Pilot Grant Awardee: Hart, J
Project Title: Exploratory Analyses of Oxidative Stress Responses to Vehicle Exhaust in a Population of Trucking Industry Workers
Award Amount: $ 11,267
Description: Specific Aim 1: Explore the distribution of oxidative stress (as measured using a fluorescent oxidation products assay) by job title and by area exposure measurements of elemental carbon (EC) and PM2.5 in the trucking industry to determine if oxidative stress varies with exposure.
Specific Aim 2: Explore the correlation of oxidative stress with levels of systemic inflammation (CRP and IL-6) and a marker of endothelial activation (sICAM-1).
Progress Report: Specific Aim 1: Ambient PM2.5 exposures on the day of blood draw and the proceeding two days were determined based on US EPA monitors in the county of the work locations, and job title was used as a proxy for occupational vehicle exhaust exposures. Generalized linear models were used to determine associations between FLOP levels and the ambient and occupational exposures.
Levels of FLOP were positively associated with average PM2.5 levels on the day of blood draw, increasing 0.32 Fl/ml (95% confidence interval (CI):0.03,0.60) for each 1mg/m3 increase in PM2.5, after adjustment for age ,use of aspirin or cholesterol drugs in the last 24 hours, and consumption of alcohol in the last 24 hours. No associations were observed between FLOP and occupational exposures to vehicle exhaust based on job title.
Our results suggest a positive association of recent ambient exposure to PM2.5 with this novel marker of systemic oxidative stress. However, we did not observe the same relationship with job titles related to vehicle exhaust exposures.
Specific Aim 2: In preliminary Spearman correlation analyses, levels of FLOP were not correlated with CRP, but were modestly correlated with both IL-6 (Spearman correlation=0.20, p-value=0.002) and sICAM-1 (spearman correlation=-0.15, p-value=0.02).
Description: This pilot project sought to establish the feasibility of genome-wide epigenetic analysis of DNA methylation marks in human fetal airway samples. The central hypothesis to be tested is that maternal environmental stressors (e.g. maternal asthma or smoking) during pregnancy cause specific and functionally important epigenetic changes in fetal airway cells developing in utero. The project began use of an extraordinary resource: a collection of normal fetal lung samples from mothers who are normal (controls), smokers, or asthmatic. The samples were collected as part of routine pathologic evaluation of tissue from elective abortion procedures, and have been assembled along with detailed maternal data as part of ongoing studies by Dr. Haley. We used laser-capture microdissection (LCM) to isolate airway epithelium from 6 paraffin embedded normal fetal lung tissue samples in offspring of normal, smoking and asthmatic mothers, respectively.
The pilot project developed methodologies and protocols needed to support future genome-wide comparison of the DNA methylation of airway cells in offspring of the three groups. First, we optimized yield and quality of the DNA harvested after LCM to establish the number of LCM airway extractions needed. Second, we validated the feasibility of DNA methylation analysis in these samples by bisulfite-conversion PCR-based sequencing of CpG sites in a panel of 5 genes linked to airway disease. The successful completion of this pilot study provided critical feasibility data to support a succcessful NIEHS ViCTER grant supplement to an existing R01. The ViCTER project will build on this pilot data to conduct a more extensive analysis of the larger number of fetal lung samples available, and to conduct genome wide methylation analysis to identify airway epigenetic targets of prenatal programming. The project also engaged two promising scientists who were not previously involved in NIEHS Center research (Drs. Sholl and Haley), and served the goal of expanding interest in environmental research to new investigators.
Resulting Grants: NIEHS ViCTER supplement to existing R01 was funded and began fall 2010 (250K per yr for two years).
Description: There is a considerable amount of concern regarding the adverse health effects resulting from chronic exposure to organophosphorus (OPs) pesticides, particularly in infants and children whom are vulnerable to the neuro-developmental toxicity of OPs. Although OPs as a group has been used for more than four decades, it is the acute toxicity in the form of inhibiting cholinesterase (ChE), an enzyme critical to the normal function of the nerve system, that is known. The chronic toxicity in humans from repetitive exposure to OPs either from occupational activities or dietary intake has never been characterized, and therefore the impact to the burden of neurodegenerative disorders in human population is extremely difficulty to quantify. In addition, several recent toxicological studies have demonstrated non-cholinergic toxicity associated with OP pesticides. Unfortunately, biomarkers tied to chronic health effects of OP pesticide exposures have not yet been identified or characterized.
