Grantee Research Project Results
2007 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 2, 2006 through March 2,2007
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 will be identified. These will be correlated with alterations in DNA oxidation in induced sputum from the lung.
Progress Summary:
Our in vitro studies use human bronchial epithelial (16HBE14o-) cells. We have shown that arsenic delays the ability of cells to form monolayers and alters the ability of the cells to “repair” and artificial wound. Arsenic increased the levels of MMP-9 which may contribute to the alterations we have seen. Calcium signaling appears to also be affected by arsenic. 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. Arsenic also altered functional tight junctions. 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. 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 analyzed altered protein expression in the lung lining fluid and airway epithelial cells 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 levels were also affected by arsenic treatment. We have also used a selective airway epithelial cell digestion to isolate and analyze altered proteins. Using MUDPIT analysis we have identified over 150 proteins changed by arsenic. We have also analyzed the most likely cell functions and disease states that are associated with the altered proteins from BAL and from the cell fractions. 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. In order to verify wound healing as a site of action of arsenic, we have examined the effect of arsenic on the time for recovery from epithelial damage caused by exposure to naphthalene. We have found that while normal recoevery from naphthalene exposure occurs within 2 weeks of exposure, damage in animals that had been exposed to arsenic was still present after 2 weeks. These data indicate that asenic can affect wound healing in an animal model.
Human population studies
Based on the results from both the in vitro and in vivo experiments, we have analyzed arsenic-induced changes in 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 ration and RAGE were significantly altered as a function of arsenic intake. In this same population, we analyzed sputum alpha-antitrypsin (AAT) by ELISA, then performed multivariate analysis to determine which predictor variables were associated with AAT, both directly and corrected for total protein. In a regression model, total urinary inorganic arsenic was negatively associated with sputum AAT (p=0.028) and AAT/protein (p=0.014). Most recently, we have found that serum MMP-9 appears to be correlated with arsenic exposures. We are sampling additional individuals to verify this finding.
Future Activities:
For the in vitro experiments, we will continue to investigate calcium signaling as a site of action. For in vivo, we will use quantitative morphological techniques to verify delays in wound healing in the distal lung of animals exposed to arsenic. We will continue to analyze our proteins identified as potential biomarkers. 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. We will also test alterations of MMP-9 in serum as a biomarker of arsenic exposure and effect.
Journal Articles on this Report : 7 Displayed | Download in RIS Format
| Other project views: | All 18 publications | 7 publications in selected types | All 7 journal articles |
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Burgess JL, Meza MM, Josyula AB, Poplin GS, Kopplin MJ, McClellen HE, Sturup S, Lantz RC. Environmental arsenic exposure and urinary 8-OHdG in Arizona and Sonora. Clinical Toxicology 2007;45(5):490-498. |
R832095 (2005) R832095 (2007) |
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Burgess JL, Kurzius-Spencer M, Poplin GS, Littau SR, Kopplin MJ, Sturup S, Boitano S, Clark Lantz R. Environmental arsenic exposure, selenium and sputum alpha-1 antitrypsin. Journal of Exposure Science & Environmental Epidemiology 2014;24(2):150-155. |
R832095 (2007) |
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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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Olsen CE, Liguori AE, Zong Y, Lantz RC, Burges JL, Boitano S. Arsenic upregulates MMP-9 and inhibits wound repair in human airway epithelial cells. American Journal of Physiology-Lung Cellular and Molecular Physiology 2008;295(2):L293-302. |
R832095 (2005) R832095 (2007) |
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States JC, Barchowsky A, Cartwright IL, Reichard JF, Futscher BW, Lantz RC. Arsenic toxicology: translating between experimental models and human pathology. Environmental Health Perspectives 2011;119(10):1356-1363. |
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,, Health, RFA, Scientific Discipline, Water, POLLUTANTS/TOXICS, Health Risk Assessment, Risk Assessments, Environmental Chemistry, Hazardous Waste, Arsenic, Biochemistry, Water Pollutants, contaminant transport, contaminated sediments, biogeochemistry, bioaccumulation, biomarker measurements, risk managementRelevant Websites:
https://cfpub.epa.gov/ncer_abstracts/index.cfm/fuseaction/display.files/fileID/7641 (9 pp, 140 K)
Progress 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.