Grantee Research Project Results
2019 Progress Report: Center for Native American Environmental Health Equity Research
EPA Grant Number: R836157Center: Center for Native American Environmental Health Equity Research
Center Director: Lewis, Johnnye Lynn
Title: Center for Native American Environmental Health Equity Research
Investigators: Lewis, Johnnye Lynn , Gonzales, Melissa , Hudson, Laurie , Cerrato Corrales, Jose Manuel , MacKenzie, Debra Ann
Institution: University of New Mexico
EPA Project Officer: Callan, Richard
Project Period: July 1, 2015 through June 30, 2020 (Extended to June 30, 2021)
Project Period Covered by this Report: July 1, 2018 through June 30,2019
Project Amount: $1,500,000
RFA: NIH/EPA Centers of Excellence on Environmental Health Disparities Research (2015) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
Project 1: Metals and metal mixtures in DNA damage and repair
Specific Aim 1. Investigate the impact of specific metals and metal mixtures in human populations using biomarkers of toxicity including measures of oxidative stress, DNA damage and PARP activity.
We will test a) DNA damage and repair capacity in peripheral blood mononuclear cells (PBMCs), b) target zinc finger protein PARP-1 activity in PBMCs, and c) measure biomarkers of oxidative stress and oxidative damage in PBMCs and urine from samples collected in the partner communities. The findings will be analyzed with respect to biomonitoring results conducted through the Environmental Monitoring and Interpretation Core. These studies will give us insights into the impact of metals and metal mixtures in the Native communities.
Specific Aim 2. Establish mechanism of specific metal disruption of zinc finger target proteins and potential metal interactions in generation of oxidative stress, DNA damage and PARP activity.
We will use controlled in vitro and cell based assays to test a) metal binding and zinc displacement from zinc finger peptides using analytical techniques, b) metal binding, zinc displacement and PARP-1 function in protein isolated from cells exposed to concentrations of metals spanning the maximum contaminant level (MCL) values and measured exceedance levels found in the communities, c) impact of metals and metal combinations of oxidative stress and oxidative damage, d) effects of metals and metal combinations on cytotoxicity, DNA damage and DNA repair and e) determine whether zinc is protective against the effects of metals. These studies will provide mechanistic insights on the actions of metals and metal mixtures at concentrations relevant to the communities.
Project 2: Development of biomarkers of autoimmunity in three tribal communities exposed to mixed metal contaminants
Aim 1: Determine whether exposure to metals or metal mixtures (measured through biomonitoring) increases the prevalence of anti-nuclear antibodies (ANA) and/or lymphocyte apoptosis in individuals from our three affected Tribal communities.
AIM 2: Determine whether exposures to metals or metals mixtures are associated with changes in cytokine profiles or T and B cell phenotypes in individuals from our three Tribal populations.
AIM 3: Determine the ability of metal mixtures relevant to our three participating Tribal communities to exacerbate or to induce autoimmune disease and immune dysregulation via a drinking water exposure in animal models.
Progress Summary:
Project 1:
We have completed DNA damage and PARP activity measurements for Cheyenne River Sioux Tribe (CRST) participants and Navajo participants (50 men and 50 women for each community). The Navajo dataset is complete with biomonitoring data from the CDC. The CRST dataset is waiting for the biomonitoring results from the Environmental Core. Samples from Crow are pending.
We worked with the statistics group in the Administrative Core on another dataset to test statistical approaches most appropriate for Aim 1, which resulted in the publication of “Metal exposure and oxidative stress markers in pregnant Navajo Birth Cohort Study participants”[ Dashner-Titus et al. 2018]. Arsenic, but not uranium, was associated with increased biomarker of oxidative stress (urinary F2 –isoprostanes) and zinc was not found to have any causal mediation of the effects of the other metals on oxidative stress.
Based on experiences using the data set described above, the statistical team identified and compared methodologies leading to a publication accepted pending minor revisions “Two-step approach for assessing the health effects of environmental chemical mixtures: Application to the Navajo Birth Cohort Study” [Luo L, et al. Environmental Health].