The most commonly used biomarker for OP exposure and health effect is to measure acetylcholinesterase (AChE) activity in red blood cells (RBC) (or butylcholinesterase, BuChE, activity in plasma) using a colorimetric method. While the degree of AChE inhibition has been generally accepted for routine monitoring of acute pesticide poisoning and for occupational health screening mandated by two state regulatory agencies (CA and WA), it suffers several significant drawbacks and limitations for establishing the link between chronic health effects to OP exposures. First of all, the post-OP exposure measurements of AChE activity only represent the enzyme inhibition at the time of venous blood collection. Considering the transit nature of the phosphorylated AChE, the enzyme activity could be either restored by spontaneous reactivation (dephosphorylation) or lost permanently due to dealkylation prior to de novo protein synthesis. Since it is likely that both spontaneous reactivation and dealkylation could be taking place simultaneously in situ, the spot measurement of AChE activity would only provide qualitative information of the magnitude of OP exposure in the form of AChE activity inhibition. Secondly, the colorimetric-based assay that is commonly used for AChE measurements poses low sensitivity and specificity, can be interfered by various unrelated chemicals in the blood, and is very sensitive to different experimental conditions, such as temperature, use of enzyme substrates/inhibitors, etc. Therefore, the state of knowledge of acute OP toxicity is limited to no more than measuring AChE activity depression in the event of elevated OPs exposure.
The fundamental toxicodynamic of OP pesticides at the molecular level of AChE, however, may shade some lights for the new biomarker development linking OP exposure to neurological disorders. It is known that OPs inhibit AChE by nucleophilic attack. The hydroxyl of serine 198 of AChE, functioned as an electronegative group, reacts with the relatively electropositive phosphorus atom of OP, resulting in an OP-AChE complex and the loss of the leaving group on the OP side. The phosphorylated AChE contains adducts that connect OPs to serine-198 in the active center of AChE by a covalent bond, and such bonding could become permanent once the phosphorylated AChE loses another alkyl group. The OP-AChE adduct therefore will remain intact and existed in RBC for as long as 120 day when erythrocytes are degraded by the spleen and in the liver. The adduct arising from the dealkylation, in theory, has a relatively long half-life compared to the depression of AChE activities. In addition, OP-AChE adducts can only form from the toxicologically active form, OP oxons, thus it would provide a surrogate measure of active or biologically-effective OP pesticide dose that is more sensitive and reliable than current direct measures of AChE inhibition. Hence, OP-AChE adduct might well be suitable as a biomarker of OPs exposure and the chronic health effect. Furthermore, the possible conformational change during the dealkylation process of OP-AChE and the nucleophilic nature of the AChE-O- monoalkylphosphonic acid may suggest the interaction of the dealkylation OP-AChE with the surrounding cells or tissues, leading to non-cholinergic, regional effects, or morphogenic activity of AChE that is independent of inhibitory effects on the enzymatic activity of AChE.
General Description: Utilizing the comprehensive Illumina HumanCVD Beadchip 50K SNP array, we aim to identify novel single nucleotide polymorphisms (SNPs) that could target novel genes of importance for predicting bronchial reactivity at the baseline survey (prior to exposure). We also aim to identify novel SNPs that predict total serum IgE at the baseline survey. We developed this aim because total IgE levels are considered a useful endophenotype for studying the genetics of atopic diseases, and there has been minimal investigation of genetic predictors of total IgE in the Chinese population. Finally, we have contributed to worthwhile consortia efforts that maximize the use of data from this pilot study.
Study: 1. A paper (see abstract below) summarizing the analysis of genetic predictors of bronchial reactivity was submitted to Thorax for review.
Rationale: No studies published to date that have examined genetic variants for asthma and associated phenotypes, utilizing high-density single nucleotide polymorphism (SNP) platforms, have been conducted in Chinese populations.
