C
Abstracts of Approved Proposals
Principal Investigators: David Batty and Beverly Roberts, University of Edinburgh
Title: Cognitive Function in Middle Age as a Predictor of Later Life Health: Analyses of the Data from the Air Force Health Study
Assets Used: Electronic data only
Abstract: This study is looking at the general association between cognitive function in midlife and later adult health: How do social and personal factors contribute to differences in human mortality, health, and health behaviors? Specifically, the researchers are examining three hypotheses. (1) Relative to their lower-performing counterparts on standard tests of cognitive function available in the AFHS (e.g., Wechsler adult intelligence scale), men with higher scores will have more favorable levels of health behaviors that have been linked to mortality, that is, they will be less likely to smoke, and more likely to be physically active, and have a healthy diet. They will also examine the links between cognition, alcohol intake, and drug use (licit and illicit). (2) Relative to their lower-performing counterparts on tests of cognitive function, men with higher scores will have more favorable levels of physiological factors that have been linked to mortality. These physiological outcomes include lower levels of blood pressure, lipids, blood glucose, insulin, and inflammatory and coagulation markers. They will separate the effects of cognition on physiological factors from those for the health behaviors. (3) Relative to their lower-performing counterparts on tests of cognitive function, men with higher scores will have longer life expectancy, that is, a lower risk of all-cause mortality and major causes of death (such as cardiovascular disease and cancer).
Principal Investigator: Kim Boekelheide, Brown University
Title: Identifying Epigenetic Molecular Biomarkers of Dioxin Exposure in Vietnam Veterans
Assets Used: Electronic data and biospecimens; pilot funding awarded
Abstract: Dioxin is a tumor promoter, alters immune function, and modifies metabolism. Environmental exposures modify epigenetic programming, including DNA methylation; therefore, measuring DNA methylation marks can provide insight into the cellular response to chemical exposures. This pilot project proposes using a new epigenome-wide molecular approach to detect dioxin exposure-related alterations in DNA methylation in biospecimens from the Air Force Health Study. One potential target for dioxin-related exposure effects is the epigenome. DNA methylation is one of the main types of epigenetic modifications, a process important to gene regulation. The pattern of DNA methylation reflects both inherited imprints and environmental conditions, allowing cells to adjust their programs in response to external cues. Immune cell function, tumor suppressor gene regulation, and metabolic gene expression are all altered by dioxin exposure, and these effects are almost certainly associated with changes in DNA methylation in cells of multiple tissues. We have published numerous studies of DNA methylation marks in normal and abnormal tissues, including adipose tissue, blood cells, and sperm. These novel results and the availability of unique biospecimens from the Air Force Health Study inspired us to collaborate on this effort to study the effects of dioxin exposure on DNA methylation marks in Vietnam veterans. Past studies have examined AFHS samples of adipose tissue, blood, and semen for their cellular and phenotypic attributes, but little molecular characterization has been performed using modern high-throughput techniques. This pilot project interrogates the DNA methylation marks of adipose tissue, blood cells, and sperm from highly exposed and comparison Air Force veterans. Importantly, all three tissue samples will be taken from the same study participant, allowing for both within individual and interindividual comparisons of dioxin-related effects across tissue type and by exposure level. DNA methylation profiling will use the Illumina Infinium HumanMethylation450 BeadChip assay that interrogates over 485,000 unique DNA methylation sites and provides CpG coverage of 99% of RefSeq genes in the human genome. Biostatistical analysis will identify alterations in DNA methylation marks associated with dioxin exposure.
