Grantee Research Project Results
2006 Progress Report: Pulmonary Biomarkers Based on Alterations in Protein Expression Following Exposure to Arsenic
EPA Grant Number: R832095Title: Pulmonary Biomarkers Based on Alterations in Protein Expression Following Exposure to Arsenic
Investigators: Lantz, R. Clark , Boitano, Scott A. , Burgess, Jefferey L.
Institution: University of Arizona
EPA Project Officer: Aja, Hayley
Project Period: January 8, 2005 through January 16, 2009
Project Period Covered by this Report: January 8, 2006 through January 16, 2007
Project Amount: $731,453
RFA: Application of Biomarkers to Environmental Health and Risk Assessment (2004) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation
Objective:
Exposure to arsenic (As) has been linked to lung cancer. Environmental exposure to these metals will result in multiple adverse effects, which can be characterized through evaluation of alterations in protein expression. We will evaluate such alterations as biomarkers of exposure and effect prior to the development of cancer. This study will use the technology of proteomics to evaluate and identify biomarkers of chronic environmental exposure to As by evaluating large numbers of proteins simultaneously. We will compare alterations in protein expression in exposed human populations in Arizona, human cell lines, and in vivo rodent studies. Patterns of alterations in protein expression, both common and unique to these different cell types, will be identified. These will be correlated with alterations in DNA oxidation in induced sputum from the lung, which we have recently found to be related to the levels of arsenic exposure.
Progress Summary:
We have initiated arsenic studies on human bronchial epithelial (16HBE14o-) cells. We have previously shown that arsenic delays the ability of cells to form monolayers. To test if arsenic altered functional tight junctions, we repeated the experiments from above, with cells grown on nuclepore filter supports and directly measured transepithelial resistance (TER) in the presence or absence of arsenic. Arsenic added at the time of plating greatly reduced the ability of the cells to form a functional monolayer. This was apparent even at 60 ppb. If cells were first allowed to form a tight monolayer prior to addition of arsenic, high levels of arsenic were still capable of reducing the epithelial resistance. Cells exposed under these conditions showed alterations in the levels and types of expression of tight junctional proteins, occuldin, and claudins. We have continued to analyze alterations in calcium signaling as a target for arsenic exposure. Arsenic reduces the number of adjacent cells that respond to a “wound” by increasing their calcium levels. In addition, the time at which intracellular calcium is elevated after a “wound” is significantly reduced in arsenic exposed cells at concentrations as low as 60 ppb. These new data further support altered wound healing and maintenance of a functional epithelial barrier as targets for arsenic exposure.
In vivo Animal Responses to Arsenic Exposure
We have continued to expand our analysis of altered protein expression in the lung lining fluid of mice exposed to arsenic in their drinking water for up to 4 weeks. Soluble proteins in the lung lining fluid were obtained through bronchoalveolar lavage (BAL). Proteins were identified using 2-D gel electrophoresis (N = 3) or multidimensional protein identification technology (MUDPIT) (N = 2) coupled with mass spectrometry (MS). A total of 44 proteins were identified. Proteins that were seen to be present in the BAL of control animals while absent in the treated animals include: glutathione-S-transferase omega-1 (GST-omega-1), contraspin, apolipoprotein A-I and A-IV, and receptor for advanced glycation end products (RAGE). Proteins up regulated by arsenic included enolase-1 and peroxiredoxin-6. Using Western Blot analysis, we have shown that levels of RAGE in the BAL decreases as a function of arsenic treatment in mice treated with arsenic in their drinking water. Previous investigators have identified GST-omega-1 as an important arsenic metabolizing enzyme. The function of RAGE in chronic inflammatory disease, wound healing, and cancer has been previously reported. In addition, alpha-1-antitrypsin (AAT) levels were also affected by arsenic treatment. We have also analyzed the most likely cell functions and disease states that are associated with the altered proteins. Using a curated analysis system (MetaCore) we have identified cell motility and alteration in wound repair as the most likely affected cellular functions and disease. This is consistent with our in vitro data.
Human Population Studies
Based on the results from both the in vitro and in vivo experiments, we have previously analyzed arsenic-induced changes in matrix metalloproteinase 9 (MMP-9) and RAGE in induced sputum in human populations. Samples were collected from 56 individuals living in Ajo, Arizona (tap water arsenic ~ 20 ppb) and from Tucson, Arizona (tap water arsenic ~ 5 ppb). First morning void urine was also collected and arsenic speciation analysis was performed. Both MMP-9/TIMP1 (tissue inhibitor of metalloproteinase 1) ratio and RAGE were significantly altered as a function of arsenic intake. In this same population, we analyzed sputum AAT by ELISA, then performed multivariate analysis to determine which predictor variables were associated with AAT, both directly and corrected for total protein. AAT has been associated with chronic obstructive pulmonary disease (COPD) and was identified as an altered protein in our in vivo animal experiments. Therefore, alteration of this protein may play a role in non-cancerous disease outcomes from arsenic exposure. In a regression model, total urinary inorganic arsenic was negatively associated with sputum AAT (p= 0.028) and AAT/protein (p= 0.014).
Future Activities:
We will continue these activities. For the in vitro experiments, we are continuing to analyze the effect of the arsenic on epithelial barrier function and are assessing whether removal of the arsenic can restore normal function. We will also continue to investigate calcium signaling as a site of action. For in vivo, one of our original objectives was to also analyze alterations in proteins in the airway epithelium. Using a technique developed by researchers at the University of California at Davis, we have been able to differentially digest the airway epithelial cells and collect the proteins from these animals. We will begin analysis of these proteins in the near future. Finally, our human studies will also continue, with the inclusion of samples collected from a Mexican population that is being exposed to 50 ppb and higher arsenic levels in their drinking water. This will broaden our current range of exposures to give us more confidence in the dose-responses we are seeing.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 18 publications | 7 publications in selected types | All 7 journal articles |
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Josyula AB, Poplin GS, Kurzius-Spencer M, McClellen HE, Kopplin MJ, Sturup S, Lantz RC, Burgess JL. Environmental arsenic exposure and sputum metalloproteinase concentrations. Environmental Research 2006;102(3):283-290. |
R832095 (2005) R832095 (2006) R832095 (2007) |
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Lantz RC, Hays AM. Role of oxidative stress in arsenic-induced toxicity. Drug Metabolism Reviews 2006;38(4):791-804. |
R832095 (2005) R832095 (2006) R832095 (2007) |
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Lantz RC, Lynch BJ, Boitano S, Poplin GS, Littau S, Tsaprailis G, Burgess JL. Pulmonary biomarkers based on alterations in protein expression after exposure to arsenic. Environmental Health Perspectives 2007;115(4):586-591. |
R832095 (2005) R832095 (2006) R832095 (2007) |
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Supplemental Keywords:
heavy metals, human health, animal, cellular, biomarkers, induced sputum, bronchial epithelial cells , Health, PHYSICAL ASPECTS, RFA, Scientific Discipline, Water, Arsenic, Biochemistry, Environmental Chemistry, Hazardous Waste, Health Risk Assessment, Physical Processes, Risk Assessments, arsenic exposure, bioaccumulation, biogeochemistry, biomarker measurements, contaminant transport, contaminated sediments, exposure,, RFA, Health, Scientific Discipline, Water, POLLUTANTS/TOXICS, Environmental Chemistry, Health Risk Assessment, Arsenic, Risk Assessments, Hazardous Waste, Biochemistry, Water Pollutants, contaminated sediments, contaminant transport, risk management, biogeochemistry, arsenic exposure, bioaccumulationProgress 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.