Objective: To identify candidate SNPs that predict bronchial reactivity using a custom high density SNP array in adult Han Chinese females in Shanghai, China.
Methods: Methacholine challenge was performed in 399 Han Chinese healthy non-smoking female subjects (median age 19.1 years). DNA was extracted from whole blood and genotyped using the Illumina HumanCVD Beadchip 50K SNP array; 30,355 SNPs passed quality control and were included for analysis. Bronchial reactivity was characterized as a continuous slope between percentage decrements in forced expiratory volume in 1 second (FEV1) per µmol methacholine on a logarithmic scale. Methacholine slope was modeled as a function of genotype for each SNP on an additive scale in linear regression. For replication, the highest ranked SNPs were extracted from imputed data from a genome-wide association study of healthy Swiss adult females (n=332); linear and logistic regressions were used to model methacholine slope and bronchial hyperresponsiveness respectively.
Results: Four SNPs were identified with uncorrected p-values ≤ 10-5 in the Han Chinese cohort, remaining statistically significant after adjustment for Benjamini-Hochberg False Discovery Rate (q<0.05), and were located in intronic regions of LEPR (rs7555955, β=0.54, p=1.1e-6; rs1046011, β=0.54, p=3.0e-6) and in 3’UTR regions of PON1 (rs845552, β=0.42, p=5.1e-6) and NLRP3 (rs10802501, β=-1.2, p=3.7e-6). Imputed rs10802501 had inverse additive effects on both methacholine slope and BHR in the replication cohort (p<0.10).
Conclusions: SNPs associated with inflammatory, immune, and oxidative stress pathways were identified as predictors of bronchial reactivity in a sample of young adult Han Chinese females. NLRP3 has emerged as a potential candidate gene and will require replication in other cohorts of Han Chinese and European ancestries.
Study 2. An abstract (see below) summarizing a preliminary analysis of genetic predictors of total serum IgE was submitted for presentation at the American Thoracic Society conference to be held in May of 2010.
Objective: To identify candidate single nucleotide polymorphisms (SNPs) as predictors of total serum IgE levels using a custom high density SNP array in predominantly young adult Han Chinese females in Shanghai, China.
Methods: Whole blood was collected from 290 healthy female non-smokers (median age 18.1 years) and total serum IgE concentration was measured using a modified classical radio immunosorbent test in this cross-sectional analysis. DNA was extracted and genotyped using the Illumina HumanCVD Beadchip 50K SNP array; 30,355 SNPs passed quality control and were included for analysis. In linear regression, total serum IgE concentration (log mg/dl) was modeled as a function of each SNP genotype in dominant and additive models and adjusted for age. SNPs with uncorrected p-values < 5e-6 were reported, and adjusted for the Benjamini-Hochberg False Discovery Rate (FDR-BH).
Results: Two SNPs were identified in the dominant model and located in an intronic region of PKCS9 of chromosome 1 (SNP #1: β=-0.83, p=4.5e-7, FDR-BH q=0.01; (SNP #2: β=-0.76, p=3.7e-6, FDR-BH q=0.05). SNP #1 was also identified in the additive model (β: -0.48, p=1.8e-6, FDR-BH q=0.08). PKCS9 codes for a serine protease whose putative function is regulation of LDL receptor levels, and is possibly involved in inflammation and stress response.
Conclusions: Two SNPs associated with hypercholesterolemia, and possibly inflammation, were identified as predictors of total serum IgE levels in this sample of predominantly young healthy adult Chinese females. SNPs in PCSK9 should be investigated in other Chinese study populations before validation in other ethnicities.