Principal Investigator: Janice Chambers, Mississippi State University
Title: A Longitudinal Study of Paraoxonase 1 (PON1) in Relationship to Type 2 Diabetes and Aging
Assets Used: Electronic data and biospecimens; pilot funding awarded
Abstract: The proposed project will investigate the serum enzyme paraoxonase 1 (PON1), a hydrolase associated with the high density lipoprotein particle. PON1 has a lipolactonase activity that allows it to be protective of the cardiovascular system and its role in cardiovascular health is well documented. PON1 is also implicated in reducing risk for type 2 diabetes. PON1 activity has been found to decline with age, but evidence for this has not resulted from a longitudinal study of the same individuals. Therefore, the AFHS offers an opportunity to investigate both the role of PON1 in risk of type 2 diabetes development and changes in PON1 activity with aging. The first hypothesis proposed for this project is: Lower serum PON1 levels are associated with the development of type 2 diabetes. The second hypothesis is: serum PON1 activities decline with age. This proposed case-control study will analyze serum spectrophotometrically for PON1 activities assayed with three substrates: paraoxon, phenyl acetate, and dihydrocoumarin. C-reactive protein (a marker of inflammation) and PON1 protein will be quantified by ELISA. The serum samples will be from males of similar age from the control group who participated in cycles 1 and 6, half of whom were diabetic in 2002. We propose a longitudinal assessment of PON1 activities and PON1 protein and C-reactive protein concentrations in the same individuals over 20 years, in relationship to occurrence of type 2 diabetes. Logistic regression models relating these measurements with clinical and demographic information will be developed. Serum and demographic/clinical information will be requested for 250 participants (125 diabetics and 125 nondiabetics). (Initially a sample activity validation study with 25 samples will be conducted to verify that activities and protein levels are sufficiently robust before the remaining 225 samples are obtained.) Outcomes anticipated from this study are (1) documentation on whether lower PON1 activity and/or protein levels are associated with occurrence of type 2 diabetes, and (2) whether PON1 activity and/or protein levels decline with age in participants without type 2 diabetes. If these associations are supported in this longitudinal study, then PON1 (with further documentation) may be a useful new biomarker for type 2 diabetes risk. We anticipate that the data and preliminary models derived from this project would be used as preliminary data to seek support for additional studies using the AFHS biospecimens and data.
Principal Investigator: Laurie Haws, ToxStrategies, Inc.
Title: Exposure-Response Relationship for Dioxin and Cancer and Non-Cancer Health Outcomes in the Air Force Health Study Cohort Using Physiologically-Based Pharmacokinetic Modeling of Exposure and Updated Mortality
Assets Used: Electronic data
Abstract: To date, very few studies have been published that describe the original TCDD exposures experienced by the Ranch Hand cohort based on the measures of TCDD in serum, and none have applied the current state-of-the-art physiologically-based pharmacokinetic (PBPK) models to estimate internal TCDD exposure over time. This exposure metric can be used with health outcome data to quantify the risk of cancer and other health outcomes, such as type 2 diabetes, in the cohort. Furthermore, the mortality experience among the Ranch Hand cohort was last assessed through 1999, and due to the age of the cohort, an updated mortality assessment would greatly improve statistical power. As such, the objectives of this study are to (1) obtain the serum TCDD data collected over time and updated mortality data for the Ranch Hand and Comparison participants; (2) use PBPK models to refine the exposure assessment; (3) conduct exposure–response modeling to assess potential mortality risk from TCDD exposures. In addition to traditional models, other approaches in exposure–response modeling will be explored such as smoothing spline regression models with the use of drsmooth statistical package in R.
Principal Investigator: George Knafl, University of North Carolina at Chapel Hill
Title: Effects of Dioxin Exposure for Male Air Force Vietnam Veterans on Reproductive Outcomes
Assets Used: Electronic data only
Abstract: The Air Force Health Study investigated the impact of herbicide exposure, especially to dioxin, on the health, survival, and reproductive outcomes of male Air Force veterans of the Vietnam War that were matched with a comparison group of Vietnam Air Force veterans not involved in those activities. Baseline data were collected in 1982. Medical records, including birth certificates, newborn clinic records, health records, and death certificates, were reviewed for all children to determine the occurrence of adverse reproductive outcomes, as well as reported adverse reproductive outcomes by parents. Serum dioxin levels were used as a measure of herbicide exposure. The effect of dioxin was based primarily on the classification of participants into Ranch Hands and comparison groups. It was concluded that the data collected prior to January 1990 provided little or no support for an adverse association between paternal dioxin exposure and reproductive outcomes. However, more complete data collected up to 2002 are available. More complete analyses of all available data have the potential for identifying adverse
dioxin exposure effects on reproductive outcomes not identified in earlier AFHS analyses. Consequently, we propose to conduct more complete analyses of the effect of dioxin exposure on reproductive outcomes for all study participants combined, not differentiated by exposure status, based on all available data, considering a wide variety of potential covariates including those accounting for missing data, and allowing for the possibility of a threshold for an adverse effect on reproductive outcomes with models determined adaptively using an objective model selection criterion.
Principal Investigator: Jack Mandel, Exponent
Title: The Reanalysis of the Ranch Hand Data
Assets Used: Electronic data only
Abstract: This study has three aims. First, examine the association between serum TCDD level and specific health outcomes in the AFHS population using a different set of assumptions and a different statistical approach than previously applied to these data, specifically testing whether the use of “as-measured” (not log transformed) serum TCDD levels and less restrictive dose–response models would provide a more precise, reliable and valid estimate of risk in the AFHS population. The second aim is to evaluate the impact of using sub-groups of the AFHS population and various comparison groups that differ from the comparisons defined in the original study protocol on the reliability and validity of the reported risks for cancer and diabetes associated with elevated serum TCDD levels. The third aim is to determine whether the rates of change in repeated laboratory measures and clinical indicators assessed in each examination cycle for an individual study participant are associated with the rate of change in the as-measured serum TCDD level.
