Grantee Research Project Results
2000 Progress Report: Arsenic-Glutathione Interactions and Skin Cancer
EPA Grant Number: R826135Title: Arsenic-Glutathione Interactions and Skin Cancer
Investigators: Snow, Elizabeth T. , Klein, Catherine B.
Current Investigators: Snow, Elizabeth T. , Frenkel, Krystyna , Klein, Catherine B. , Mirochnitchenko, Oleg I. , Bosland, Maartin , Steinberg, Mark
Institution: New York University Medical Center , Deakin University
Current 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 Period Covered by this Report: October 1, 1999 through September 30, 2000
Project Amount: $822,293
RFA: Arsenic Health Effects Research (1997) RFA Text | Recipients Lists
Research Category: Drinking Water , Human Health , Water
Objective:
We proposed 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 (As) on glutathione regulating enzymes in human keratinocytes in vitro, and in mice in vivo. We predicted that exposure to physiologically relevant, low doses of As will result in the activation of enzymes such glutathione S-transferase (GST), glutathione reductase (GR), g-glutamylcysteine synthetase (g-GCS), and glutathione peroxidase (GPx) due to changes in cellular phosphorylation and/or redox status. This activation may either potentiate or ameliorate the induction of cellular stress responses. Our three specific aims are to: (1) examine the effect of arsenic on the activities of GSH-metabolizing enzymes in vitro: glutathione transferase-pi (GST-p), 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), and 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 GSH levels on the rate of papilloma induction in a mouse skin tumorigenesis model using normal mice and mice which over express human GPx.Progress Summary:
In Year 3 of this grant, we continued work on each of the three aims, focusing extensively on Aims 2 and 3, and bringing to completion Aim 1. The initial enzyme kinetics experiments are now complete and have been submitted for publication in Chemical Research in Toxicology. Our research on GSH and GSH-dependent enzyme levels in cultured human epidermal keratinocytes and epidermal fibroblasts is in its final stages. A large portion of this work has been written up for publication and will be submitted shortly. We have also completed two phases of the animal carcinogenesis experiments using the normal (C57Bl/6 x CBA/J)F1 mice and a third group of animals are now being treated with a larger dose of TPA to ensure the production of papillomas. The animals exposed to DMBA and TPA in this experiment are finally showing a positive response, but preliminary data suggest that arsenic in the drinking water may reduce skin papilloma formation in the DMBA and TPA-treated mice rather than enhancing the carcinogenesis process, as was predicted. The final set of experiments using transgenic GPx-overproducing mice will begin in January/February 2001 when we should have sufficient animals to achieve statistically significant results. Preliminary Results. In Aims 1 and 2, we have shown that physiologically relevant concentrations of arsenic (less than 100 µM) do not directly inhibit GSH metabolizing enzymes. However, low concentrations of AsIII can cause 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. These results show that even low, relatively non-toxic concentrations of arsenic can directly modulate cellular redox activity which, in turn, may alter cellular signaling and other aspects of intermediary metabolism and thereby contribute to the carcinogenic process. However, we have also found, contrary to earlier hypotheses, that these changes are not brought about by the direct inhibition of redox-related enzymes by inorganic As or arsenic metabolites. Most enzymes are quite insensitive to physiological concentrations of arsenic. Direct enzyme inhibition is only seen at high arsenic concentrations or with arsenic complexes found at very low concentrations in skin cells. It is proposed instead that arsenic produces alterations in the activity of these redox regulating enzymes by initiating changes in the regulation of the genes. The means whereby arsenic initiates these changes is not yet established and we are now investigating the specific role of reactive oxygen species in the induction of gene expression and in As-induced cellular toxicity. However, two findings are most relevant for assessment of risk due to exposure to inorganic arsenic: (1) the dose response for redox-related gene expression is highly non-linear (the expression generally peaks at sub-toxic concentrations of AsIII, and then drops at higher concentrations); and (2) the changes in enzyme activity are due predominantly to changes in gene expression 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, we are examining the hypothesis that arsenic acts as a progressor or co-promoter in the production of skin cancer and evaluating the role of GSH in this process. We have completed two sets of carcinogenesis experiments using a relatively low concentration of TPA in the belief that the As would act as a co-promoter. As a result, no overt papillomas were observed on either the DMBA/TPA treated positive controls or on the As-treated animals of any group. A third experiment is now in progress having started in late July 2000 and preliminary data at week 12 after DMBA exposure shows significant papilloma formation in the DMBA/TPA treated animals. However, these same data indicate that arsenic may actually cause a significant reduction in papilloma formation. This suggests 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 an anti-carcinogen. 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.Future Activities:
Research related to Aim 2 is still continuing as we evaluate the mechanism of gene induction by AsIII. Aim 3 is only 50 percent complete and is also continuing. Samples of skin and other tissues from the first three sets of animal experiments have been stored at ?80?C and other samples have been preserved in paraffin blocks. During this next year, we will use these samples to investigate tissue arsenic levels and the levels of GSH-related proteins and mRNA in the skin, bladder and other tissues from the As-treated and control animals. An additional group of animal experiments that have not yet been completed will use human hGPx-overproducing transgenic mice. GPx is one of the few genes we have identified that is down-regulated by a low dose of AsIII and these animals are also highly sensitive to DMBA/TPA-induced skin cancer. They should provide an interesting additional test of As as a co-promoter or progressor. We will initiate this next set of carcinogenesis experiments in January or February 2001, as soon as sufficient numbers of hGPx animals are generated.Journal Articles:
No journal articles submitted with this report: View all 25 publications for this projectSupplemental 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, Genetics, Environmental Chemistry, Health Risk Assessment, Arsenic, Risk Assessments, Disease & Cumulative Effects, Water Pollutants, Biology, Drinking Water, 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.