Grantee Research Project Results
Final Report: Arsenic-Glutathione Interactions and Skin Cancer
EPA Grant Number: R826135Title: Arsenic-Glutathione Interactions and Skin Cancer
Investigators: Snow, Elizabeth T. , Frenkel, Krystyna , Klein, Catherine B. , Mirochnitchenko, Oleg I. , Bosland, Maartin , Steinberg, Mark
Institution: New York University Medical Center
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1997 through September 30, 2000 (Extended to September 30, 2001)
Project Amount: $822,293
RFA: Arsenic Health Effects Research (1997) RFA Text | Recipients Lists
Research Category: Drinking Water , Human Health , Water
Objective:
This research project was designed to test the hypothesis that arsenic-induced cancer is the result of changes in cellular redox control mediated by altered glutathione (GSH) levels by exploring the effect of arsenic on glutathione regulating enzymes in human keratinocytes in vitro and in mice in vivo. It was predicted that exposure to physiologically relevant, low doses of As would result in the activation of enzymes such as glutathione S-transferase (GST), glutathione reductase (GR), -glutamylcysteine synthetase (-GCS), and glutathione peroxidase (GPx) due to changes in cellular phosphorylation and/or redox status. This activation would either potentiate or ameliorate the induction of cellular stress responses. Subsequent to an oxidative stress response in certain cell types, such as keratinocytes, arsenic can induce hyperproliferation and inhibit DNA repair. Three specific aims of the project were to: (1) examine the effect of arsenic on the activities of GSH-metabolizing enzymes in vitro, glutathione transferase-pi (GST-), GR, and GPx; (2) assess GSH-dependent redox status in arsenic-treated keratinocytes by determining the activities of GR and GPx in human cells treated in culture with a low dose of inorganic arsenite (AsIII), by monitoring the production of reactive oxygen species (ROS), GSH, and GSSG levels; and (3) evaluate the role of GSH in arsenic-induced carcinogenesis by examining the effect of varied arsenic and GSH levels on the rate of papilloma induction in a mouse skin tumorigenesis model using normal mice and mice that overexpress human GPx.Summary/Accomplishments (Outputs/Outcomes):
The results of Aim 1 have been published (Chouchane and Snow, 2001), and three additional papers related to Aim 2 are either published or in press (Snow, et al., 1999; 2001; Hu, et al., 2002). An additional five publications related to this grant are either recently submitted or in the later stages of preparation. Portions of Aim 3 have been completed, that is, the animal carcinogenesis experiments using the normal (C57Bl/6 x CBA/J)F1 mice, the results of which currently are being analyzed (Klein and Snow, in preparation). Preliminary results show that low concentrations of arsenic (V) in the drinking water (0.2 to 2.0 mg/L) significantly reduce papilloma formation and subsequent skin cancer in the DMBA and TPA-treated mice. The final sets of proposed experiments using transgenic GPx-overproducing mice were not possible because the transgenic mice failed to reproduce adequately.Research on GSH and GSH-dependent enzyme levels in cultured human epidermal keratinocytes and epidermal fibroblasts has been completed; however, related experiments are still ongoing. As described in our published papers, physiologically relevant concentrations of arsenic did not directly inhibit either GSH metabolizing enzymes (Chouchane and Snow, 2002) or DNA repair enzymes (Hu, et al., 1998). However, low concentrations of inorganic arsenic caused significant changes in cellular GSH levels and in the relative activity and gene expression of a variety of redox active enzymes either in cultured human keratinocytes or human fibroblasts (Snow, et al., 1999 and 2001; Schuliga, et al., 2002). These results showed that even low, relatively nontoxic concentrations of arsenic can directly modulate cellular redox activity. This altered redox activity, in turn, alters cellular signaling (Hu, et al., 2002) and other aspects of intermediary metabolism and thereby contributes to the carcinogenic process. Arsenic produces the observed alterations in the activity of these redox regulating enzymes by initiating changes in the regulation of the genes (Schuliga, et al., 2002) in part by modulating the regulation of two key proteins, thioredoxin and Ref-1 (Hu, et al., 2002). The specific role of reactive oxygen species is being investigated in the induction of gene expression and in As-induced cellular toxicity.