All metal cytotoxicity studies (individual and combined) have been completed in human monocytes (THP-1 cells) and human T-cells (Jurkat cells) and we do not detect any interactions (enhanced cytotoxicity) in mixed metal exposures (Aim 2). We have also tested single metals in normal human peripheral blood mononuclear cells (PBMC) and normal human T-cells and the normal human cells are no more sensitive to arsenic or uranium than the cell lines (THP-1 and Jurkat).
Uranium did not promote an oxidative stress response in THP-1 or Jurkat cells. Expression of the heme oxygenase (HMOX1) gene is a sensitive indicator of oxidative stress and arsenic, but not uranium, induced HMOX mRNA (Figure 1). This finding was confirmed at the protein level (not shown). Other measures included detection of reactive oxygen species with a fluorescent dye and oxidative stress nuclear signaling with similar results.p
Arsenic, but not uranium, induced direct DNA damage as detected by pH2AX staining, a marker of DNA strand breaks. Similarly, uranium did not inhibit the activity of the DNA repair protein PARP-1 although PARP inhibition by arsenic was observed in both cell lines (Figure 2). p
In keeping with this observation, arsenic potentiated the cytotoxicity of a DNA damaging agent (etoposide), but uranium did not. These findings were unexpected because uranium has been reported to induce oxidative stress and inhibit DNA damage repair in human keratinocytes and bronchial epithelial cells. We are planning to explore a mechanism based on differential expression of NADPH oxidases to address the observed cell type differences.
Interestingly, the observation that uranium does not inhibit PARP activity or induce DNA strand breaks is consistent with the lack of oxidative stress generation by uranium in these cells. We reported that arsenic inhibition of PARP-1 requires both arsenic binding to the zinc finger motif of PARP-1 and oxidation of the cysteine residues of the PARP-1 zinc fingers and the oxidation was mediated by arsenic stimulation of NADPH oxidase activity. Although uranium interacts with a PARP zinc finger peptide using analytical techniques, the absence of oxidative stress and cysteine modification would account for the lack of PARP inhibition. This observation may be important for understanding differential sensitivity ofp different cell types to uranium based on metal generation of oxidative stress. To test other mechanisms by which uranium might interfere with DNA repair, we tested protein levels of ten DNA repair proteins that had been reported in the literature to be regulated by metals. Arsenic decreased OGG1, PARP-1, XRCC1 and MLH as detected by western blot analysis. Uranium increased OGG1 and decreased XRCC1 and XPC. We plan to expand this analysis to metal mixtures in the coming year.
Project 2:
In addressing the objectives outlined in aim 1, we have found using a multiple linear regression analysis a significant positive association between urine uranium levels and the presence of ANA in male participants of the Navajo Birth Cohort Study (NBCS).p Among the ANA positive sera from Navajo Birth Cohort Study (NBCS) male participants, we observe an increased staining intensity (>2+ at nuclear site at a 1:160 dilution) compared to previous clinical literature information and to the NHANES published information. In addition, cytoplasmic staining was also observed among the positives indicative of possible extensive molecular changes involved by metal exposures.p We have found that among Cheyenne River Sioux Tribal members, around 1/3 of them had ANA detected, again higher than national averages. We previously reported higher than expected (vs published national averages) levels of antibodies against denatured DNA and other nuclear components (histone, native DNA, chromatin).p Overall, the specificity profiles are consistent with those associated with environmental damage and are not typically used in autoantigen clinical panels.p We continued to examine the increased prevalence of serum autoantibodies during the current reporting period by examining other autoantigen productions. Using serum samples from CRST participants with sufficient sample volume, we looked for the presence of anti-cyclic citrullinated protein immunoglobulin (CCP).p The laboratory determination was conducted at a CLIA-certified immunological laboratory as this assay has clinical significance in rheumatoid arthritis (RA) diagnosis. The anti-CCP immunoglobulins react with a RA-specific antigen, specifically a citrullinated (post-translationally altered) form of filaggrin. A majority of samples were within the normal ranges of 0- 2.9 U/ml. Only a smaller percentage of examined sera had (3/161) elevated CCP results indicative of disease-specific (RA) production. Active cigarette smoking was not a predictor of these results.
