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). Our microarray 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. 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 proliferative 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, caspase activity, and NFkappaB activity at critical time points in B6C3F1 mice exposed to TCDD in the presence or absence of endotoxin. Combined TCDD/endotoxin treatment altered the kinetics of TCDD-induced hepatotoxicity, with peak serum enzyme levels occurring 3-4 days earlier than with TCDD alone. An increased percentage of hepatocytes from TCDD-treated mice displayed the classical apoptotic phenotype, compared to controls. Consistent with induction of apoptosis, TCDD stimulated expression of TNFalpha at days 10 and 14 in both LPS- and saline-treated mice. In contrast, Fas mRNA expression was modulated rapidly following TCDD exposure. At 40 mg LPS, caspase 3 activity was stimulated in TCDD exposed mice at 3 and 7 days, and then suppressed at 10 and 14 days. TNFR1, TNFR2 and NFkappaB 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. The protective effects of cycloheximide, alterations in TNFalpha gene expression, and lack of involvement of NFkappaB suggest that TCDD-induced apoptosis may be mediated by TNF via the TNFalpha/TNFR2/TRAF pathway, but that it is not the result of suppression of NFkappaB.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Intramural Research (Z01)
Project #
1Z01ES030107-05
Application #
6681926
Study Section
(LT)
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2002
Total Cost
Indirect Cost
Name
U.S. National Inst of Environ Hlth Scis
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Kadiiska, M B; Gladen, B C; Baird, D D et al. (2005) Biomarkers of oxidative stress study II: are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning? Free Radic Biol Med 38:698-710
Gentry, P Robinan; Covington, Tammie R; Lawrence, Greg et al. (2005) Comparison of tissue dosimetry in the mouse following chronic exposure to arsenic compounds. J Toxicol Environ Health A 68:329-51
Xie, Yaxiong; Trouba, Kevin J; Liu, Jie et al. (2004) Biokinetics and subchronic toxic effects of oral arsenite, arsenate, monomethylarsonic acid, and dimethylarsinic acid in v-Ha-ras transgenic (Tg.AC) mice. Environ Health Perspect 112:1255-63
Patterson, Rachel; Vega, Libia; Trouba, Kevin et al. (2004) Arsenic-induced alterations in the contact hypersensitivity response in Balb/c mice. Toxicol Appl Pharmacol 198:434-43
Trouba, K J; Germolec, D R (2004) Micromolar concentrations of sodium arsenite induce cyclooxygenase-2 expression and stimulate p42/44 mitogen-activated protein kinase phosphorylation in normal human epidermal keratinocytes. Toxicol Sci 79:248-57
Carey, Michelle A; Germolec, Dori R; Langenbach, Robert et al. (2003) Cyclooxygenase enzymes in allergic inflammation and asthma. Prostaglandins Leukot Essent Fatty Acids 69:157-62
Carey, Michelle A; Germolec, Dori R; Bradbury, J Alyce et al. (2003) Accentuated T helper type 2 airway response after allergen challenge in cyclooxygenase-1-/- but not cyclooxygenase-2-/- mice. Am J Respir Crit Care Med 167:1509-15
Patterson, Rachel M; Stachlewitz, Robert; Germolec, Dori (2003) Induction of apoptosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin following endotoxin exposure. Toxicol Appl Pharmacol 190:120-34
Trouba, Kevin J; Hamadeh, Hisham K; Amin, Rupesh P et al. (2002) Oxidative stress and its role in skin disease. Antioxid Redox Signal 4:665-73
Detweiler, Charles D; Deterding, Leesa J; Tomer, Kenneth B et al. (2002) Immunological identification of the heart myoglobin radical formed by hydrogen peroxide. Free Radic Biol Med 33:364-9

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