In particular, the regulation of multiple glutathione-related enzymes, including GR and GST that play key roles in cellular redox metabolism, is strongly modified by exposure of human cells to low levels of inorganic arsenite (AsIII). Substantial increases in GR enzyme activity and mRNA levels were shown in human fibroblast and keratinocyte cells after exposure to low micromolar levels of AsIII for 24 hours (concentrations of AsIII that did not significantly reduce cell viability). Arsenite upregulation of GSH synthesis was found to parallel the upregulation of GR in cultured keratinocytes as shown by increases in intracellular GSH levels, -glutamylcysteine synthetase (-GCS) enzyme activity and mRNA levels and cystine uptake. Relative GSH levels were increased only slightly in PMC42 breast tumor cells that have higher constituitive levels of GSH. GST activity also was shown to increase slightly in keratinocytes, but not in fibroblasts or breast tumor cells, upon exposure to low micromolar AsIII for 24 hours. If the levels of GSH in keratinocyte cells were depleted by pretreatment with L-buthionine sulfoximine or chloroethanol, the cells became more sensitive to treatment with AsIII, indicating that the increased levels of GSH and GR are protective. Overall, the results show that sublethal arsenic induces a multicomponent response in human keratinocytes that involves the GSH system and counteracts the acute toxic effects of iAs. The modulation of GR is an integral part of this response, as GR maintains the reduced status of GSH, facilitating the function of GSH as an anti-oxidant (Schuliga, et al., 2002a).
The AsIII-dependent regulation of glutathione peroxidase (GPx), an antioxidant selenoprotein that is involved in the reduction of organo- and hydro-peroxides, also was investigated as part of this aim. The levels of GPx1 mRNA (the major GPx isoform) were significantly decreased in both fibroblast and breast tumor cell lines, whereas they were increased in keratinocyte cells after exposure to AsIII for 24 hours. Changes in cytosolic glutathione peroxidase (cGPx) enzyme activity paralleled changes in the levels of GPx1 mRNA in the fibroblast cells. As expected, selenium supplementation stimulated cGPx enzyme activity in fibroblasts without affecting the level of GPx1 mRNA. Interestingly, selenium also inhibited the negative regulatory effect of AsIII on the level of GPx1 mRNA. Previous studies have shown that exposure to AsIII induces oxidative stress in mammalian cells by causing a rapid burst in reactive oxygen species (ROS). However, hydrogen peroxide, an agent that also promotes oxidative stress, induced an increase instead of a decrease in the level of GPx1 mRNA in fibroblasts. In addition, antioxidants were found to have little effect in preventing the downregulation of GPx1 expression by AsIII even when they significantly reduced increases in ROS. Studies using 4-(N-(S-glutathionylacetyl)) aminophenylarsenoxide (GSAO), an arsenical that is impermeable to cellular membranes, demonstrated that the regulatory effect of As on the expression of GPx1 involves a membrane component. Overall, this study shows that As regulates the expression of GPx at a pre-translational level, is dependent on cell type and the availability of selenium, and is not directly dependent on the As-induced increase in ROS. These data provide further insights into the pathogenesis of As-related disease and the antagonistic interaction between As and selenium compounds (Schuliga, Donoghue, and Snow, in preparation).
This research project was expanded to include thioredoxin (Trx) and thioredoxin reductase (TR). TR and Trx operate together as a protein disulfide reducing system (Trx system) affecting redox control over cell signaling pathways involved in oxidative stress. In addition to GSH and GR, the level of Trx mRNA is unregulated in cultured human cells after exposure to AsIII. HaCaT keratinocytes were the most responsive cell type examined, with the level of Trx mRNA peaking at approximately 150 percent above the basal level after exposure to 3µM AsIII for 24 hours. Significant increases in the level of Trx mRNA were observed at nontoxic concentrations of AsIII, as measured by the uptake of neutral red dye, in all cell types examined. The upregulation of Trx protein expression was found to parallel the upregulation of Trx mRNA levels, and the Trx levels were found to parallel an increase in the level of TR mRNA. These changes in gene regulation were preceded by a rapid increase in ROS. However, AsIII, unlike H2O2, does not induce the translocation of Trx into the nucleus. Overall, the results show that the upregulation of the Trx system also is associated with the cellular stress response provoked by low dose AsIII (Schuliga and Snow, in preparation).
