The US military used burn pits at large and small forward operating bases during Operation Enduring Freedom (2001 to 2014) in Afghanistan and Operation Iraqi Freedom (2003 to 2011) in Iraq (OEF/OIF) and although less common, their use continues in some forward-deployed locations in Afghanistan today.1,2 Burn pits have been used to dispose of solid waste materials including plastics, metals, rubber, paints, solvents, munitions, and wood, depending on waste segregation practices at the particular base.1,2
The Department of Defense (DoD) efforts to assess deployment exposures of military personnel and civilian employees to burn pits, have usually consisted of only obtaining ambient sampling data, but there were few systematic efforts to collect breathing zone measurements1,2 Ambient sampling at the outset of OEF/OIF measured particulate matter (PM), volatile organic compounds (VOCs), polycyclic aromatic hydrocarbons (PAHs), heavy metals, and other common deployment exposures. These ambient sampling plans were poorly designed and failed to include several types of pollutants.3,4 In a few instances breathing zone sampling data were systematically collected, but breathing zone air sampling data is particularly difficult to obtain in the setting of a military deployment due to both logistical and operational concerns.
Recent epidemiological studies that have examined the contributions of deployment environmental exposures on post-deployment health have reported an increased risk of respiratory illness related to deployment environmental exposures in Iraq and Afghanistan.5–9 Garshick et al10 reviewed the published literature and reported an overall 20% increased incidence of respiratory illness related to environmental exposures compared with non-deployed controls for respiratory symptoms, new onset asthma, and other respiratory illness at the 2018 American Thoracic Society Deployment and Respiratory Health Workshop held in San Diego, CA.
This manuscript is intended to provide an overview of a large, ongoing, important study of biomarkers in deployed US Forces. The Military Biomarkers Research Study (MBRS) is a long-term study that consists of four phases and much work has already been published, to include publication of a journal supplement.
The MBRS assessed the use of serum biomarkers to predict health risks in deployed US Service Members and consisted of four phases. In Phase I of the study, we did a pilot study to assess the feasibility of using serum to detect biomarkers of internal dose, and biomarkers of effect. In phase II of the study, we examined the environmental sampling data to see if there were correlations between exposure data and serum biomarkers of internal dose and biomarkers of effect. In Phase III of the study, we assessed whether serum biomarkers of burn pit exposures are associated with health outcomes in deployed US service members. In Phase IV, we validated our Phase III study findings by exposing pulmonary cell lines to PAHs and dioxins and observed the changes in serum metabolomics and biomarkers.
Because breathing zone air sampling data are difficult to obtain and concern about long-term health effects2 from exposures have persisted, DoD leaders wanted to assess whether it was feasible to use serum biomarkers to predict risks of adverse health outcomes using samples retrieved from the DoD Serum Repository (DoDSR).2,11 In Phase I of the MBRS, the Defense Medical Surveillance System (DMSS)11 was used to obtain Service member demographic data and medical outcomes data that was linked to the serum samples retrieved from the DoDSR. This powerful epidemiological resource allowed us to investigate the relationship between deployment exposures, and serum biomarkers.11 Ambient air sampling was conducted at Joint Base Balad in Iraq and these data were used by our team to develop a health risk assessment for adverse health outcomes in service members assigned to burn pits at Balad Air Base.12,13 Similarly, we used breathing zone sampling data collected at Bagram Air Base in Afghanistan on service members who provided security around the burn pits in Afghanistan.14 The DoDSR serum, demographic data from DMSS, and air sampling data were linked by personnel at the DoD Armed Forces Health Surveillance Center, Silver Spring, MD and then turned to the research team for analysis under a DoD approved data sharing agreement.