To address aim 2, we used a Bayesian clustering technique to assign study participants into groups based on their metals exposure profiles. Through this method, participants “clustered” into 6 groups based on whether their metals levels were in the first through 4th quartile for that metal.p About ¼ of study participants were in the lowest quartile (group 1) and about the same proportion were in the highest exposure cluster (group 6).p Strikingly, participants who fell into the three highest exposure clusters have elevated circulating levels of the inflammatory cytokine, IFNγ.p Analysis is underway to determine if cluster assignments and cytokine expression are associated with other study outcomes such as biomarkers of autoimmunity (ANA) and lymphocyte phenotype.
In Aim 3,p we are using animal models to address immunologic alterations resulting from low-dose, chronic exposure to mixed metals in drinking water which represent the primary contaminants of concern on the Sioux, Navajo and Crow Reservations. To model “lifetime” exposure, beginning in utero, adult female C57BL/6N mice were exposed to drinking waterp(control or As+U+Mn or U+Mn water) for 7 days and then paired with male C3H/HeJ mice.p Drinking water exposure continued through pregnancy and up to weaning.pThe pups, B6C3F1, were then continuously exposed to the metal mixtures in drinking water until 12 weeks of age. At 12 weeks we measured serum antinuclear autoantibodies (ANA); splenic lymphocytic subpopulations including activated T cells, mature and immature B cells, T regulatory cells (Treg) by flow cytometry; IgM antibody production to a T cell dependent antigen; total IgM and IgG levels; proteinuria; creatinuria; global methylation in brain tissue; serum autoantibodies to glomerular antigens; body and organ weights.p The most remarkable findings include significant changes observed in maturation and activation of T and/or B lymphocytic populations related to mixed metal exposure.p The change in these populations could reflect a metals-induced immune dysregulation phenotype similar to that observed in our human population studies and as such warrant further investigation to better understand the impact of these changes on immune function.p It will also be important to determine the role of mixed metal exposures and the mechanism by which this exposure alters immune responses.
Future Activities:
Project 1:
For Aim 1 we will continue analysis of participant samples as outlined in the aim. We will coordinate with Research Project 2 in data analysis and work with the Environmental Core to obtain biomonitoring results necessary to complete analysis of the CRST samples. Population statistics analysis is conducted in collaboration with the P50 Biostatistics Team. We will work with the CEC and RTC to properly communicate findings back to the community after analysis is complete.
For Aim 2 we will focus on completing the metal mixtures analysis using the approaches described in the aim and further investigate metal impact on DNA repair protein expression. We will also conduct careful analysis of the ability of different metals or metal mixtures to generate oxidative stress as this appears to be a critical component DNA damage and cell toxicity based on our data. All research is conducted within a QA/QC program to ensure rigorous, robust and unbiased results.
Project 2:
We will continue to assess the impact of mixed metal exposure on immune parameters-cytokine profiles, lymphocyte phenotypes, and ANA within the three tribal communities (Navajo, CRST, and Crow Nation). We have already received Crow Nation participants’ biospecimen samples and their immunological examinations are now underway. The results from these studies will form a solid basis to apply for additional research funding to allow us to continue our work to better understand the impact of metals exposure on immune activity and further address community concerns of various heavy metal exposures.