To determine the underlying mechanism by which cellular levels of these redox proteins are regulated by AsIII, AP-1 and NF-kB DNA binding activity was examined in human fibroblast cells exposed to both acute (24 hours) and chronic (10-20 week) low dose AsIII. Short-term treatment with 0.1 to 5 µM AsIII activates both AP-1 and NF-kB binding and upregulates expression of c-Fos and c-Jun and the redox regulators, Trx and Redox factor-1 (Ref-1). In contrast, chronic, 10-week exposure to 0.1 or 0.5 µM AsIII decreased AP-1 and NF-kB binding activity and c-Jun, c-Fos and Ref-1 protein levels, but increased Trx expression. Short-term exposure to TPA, a phorbol ester tumor promoter, or H2O2 also activated AP-1 and NF-kB binding. However, pretreatment with AsIII prevented this increase. These results suggest that AsIII may alter AP-1 and NF-kB activity, in part by upregulating Trx and Ref-1. The different affects of short- versus long-term AsIII treatment on the acute-phase response to oxidative stress reflects changes in the expression of Ref-1, c-Fos, and c-Jun, but not Trx (Hu, Jin, and Snow, 2002).
Finally, the effect of AsIII on Trx and TR gene expression in mouse 3T3 fibroblast cells were studied after both short-term and long-term exposure to low dose AsIII. The results showed that AsIII also upregulates the expression of Trx and TR in mouse fibroblast cells, especially after long-term exposure to submicromolar AsIII (Hu, Jin, and Snow, in preparation).
In conclusion, low doses of AsIII cause a significant and lasting change in the expression and activity of a number of proteins and enzymes critically involved in the regulation of cellular redox levels. The mechanism of how arsenic causes these changes is not yet fully understood. However, two findings are most relevant for assessment of risk due to exposure to inorganic arsenic: (1) the dose response for gene expression is highly nonlinear, the response has a clear peak at exposure levels that are less than approximately 50 percent toxic for human cells; and (2) the changes in enzyme activity are due predominantly to changes in gene expression (or mRNA stability) and are not the result of direct inhibition of the enzymes. These findings need to be incorporated into any reasonable mechanism-based model for risk assessment.
For Aim 3, the hypothesis was examined that arsenic acts as a progressor or co-promoter in the production of skin cancer, and the role of GSH in this process was evaluated, using the DMBA/TPA initiation/promotion model for mouse skin carcinogenesis. For some groups of animals, N-acetyl cysteine (NAC) was added to the water at a concentration of 10 mg/L 1 week prior to treatment with DMBA and continued throughout the experiment to increase GSH levels and add antioxidant protection. For others, AsV is added (? NAC) to the drinking water at a concentration of either 0.2 or 2.0 mg/L. It was expected that the As would act as either a co-promoter or sensitizer. Three sets of experiments were completed (13 experimental groups with 9 animals in each group in the first experiment; 12 animals per group in the second experiment consisted mainly of critical negative controls; and in the third experiment, 18 animals per group and a higher dose (5 µg/mL) of TPA were used). These experiments are now terminated. No overt papillomas were observed in any of the negative controls or in the animals treated with AsV or NAC alone.