Principal Investigator: Allan Mazur, Syracuse University
Completed
Assets Used: Electronic data only
Mazur, A., R. Westerman, A. Werdecker, and U. Mueller. 2014. Testosterone and Type 2 Diabetes in Men. Aging Male 17(1):18-24.1
Abstract: Objective: To assess from observational data if low testosterone in men is an independent risk factor for high fasting glucose (FG) and for a diagnosis of type 2 diabetes (T2D). Methods: Multivariate analysis of data from 991 male U.S. Air Force veterans who completed six medical examinations over 20 years. Results: Low testosterone was moderately related to high FG, independent of age and obe-
_______________
1Mazur, A., R. Westerman, A. Werdecker, and U. Mueller. Testosterone and type 2 diabetes in men, copyright © 2014, Informa Healthcare.
sity. Low testosterone is a very weak predictor of a diagnosis of T2D. Conclusions: In men, low testosterone is an independent risk factor for high FG, comparable to aging and obesity. Low testosterone is a weak predictor of a diagnosis of T2D.
Mazur, A., R. Westerman, and U. Mueller. 2013. Is Rising Obesity Causing a Secular (Age-Independent) Decline in Testosterone Among American Men? PLoS ONE 8(10):e76178. doi:10.1371/journal.pone.0076178.
Abstract: The testosterone of men in industrial societies peaks in their twenties and tends to decline with increasing age. Apart from this individual-level decline, there have been reports of a secular (age-independent population-level) decline in testosterone among American and Scandinavian men during the past few decades, possibly an indication of declining male reproductive health. It has been suggested that both declines in testosterone (individual-level and population-level) are due to increasing male obesity because men in industrial society tend to add body fat as they age, and overall rates of obesity are increasing. Using an unusually large and lengthy longitudinal dataset (991 U.S. Air Force veterans examined in six cycles over 20 years), we investigate the relationship of obesity to individual and population-level declines in testosterone. Over 20 years of study, longitudinal decline in mean testosterone was at least twice what would be expected from cross-sectional estimates of the aging decline. Men who put on weight intensified their testosterone decline, some greatly so, but even among those who held their weight constant or lost weight during the study, mean testosterone declined 117 ng/dl (19%) over 20 years. We have not identified the reason for secular decline in testosterone, but we exclude increasing obesity as a sufficient or primary explanation, and we deny the supposition that men who avoid excessive weight will maintain their youthful levels of testosterone.
Principal Investigator: Adam Mitchell, Emory University and Atlanta VA Medical Center, Department of Veterans Affairs
Title: Identifying Novel Biomarkers of Vulnerable Coronary Artery Disease: The Air Force Health Study
Assets Used: Electronic data and biospecimens
Abstract: Identification of patients at high risk for primary cardiovascular events (myocardial infarction and coronary death) and innovative prevention strategies could reduce the incidence of ischemic heart disease, a leading cause of morbidity and mortality. This project aims to identify novel epigenetic and metabolic biomarkers associated with future cardiovascular events. MicroRNAs are short, noncoding RNAs that function as posttranscriptional gene regulators through targeted binding of messenger RNAs. Because microRNAs circulate in the blood and are altered in disease states, they may serve as useful biomarkers. The serum metabolome also shifts with disease, and other groups have found
metabolites associated with adverse cardiovascular outcomes. We hypothesize that changes in circulating microRNAs and metabolomics profiles accompany, and likely contribute to, the development of vulnerable atherosclerotic disease. Using a nested case-control study design within the AFHS, we will characterize circulating microRNAs and metabolomics signatures associated with impending cardiovascular events. Study subjects identified as having medical record verified myocardial infarction or coronary death will serve as cases, and serum samples will be obtained from the clinical visit that immediately preceded their coronary event. Control subjects will be selected using incidence density sampling whereby a control subject with a calculated cardiovascular risk similar to each case will be sampled at the same time point as the matched case. Comprehensive microRNAs profiling will be performed using deep sequencing on 50 cases and 50 controls to discover microRNAs associated with cardiovascular events. The top 20 differentially expressed microRNAs will be validated in the remainder of the cohort with qRT–PCR. High-Performance Metabolic Profiling capable of measuring > 20,000 metabolites will be performed on all samples. The effect of serum microRNAs and metabolite levels on the occurrence of cardiovascular events will be compared within case–control matched sets using conditional logistic regression analysis. Moreover, development of an aggregate score by combining multiple microRNAs and metabolites that potentially improves the prediction of cardiovascular events will be explored by discrimination analysis. At the conclusion of these studies, we will have comprehensively characterized the circulating microRNAs transcriptome and metabolome in humans vulnerable to primary cardiovascular events. We believe these data will allow us to identify a signature of cardiovascular risk that could enhance current cardiovascular disease risk prediction models. In addition, identification of microRNAs and metabolic derangements could uncover new signaling pathways that are important in the progression of atherosclerosis proximal to the development of adverse clinical outcomes.