Journal Articles: 13 Displayed | Download in RIS Format
Other center views: | All 89 publications | 13 publications in selected types | All 13 journal articles |
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Dasher-Titus EJ, Hoover J, Luo L, Lee J-H, Du R, Liu KJ, Traber MG, Ho E, Lewis J, Hudson LG. Metal exposure and oxidative stress markers in pregnant Navajo Birth Cohort Study participants. Free Radical Biology and Medicine 2018;124:484-492. |
R836157 (2018) R836157 (2019) |
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Doyle JT, Kindness L, Realbird J, Eggers MJ, Camper AK. Challenges and opportunities for tribal waters:addressing disparities in safe public drinking water on the Crow Reservation in Montana, USA. International Journal of Environmental Research and Public Health 2018;15(4):567. |
R836157 (2018) R836157 (2019) R835594 (2018) R835594 (Final) |
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Eggers MJ, Doyle JT, Lefthand MJ, Young SL, Moore-Nall AL, Kindness L, Other Medicine R, Ford TE, Dietrich E, Parker AE, Hoover JH, Camper AK. Community engaged cumulative risk assessment of exposure to inorganic well water contaminants, Crow Reservation, Montana. International Journal of Environmental Research and Public Health 2018;15(1):76. |
R836157 (2019) R835594 (2017) R835594 (2018) R835594 (Final) |
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Eggers MJ, Doyle JT, Lefthand MJ, Young SL, Moore-Nall AL, Kindness L, Other Medicine R, Ford TE, Dietrich E, Parker AE, Hoover JH, Camper AK. Community engaged cumulative risk assessment of exposure to inorganic well water contaminants, Crow Reservation, Montana. International Journal of Environmental Research and Public Health 2018;15(1):76 (34 pp.). |
R836157 (2018) |
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Girlamo C, Lin Y, Hoover J, Beene D, Woldeyohannes T, Liu Z, Campen M, MacKenzie D, Lewis J. Meteorological data source comparison-a case study in geospatial modeling of potential environmental exposure to abandoned uranium mine sites in the Navajo Nation. ENVIRONMENTAL MONITORING AND ASSESSMENT 2023;195(7):834 |
R836157 (2020) |
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Gonzales M, King E, Bobelu J, Ghahate DM, Madrid T, Lesansee S, Shah V. Perspectives on biological monitoring in environmental health research: a focus group study in a Native American community. International Journal of Environmental Research and Public Health 2018;15(6):1129 (8 pp.). |
R836157 (2018) R836157 (2019) |
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Harmon ME, Lewis J, Miller C, Hoover J, Ali AS, Shuey C, Cajero M, Lucas S, Zychowski K, Pacheco B, Erdei E, Ramone S, Nez T, Gonzales M, Campen MJ. Residential proximity to abandoned uranium mines and serum inflammatory potential in chronically exposed Navajo communities. Journal of Exposure Science & Environmental Epidemiology 2017;27(4):365-371. |
R836157 (2018) R836157 (2019) |
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Hoover JH, Coker E, Barney Y, Shuey C, Lewis J. Spatial clustering of metal and metalloid mixtures in unregulated water sources on the Navajo Nation – Arizona, New Mexico, and Utah, USA. Science of The Total Environment 2018;633:1667-1678. |
R836157 (2018) R836157 (2019) |
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Hoover J, Gonzales M, Shuey C, Barney Y, Lewis J. Elevated arsenic and uranium concentrations in unregulated water sources on the Navajo Nation, USA. Exposure and Health 2017;9(2):113-124. |
R836157 (2016) R836157 (2017) R836157 (2019) |
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Hoover J, Coker E, Erdei E, Luo L, MacKenzie D, Lewis J. Preterm Birth and Metal Mixture Exposure among Pregnant Women from the Navajo Birth Cohort Study. ENVIRONMENTAL HEALTH PERSPECTIVES 2023;131(12). |
R836157 (Final) |
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Lewis J, Hoover J, MacKenzie D. Mining and environmental health disparities in Native American communities. Current Environmental Health Reports 2017;4(2):130-141. |
R836157 (2017) R836157 (2019) |
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Rodriguez-Freire L, Avasarala S, Ali AS, Agnew D, Hoover JH, Artyushkova K, Latta DE, Peterson EJ, Lewis J, Crossey LJ, Brearley AJ, Cerrato JM. Post Gold King Mine spill investigation of metal stability in water and sediments of the Animas River watershed. Environmental Science & Technology 2016;50(21):11539-11548. |
R836157 (2016) R836157 (2017) R836157 (2019) |
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Saup CM, Williams KH, Rodríguez-Freire L, Cerrato JM, Johnston MD, Wilkins MJ. Anoxia stimulates microbially catalyzed metal release from Animas River sediments. Environmental Science: Processes & Impacts 2017;19(4):578-585. |
R836157 (2017) R836157 (2019) |
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Supplemental Keywords:
metals, DNA repair, arsenic, uranium, metals, ANA, Immune activity, Native AmericanRelevant Websites:
Navajo Birth Cohort Study A beautiful life for mother and baby. Exit
Progress and Final Reports:
Original Abstract Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R836157C001 Metals and metal mixtures in DNA damage and repair
R836157C002 Development of biomarkers of autoimmunity in 3 tribal communities exposed to mixed metal contaminants
The 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.
Project Research Results
- Final Report
- 2020 Progress Report
- 2018 Progress Report
- 2017 Progress Report
- 2016 Progress Report
- Original Abstract
13 journal articles for this center