In the first two experimental groups, papillomas were not raised in these mice using a DMBA carcinogen protocol followed by biweekly TPA (3 µg/mL). However, in the third experimental group, animals exposed to the standard DMBA/TPA protocol showed high numbers of papillomas. More than 90 percent of the animals treated with the standard DMBA/TPA protocol showed evidence of papilloma formation. Mice that were treated with the antioxidant, N-acetylcysteine, in the drinking water at a concentration of 10 mg/L prior to and after the DMBA exposure exhibited a 40 percent decrease in papilloma formation. However, there was an even more striking 50 percent decrease in papilloma formation when the mice were given drinking water with the low dose of AsV (0.2 mg/L), in addition to the standard DMBA/TPA treatment. Exposure to low dose AsV plus NAC gave an additive response, with an overall level of papilloma formation that was only 25 percent of that seen in the animals exposed to the DMBA/TPA regimen alone. The mice that were exposed to the higher (but still relatively low) level of AsV in the drinking water (2 mg/L) also exhibited a further 50 percent decrease in papilloma formation relative to the mice exposed to either NAC (plus DMBA/TPA) alone or to low dose As (plus DMBA/TPA). However, these data clearly show that low doses of AsV and/or N-acetylcysteine can cause a significant reduction in papilloma formation caused by an initiator plus promoter. This indicates that in a mouse model for skin cancer, AsV in the drinking water at concentrations that are similar to those found in highly exposed human populations may act as antipromoter or anticarcinogen.
This is consistent with the lack of As-induced carcinogenesis in other animal models, and again indicates that the biological response of humans to As is quite different from that of rodents. These results also are consistent with our finding that long-term exposure to low dose As can specifically downregulate the cellular response to TPA (Hu, Jin, and Snow, 2002). Samples of skin and other tissues from these experiments have been stored at -80?C, and other samples have been preserved in paraffin blocks. Preliminary experiments have begun to evaluate levels of GSH and the relative gene expression of Trx, TR, GPx, and GR in the tissues of As-exposed and unexposed mice. The levels of As species also will be determined in the skin, bladder, liver, and lungs from the As-treated and control animals.
Journal Articles on this Report : 6 Displayed | Download in RIS Format
Other project views: | All 25 publications | 6 publications in selected types | All 6 journal articles |
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Alam MG, Snow ET, Tanaka A. Arsenic and heavy metal contamination of vegetables grown in Samta village, Bangladesh. The Science of The Total Environment 2003;308(1-3):83-96. |
R826135 (Final) |
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Chouchane S, Snow ET. In vitro effect of arsenical compounds on glutathione-related enzymes. Chemical Research in Toxicology 2001;14(5):517-522. |
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Hu Y, Jin XM, Snow ET. Effect of arsenic on transcription factor AP-1 and NF-kappaB DNA binding activity and related gene expression. Toxicology Letters 2002;133(1):33-45. |
R826135 (Final) |
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Schuliga M, Chouchane S, Snow ET. Upregulation of glutathione-related genes and enzyme activities in cultured human cells by sublethal concentrations of inorganic arsenic. Toxicological Sciences 2002;70(2):183-192. |
R826135 (Final) |
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Snow ET, Sykora P, Durham TR, Klein CB. Arsenic, mode of action at biologically plausible low doses: what are the implications for low dose cancer risk? Toxicology and Applied Pharmacology 2005; 207(2 Suppl 1):557-564. |
R826135 (Final) |
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Sykora P, Snow ET. Modulation of DNA polymerase beta-dependent base excision repair in cultured human cells after low dose exposure to arsenite. Toxicology and Applied Pharmacology 2008;228(3):385-394. |
R826135 (Final) |
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Supplemental Keywords:
drinking water, risk assessment, health effects, metabolism, dose-response, human, animal, susceptibility, heavy metals, genetics, biology, pathology, cancer., RFA, Health, Scientific Discipline, Toxics, Water, POLLUTANTS/TOXICS, National Recommended Water Quality, Environmental Chemistry, Health Risk Assessment, Genetics, Arsenic, Risk Assessments, Disease & Cumulative Effects, Water Pollutants, Drinking Water, Biology, Pathology, health effects, human health effects, keratinocytes, exposure and effects, dose response, dose-response, glutathiones, exposure, skin keratoses, effects, human exposure, genotoxicity, cancer, GSH, protein sulfhydryls, metabolism, water quality, drinking water contaminants, arsenic exposure, heavy metalsProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.