In Phase I of the MBRS which was completed earlier, researchers from the Uniformed Services University partnered with researchers from Emory University, University of Rochester and Clarkson University to do an exploratory study of stored sera to assess the feasibility of using serum to detect biomarkers of internal dose and biomarkers of effect on 30 orphan serum samples where the sample source was not known.15–18 In phase II of the MBRS, we assessed the environmental sampling data to see if there were correlations between exposure data and serum biomarkers on internal dose and biomarkers of effect in pre- and post-deployment serum samples collected for deployers and controls who did not deploy. We also assessed the impact of deployment exposures on metabolic pathway alterations and alterations in biomarkers including microRNA and cytokines.19–22 In Phase III of the MBRS, we assessed whether serum biomarkers of burn pit exposures (cytokines, chemokines, and microRNAs and alterations in metabolic pathways) are associated with health outcomes in deployed US service members and controls.23,24 We compared deployers with respiratory health outcomes with non-deployers and examined associations with changes in biomarker expression to determine if deployment exposures increased the risk of adverse health outcomes.25–30 In Phase IV of the MBRS, we validated our Phase III study findings by exposing pulmonary cell lines to PAHs and dioxins30,31 in an effort to validate the changes in serum metabolomics and miRNA biomarkers.32
Phases I and II
Phase I was a pilot study which established the feasibility of using serum from the DoDSR to retrospectively identify and measure biomarkers of internal dose and effect. This pilot used 30 “orphan” samples, which could not be associated with a specific individual, to verify that the specimens in the DoDSR could successfully be used for biomarker analysis, despite their small volume size and storage conditions.15–18
In phase II, we explored the use of alternatives to measuring breathing zone samples of external dose by analyzing previously collected serum in the DoDSR to see if changes occurred in the serum after deployment and compared these changes with those in non-deployed controls. The analytical methods used to test the DoDSR samples for biomarkers have been described in detail previously.15–22
We looked to see if correlations between environmental sampling data and serum biomarkers of internal dose and effect could be identified.19,20 The study cohort consisted of 200 “cases” who had deployed to either Bagram Air Base or Joint Base Balad during burn pit operations that were matched with 200 controls who were in the military but had not deployed. The analysis looked for changes in deployers and in controls before and after deployment and the results in the 200 deployers were compared with the results in the 200 non-deployed controls.19–22
The DMSS provided service member demographic data linked to the serum samples retrieved from the DoDSR and the de-identified serum and demographic data were provided to each research center. We did a retrospective analysis of deployed Service members to test for associations between serum levels of PAH and dioxin18 and serum biomarkers of metabolic pathway alterations19,20 and changes in miRNAs.21,22 Two new studies were completed for Phase II by our team that examined molecular profiling of deployed service members25 and post-deployment serum alterations due to environmental chemical exposures.26
In Phase III, we assessed whether serum biomarkers of exposure to burn pit emissions are associated with health outcomes in deployed US service members.23,24 The DMSS was used to obtain service member demographic data and medical outcomes data that was linked to the serum samples retrieved from the DoDSR. We linked serum biomarker analysis results with health outcomes in deployers and non-deployers and changes in respiratory health for deployers and controls.23 We identified respiratory health outcomes for deployers using the International Classification of Diseases-9 coded, medical encounter data and compared the results with controls after adjusting for demographic variables including sex, race, branch of service, military component, deployment history, military occupational code, and smoking status.
In this supplement, we report new Phase III analysis work that examined whether any of the serum biomarker changes were different between the deployers and non-deployers.27,28 Thakar et al29 identified deployers with diseases of the respiratory system and circulatory system and looked for associations in altered metabolomic and microRNA levels compared with non-deployed controls.
In Phase IV, we validated the serum biomarker findings in our Phase III study findings by exposing pulmonary cells to polycyclic aromatic hydrocarbons (PAHs), naphthalene, and Dioxin,30 and Benzo[a]pyrene in the laboratory and examining changes in serum metabolomics31 and miRNA biomarkers,32 and comparing the results with changes previously observed in the post-deployment samples from the DoDSR regarding the metabolic pathway alterations20 and up and down regulation of several miRNAs.21 The above studies by Smith et al.30–32
ARTICLES IN THIS SUPPLEMENT
Phase II Study Results
Krahl et al33reviewed the environmental sampling methodology currently employed and presented information regarding emerging technologies that will enhance the ability of the DoD and private sector to obtain personal breathing zone environmental sampling data. Krahl et al also highlights the DoD's need to integrate the exposure sampling data into a Risk Management decision making regarding prevention of exposures to unsafe levels of environmental and occupational hazards. Dr Krahl notes exposure assessment has undergone a paradigm shift in the past 15 years, moving to consider the totality of exposures over the course of a lifetime that is defined as the exposome. The DoD seeks to advance exposure assessment through the adoption of new biomonitoring sensors that are light-weight, ruggedized, and wearable by personnel that enable the capture of individual-level occupational and environmental exposure data, permit rapid analysis of biospecimens, and permit real-time operational risk assessments that permit characterization of high, medium, or low exposure risk and related health effects. The data generated must be archived in a way that permits longitudinal health assessments in the future.
