Growth Factors /Inflammatory Mediators /Target-organ Tox

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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.