appears to be necessary for development of the apoptotic phenotype. The protective effects of cycloheximide and alterations in TNFalpha, and NFkappaB gene expression suggest that TCDD treatment may result in alterations in TNF-induced activation of NFkappaB via the TNFalpha/TNFR2/TRAF pathway. The alterations in Fas and Caspase 3 gene expression indicate initiation of apoptosis and suggest involvement of the Fas Ligand/Fas/Caspase pathway.
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