Khan et al25 did a retrospective analysis of pre and post-exposure serum samples. They identified exposure to tobacco smoke using analytes obtained from post-exposure serum samples. The molecular analysis was done on 800 human serum samples from the DoDSR. Subjects were classified as smokers or non-smokers based on direct measurement of serum cotinine levels. A machine-learning pipeline with supervised support vector machines (SVM) was used to examine the molecular profiles of smokers and non-smokers. The SVM predicted smokers and non-smokers with accuracy that was increased using recursive feature elimination. Correcting for age and sex did not significantly impact the results and selected miRNAs were associated with tobacco smoke and supported the validity of the computational approach.
Smith et al26 examined changes in post-deployment serum samples compared with the same individuals pre-deployment serum samples and compared the results of post-exposure serum samples with environmental chemicals present in serum samples from non-deployed controls. De-identified pre and post-deployment serum samples and control serum samples were analyzed by high-resolution metabolomics. A total of 271 environmental chemicals were identified. Pairwise comparisons were performed to test for differences in the pre- and post-deployment serum. Several pesticides and other environmental chemicals were detected in the post-deployment serum. Only small differences were observed in the serum of deployers to Balad and Bagram.
Go et al27 used high-resolution metabolomics to identify changes in the serum of military personnel deployed to Balad, Iraq, or Bagram, Afghanistan. Pre- and post-deployment samples were obtained from the DoDSR. High resolution metabolomics and bioinformatics were used to identify metabolic differences between deployers and non-deployers. Differences were observed at baseline in the pre-deployment serum samples of personnel deployed to Bagram and Balad and with non-deployed controls. Deployment to Balad was associated with alterations to amino acid and lipid metabolism, consistent with inflammation and oxidative stress, and pathways linked to metabolic adaptation and repair. Deployment to Bagram was associated with metabolic changes in lipid pathways linked to cell signaling and inflammation. Dr Go observed that metabolic changes post-deployment are consistent with responses to air pollution and other environmental stressors.
Thatcher et al28 examined the health risks of deployment to sites with open burn pits. They investigated whether post-deployment serum biomarkers were altered following exposure to burn pits. A regression model was developed using a supervised vector machine to identify serum biomarkers with significant associations to both exposures and deployment. They found serum polycyclic aromatic hydrocarbons (PAHs), dioxins, or furans were altered and associated with changes in miRNA biomarkers.
A signature of exposure to open burn pits was discovered that provides a framework for using post-exposure sera to identify exposures when contemporaneous monitoring is inadequately powered or of poor quality.
These studies are reported in this supplement.
Phase III Study Results
Thakar et al29 designed a novel computational framework to examine associations across different datasets and link deployment related exposures, molecular responses, and associated health outcomes in service members after burn pit exposures in deployed US Service Members. They observed associations with changes in serum microRNAs and metabolic pathway alterations and health outcomes in security force personnel who deployed to Balad, Iraq, or Bagram, Afghanistan, who had burn pit exposures. The results were compared with the findings in matched controls never deployed. The results show respiratory affects were broadly linked to environmental chemicals while sinusitis and rhinitis were linked to cotinine levels and tobacco use. They also observed both the up and down regulation of select microRNAs in response to deployment exposure to PAHs and dioxins from burn pit exposure. These findings show that integrative analysis can identify molecular links between exposures, biologic responses, and disease outcomes.