Principal Investigator: Kenneth Ramos, University of Louisville
Title: Detection of L1 Protein in Ranch Hand Biospecimens
Assets Used: Electronic data and biospecimens; pilot funding awarded
Abstract: The Air Force Health Study is one of the most exhaustive epidemiologic investigations of adverse human health effects in Vietnam veterans possibly related to herbicide exposure during aerial application of herbicides. While several herbicides were used for crop destruction, most studies have focused on Agent Orange. Questions related to the health effects of chemicals present in Agent Orange and the mechanisms involved in pathogenesis remain unanswered. Of particular interest are findings linking Agent Orange exposure to various types of cancer. As such, the study of cancer biomarkers would be highly beneficial given that veterans exposed to Agent Orange may be at heightened risk for malignancy. In this applica-
tion, studies are proposed to measure L1 ORF1p (Open Reading Frame 1 protein encoded by the Long Interspersed Nuclear Element-1) as a serum biomarker of genomic instability and cancer outcomes in veterans exposed to Agent Orange. An ELISA (enzyme-linked immunosorbent assay) platform will be used to measure the abundance of L1 ORF1p in serum specimens of veterans who participated in the AFHS. ORF1p is a 40 kDa RNA binding protein that is essential for L1 retro-element mobilization and pathogenesis. Our recent studies have shown that levels of L1 ORF1p are elevated in plasma samples of cancer patients and therefore, we are interested in further evaluating the utility of this biomarker in cancer evaluation. L1 reactivation causes genetic instability secondary to DNA strand breaks, insertion mutations, altered splicing, increased frequency of recombination, and loss of transcriptional control of genes in the vicinity of insertions. One or more of these alterations has been associated with cancer. We have shown that environmental hydrocarbons reactivate L1 in somatic cells via epigenetic mechanisms that involve removal of retinoblastoma-associated repressor complexes and recruitment of proteins that mediate transcriptional activation of L1. Epigenetic reactivation of L1 reprograms the genome to induce oncogenic phenotypes and/or facilitate insertion mutations that disrupt genome architecture and function. Thus, measurement of ORF1p may be a sensitive biomarker of L1 activity and a proxy of genomic instability and cancer predisposition in human populations. The appearance of ORF1p in human serum reflects protein leakage into the extracellular compartment after apoptotic or necrotic cell death following toxic injury or disease. We hypothesize that human exposure to Agent Orange is associated with increased levels of L1-encoded ORF1p in serum, which in turn, correlates with increased incidence of cancer outcomes in Ranch Hand veterans. This hypothesis will be tested in two specific aims designed to measure L1 ORF1p in serum samples from cycles 1 and 4 of veterans exposed to Agent Orange and correlate serum levels of L1 ORF1p with TCDD levels, history of malignancy, and clinical outcomes. Measurement of L1ORF1p in serum may identify participants who have been diagnosed with cancer or those who are at increased risk of cancer development or malignant progression.
Principal Investigator: Web Ross, Pacific Health Research and Education Institute, Department of Veterans Affairs
Title: Parkinson’s Disease and Premotor Features of Parkinson’s Disease in the Air Force Health Study
Assets Used: Electronic data only; pilot funding awarded
Abstract: Evidence from epidemiologic and basic science research suggests pesticides contribute to Parkinson’s disease risk. Specifically, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), the two constituents of Agent Orange, have been implicated in two separate studies, prompting the Institute of Medicine to acknowledge a suggested association of pesticides with
Parkinson’s disease. Additionally, the Department of Veterans Affairs (VA) has added Parkinson’s disease to the list of illnesses possibly caused by Agent Orange making Parkinson’s disease a service-connected condition for Vietnam veterans. There have been no studies of Agent Orange exposure and Parkinson’s disease in Vietnam veterans. The AFHS data provide a unique opportunity to address the association of Agent Orange exposure in Vietnam with Parkinson’s disease as well as with premotor features of Parkinson’s disease. The four aims seek to address key questions related to Agent Orange exposure and Parkinson’s disease risk using data from the AFHS. (1) determine prevalence of Parkinson’s disease in exposed Ranch Hand participants versus nonexposed comparison participants; (2) examine the association of serum dioxin with Parkinson’s disease in AFHS participants; (3) determine prevalence of premotor Parkinson’s disease features in Ranch Hand participants versus comparison participants; and (4) examine the association of serum dioxin with premotor Parkinson’s disease features in AFHS participants. To accomplish this, the researchers will determine the prevalence of Parkinson’s disease in Ranch Hands and comparison participants from self-report at exam 6 and, after 2002, from VA and Medicare datasets, and use data from exam cycles 1–6 to determine the prevalence and association of serum dioxin with premotor Parkinson’s disease features in the cohort.