Resulting Publications: 1. Amar J Mehta, Xihong Lin, Feng Chen, Ellen A Eisen, Hongxi Zhang, He-lian Dai, Li Su, Medea Imboden, Ivan Curjuric, Thierry Rochat, Nino Künzli, Nicole Probst-Hensch, Hakon Hakonarson, David C Christiani. Genetic Predictors of Bronchial Reactivity in Adult Chinese Females. Submitted to Thorax for review
2. Matthew B Lanktree, Yiran Guo, Muhammad Murtaza, et al. Meta-analysis of gene-centric association studies identifies new genes for adult height. Formally accepted for publication by American Journal of Human Genetics
Description: Traffic related air pollution, especially urban outdoor pollution, is a global public health problem. While the cardiorespiratory effects of air pollution are well described, relatively little is known regarding its neurotoxicity. Given the prevalence of such exposure, even subtle effects may have large public health implications. To our knowledge, there are only 2 population based epidemiologic studies of air pollution and cognition in children, one found associations between prenatal exposure to polycylic aromatic hydrocarbons in mothers and cognition at age 3, the other found an association between childhood exposure and cognition at age 9 years.1,2 Among the critical public health questions still to be addressed are 1) the role of exposure timing (i.e. at which developmental window are children most susceptible to exposure, 2) are there effects on behavior as well as cognition, 3) what are the genetic and epigenetic susceptibility factors, and 4) does social context modify air pollution toxicity.
This proposal represents an exciting opportunity to address these issues and a create new direction for a well-established birth cohort in Boston (“Project ACCESS” – Asthma Coalition on Community, Environment and Social Stressors). As such, it fulfills the primary goals of the NIEHS Center Pilot Program by establishing new collaborations (U of Adelaide) and new lines of research for Center investigators. The primary aims of the parent study are to address the effects of psychosocial stress as a predictor of asthma incidence during early childhood. As air pollution is a potential confounding factor for asthma, extensive population based efforts have been made to measure this factor longitudinally. We thus have already characterized measures of air pollution in prenatal and early childhood developmental windows in ACCESS. We can therefore address the role of exposure timing in determining the dose response slopes in relation to neurodevelopmental outcomes as well.. We are uniquely positioned to address this important research question prospectively at a relatively modest cost. To conduct this work, we will need to phenotype the ACCESS cohort, a process which will be expensive and will require several years of work. We propose to lay the ground work for the RO1 by phenotyping 80 children in this pilot proposal, which will serve as the preliminary data for the larger study involving the entire cohort (n=814 active participants). The advantage of extending this work in ACCESS is that 1) it is on-going, 2) will allow for measures of behavior as well as intelligence, 3) we have archived DNA for both genetic and epigenetic analyses, 4) extensive measures of the social environment (a significant confounder/modifier of neurodevelopmental outcomes) and 5) because our team has considerable expertise in both air pollution epidemiology (RJ Wright and J. Schwartz), stress research (RJ Wright) as well as neurodevelopment (RO Wright, N. Burns and D. Bellinger).
Description: Environmental exposure to air pollution during prenatal and early postnatal development has been linked to a growing number of childhood diseases, including neurological effects (Perera et al 2006; Franco Suglia et al in press). Biologic markers are useful as quantitative dosimeters of dose and effect associated with fetal/early childhood exposures, which can facilitate evaluation of interindividual variability in response and susceptibilities (Whyatt and Perera 1995). Reactivity measures of the autonomic nervous system (ANS), an individual’s physiologic response to a discrete environmental stimulus, have received increasing attention in environmental epidemiology given links to health outcomes specifically related to air pollution exposure (Park et al 2005). However, the work to date has focused primarily on adults. Recently evidence of the prenatal period and early childhood as critical stages of development has emerged. This has led to efforts to develop methodology to assess individual stability and developmental changes in autonomic resting and reactivity measures related to environmental exposures in these periods. Such work may provide new insight into pathways to later physical and mental health problems. Specifically, if fetal origins of disease are related to exposure to ambient pollutants, studies will need to address how fetal exposure to air pollution may influence later life infant autonomic reactivity, whether such effects are independent of postnatal exposures, and how these pathways may, in turn, influence subsequent physical and neurodevelopmental health. Incorporating measures of ANS into developmental epidemiological studies thus has great potential (Alkon et al 2006).