Phase IV Study Results
Smith et al30 conducted an in vitro study to identify metabolic changes related to benzo(ghi)perylene (BghiP) and 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD) exposure in human lung fibroblasts which served to validate biological associations previously reported.20
They exposed human lung fibroblasts to BghiP and HpCDD in vitro and analyzed extracts by MWAS to identify alterations in metabolic pathways, and changes in gene expression were measured by real time polymerase chain reaction. They found metabolic perturbations in amino-acid, oxidative stress, and fatty-acid pathways following exposure to both BghiP and HpCDD. HpCDD was also noted to increase gene expression. They concluded that exposure to PAH or dioxin perturbs amino acid pathways by different mechanisms. Further, these findings show an effect on central homeostatic systems by environmental exposures that can affect disease susceptibility.
Smith et al31 examined in the serum of deployers for Benzo[a]pyrene (BaP) which is a PAH found in smoke and noted to be a carcinogen. A broad range of other adverse health effects-have been linked to air pollution particles containing BaP. Recent studies show that metabolic effects of BaP include disruption of fatty acid and lipid metabolism. In the present study, human lung epithelial (A549) cells were treated with BaP, and extracts were analyzed in a metabolome-wide association study (MWAS). The results show that BaP alters lipid metabolism and changes fatty acid, glycerol, sphingolipids, and 27-hydroxycholesterol pathways. Additional associations with amino acid metabolism were found in pulmonary cells that validated earlier findings in human sera. These findings suggest that air pollution may exacerbate disease processes by altered mitochondrial and amino acid metabolism.
Woeller et al32 observed previously21 that deployment to sites with open burn pits was associated with altered serum microRNA (miRNA) levels. In the current study, they examined whether miRNA expression changes were seen post-deployment in vitro in primary human lung fibroblasts (HLFs) after exposure to the PAHs naphthalene and benzo(ghi)perylene (BgP) and the polychlorinated dibenzodioxin 1,2,3,4,6,7,8-Heptachloro-dibenzo-p-dioxin (HpCDD). Changes in miRNA activation and mRNA levels were detected by quantitative polymerase chain reaction. The results showed that miRNAs linked to burn pit exposure were altered by HpCDD and alterations in the AhR attenuated these findings. These studies confirm our earlier findings that specific miRNAs are altered by HpCDD exposure.
Our Military Biomarkers Research Study began with an initial attempt to evaluate the utility of the DoDSR with respect to precision medicine and use of exposure biomarkers. Given the lack of environmental sampling data and the logistical issues related to the collection of air sampling data, there is a need for the DoD to develop ways of capturing deployment exposure information that can be used in a meaningful fashion to help guide the health risk assessments of deployed troops. Our team took a broad approach in assessing the utility of sera in the DoDSR with regard to serum quality and health parameter measurements in terms of metabolomics, cardiovascular and inflammatory markers, and miRNA levels. We were able to identify novel biomarkers that are associated with deployment exposures. Further, we were able to link in a global way the results of metabolomics, cytokines, micro-RNAs, and environmental chemicals to deployment environmental exposures and health risk. Our studies reported in our first supplement23 and in this journal supplement demonstrate29 the utility of the current DoDSR in linking exposures with health outcomes. The studies emphasize the need to maintain the current DoDSR while expanding its specimen profile to improve its operational capabilities. The addition of other specimens to the DoD repository, possibly blood, urine, and DNA, would enhance its usefulness. However, exposure detection and characterization, particularly in deployed, hostile areas, must receive top priority because it represents an extremely challenging and complex series of problems to address. The studies of biomarkers in repository specimens reported here provide optimism that this approach will continue to contribute meaningful information. However, this approach must not be viewed as the only solution to the problem of inadequate exposure identification and characterization in deployed Service Members.
The results presented here should not diminish other current efforts to improve timely exposure identification and characterization, to include informing military leaders of their responsibilities for protecting their troops, developing more efficient means for conducting personal and area sampling, and refining the art and science of obtaining better information from questionnaires and medical encounters and modeling of exposures.
Further, the results from our studies provide the justification for a much larger study that would use a larger cohort of exposed and unexposed service members that would have sufficient power to detect significant differences between the exposed and control subgroups. This would permit an opportunity to do targeted breathing zone exposure assessments that could also be used to validate the internal biomarkers of dose and help to better link internal dose measurements with specific health outcomes. A National Academy workshop found our approach to be sound and deserves the funding to execute the study in a much larger population with a longer follow-up period.34
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