Principal Investigator: David Roth, San Diego Healthcare System, Department of Veterans Affairs
Title: Caveolin’s Role During Healthy Aging
Assets Used: Electronic data and biospecimens; pilot funding awarded
Abstract: Heart failure is the most common cause of death in older patients and is often caused by cardiovascular disease. Cholesterol is an important component of the cell membrane and is an essential element of membrane lipid rafts. Caveolae, a subset of membrane lipid rafts, serve as signaling centers and contain caveolin proteins, cholesterol, and sphingolipids. This study aims to determine if there is an association between caveolin protein expression levels in serum and adipose tissue and the development of heart failure. The role of caveolin and membrane lipid raft expression and serum cholesterol levels on the development of heart failure will be tested by investigating serum and adipose tissue samples from a pilot cohort of nonherbicide exposed participants: 15 participants that developed heart failure and 15 matched control participants who did not develop heart failure. Investigation will also include the role of serum cholesterol and tissue cholesterol levels on the development of heart failure by testing the total cholesterol content in whole serum and in a subset of whole adipose tissue samples. Correlation analysis between caveolin protein expressions, cholesterol levels, and the incidence of heart failure will be performed.
Principal Investigator: David Seldin, Amyloid Treatment & Research Program, Boston University School of Medicine and Boston Medical Center
Title: Incidence of Abnormal Free Light Chains and Other Markers of Light Chain Amyloidosis in Veterans Exposed to Agent Orange: A Pilot Study
Assets Used: Electronic data and biospecimens; pilot funding awarded
Abstract: The researchers propose detection of preclinical markers of amyloidosis in the serum of veterans exposed to Agent Orange can be used to facilitate early diagnosis and treatment. Participants that have both serum samples and adipose tissue specimens from the cycle 5 exam will be used to analyze immunoglobulin free light chains and other serum biomarkers of clonal plasma cell disease in participants with elevated free light chains. These tests will be used to attempt to determine if these participants have developed clinical features of amyloid disease by measuring known biomarkers of amyloid cardiomyopathy in the serum confirmed with adipose tissue as well as linking to mortality cause information suggests they may have died due to amyloidosis.
Principal Investigator: Youn Shim, Centers for Disease Control and Prevention
Title: Monoclonal Gammopathy of Undetermined Significance and MicroRNAs in Ranch Hand Veterans
Assets Used: Electronic data and biospecimens; pilot funding awarded
Abstract: Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant disorder resulting from unregulated clonal expansion of antibody-secreting B-cells. Individuals with MGUS can be identified by detecting monoclonal immunoglobulins (M-protein) and unbalanced free light chains in serum. The prevalence of MGUS increases with age and is about 4.8% in men aged 50 years and older. MGUS confers significantly increased risk for multiple myeloma, progressing at a rate of 1% per year. Multiple myeloma is considered to have suggestive evidence of an association with exposure to herbicides, including Agent Orange and its contaminant TCDD, among Vietnam veterans. TCDD is known to affect microRNA levels by binding of the miR-191 promoter to the AhR transcription factor. TCDD may alter microRNA levels that are involved in regulating apoptosis and other critical processes of cell proliferation. Malignant cells have been found to selectively package their microRNAs into exosomes for secretion, and altered levels of certain microRNAs are reported in multiple myeloma as well as in MGUS.
MicroRNAs, free light chains, and M-protein have long-term stability in serum stored under a wide range of conditions. Our study will be the first to investigate MGUS and microRNAs using sensitive state-of-the-art laboratory methods to analyze stored serum specimens of Air Force Health Study (AFHS) participants. The purpose of this study is to determine whether exposure to Agent
Orange and TCDD increased the risk for MGUS in Ranch Hand veterans, by using serum samples stored by the AFHS during the 2002 investigation. We will also explore whether TCDD influences the progression of MGUS by testing for three novel biomarkers: M-protein concentration, free light chain ratio, and a microRNA panel. For this purpose, we will use serum specimens stored at three time points (1982, 1992, and 2002).