The goal of this pilot study is to further develop new technology for assessing infant autonomic reactivity that can be incorporated into ongoing and future developmental environmental epidemiological studies related to air pollution and neurodevelopment. Data derived from the pilot studies using this new technology will be invaluable in seeking funding from the National Institutes of Health to examine the effects of air pollution in the perinatal/early childhood periods on neurodevelopment and begin to explore underlying mechanisms in an ongoing Boston pregnancy cohort, Project ACCESS (RJ Wright, PI) (Wright et al 2008). We are uniquely positioned to make significant advances in this regard with minimal additional funding. This study will allow continued development of methodology using the Vivometric’s LifeShirt® System as well as newly developed software to facilitate data reduction and analysis while accounting for unique challenges in infant data. The LifeShirt® System is a unique, advanced, non-invasive ambulatory monitoring system that collects and stores continuous measures of multiple physiological parameters, with a particular focus on detailed analysis of cardiac and respiratory functioning. Because the system is ambulatory, it has significant potential for use both in the laboratory and in the field.
Aim 1: Examine associations among pre- and postnatal exposure to ambient air pollution (long- term average black carbon particles and other pollutants at home address derived by spatial modeling) and autonomic function in 6-month old infants assessed using the infant LifeShirt® system in a controlled laboratory setting as indexed by:
1a. Log-transformed frequency domains of heart rate variability (HRV) including low frequency power (LF), high frequency power (HF) and the ratio of LF/HF power.
1b. Respiratory sinus arrhythmia (RSA)
Aim 2: Assess the feasibility of performing ambulatory monitoring in preschool-aged children (ages 4-5 years) using the Pediatric LifeShirt® system and a personal monitor for real-time black carbon exposure assessment in this urban sample.
2a. Conduct preliminary analysis of the relationship between real-time black carbon exposure and sympathovagal indices in pre-school aged children compared to long-term annual average exposure derived from spatiotemporal land-use regression modeling and autonomic function.
Pilot Grant Awardee: Banks, M
Project Title: Contaminant Exposure Assessment of a Cement Factory Based Community in Ravena, New York
Amount of Support: $ 17,500
Description: Little is known about the effects of cement factory pollution, emissions, and kiln dust on contaminant exposure in human populations that live in close proximity to these point-sources. In Ravena, NY, and vicinity, environmental pollution from a local cement plant (Lafarge Building Materials Inc.) is considered significant. Approximately 2,300 children attend school each day within a 4-mile radius of the cement factory. Moreover, of the 2,300 children, 1,250 attend educational facilities across the street from the cement plant at the local middle and high schools. Community Advocates for Safe Emissions (CASE) came together in response to widespread reports of children in the Ravena, NY community, and vicinity, who were suffering from the effects of mercury and lead poisoning, and who were battling rare childhood cancers. Here we propose to measure a variety of hazardous substances in both environmental and human sample matrices and use multiple biomarkers, including heavy metals, PAH’s, and dioxin levels, to indicate exposures. Because of the direct nature, and large number of children and adults being exposed to numerous hazardous substances, this meritorious investigation offers several scientific and public health action opportunities that will likely have wide applicability for other environmental and community health studies confronting similar pollution issues.
- Changes to US-EPA Policy on Cement Factory Regulations for Mercury, Nitrogen and Sulfur pollution (January 2010, and August 2010).
- HSPH Scientific Public Presentation in Ravena, NY (January, 2011)
Description: Preterm birth is a major public health problem that disproportionately affects poor and minority populations. Arsenic and lead have been shown to be risk factors for preterm birth. Environmental metal exposures tend to correlate with socioeconomic gradients and it is unknown how much of the social disparities are attributable to such exposures. Lead and arsenic have also been shown to alter epigenetic markers such as DNA methylation. The target tissue of interest in this proposed study is the cervix. It is unknown whether lead or arsenic-induced epigenetic changes in the cervix could predict a woman’s susceptibility to delivering preterm.. Early cervical foreshortening places women at risk of delivering preterm and preterm labor is characterized by premature cervical changes. We propose to enroll 100 participants from Dr. Wright’s larger, “Metal Mixtures and Neurodevelopment” birth cohort in Mexico City into a nested prospective cohort study. We plan to collect their cervical DNA during routine prenatal Pap Smears and will explore the association between global DNA methylation patterns as well as the gene-specific DNA methylation of the Prostaglandin E Receptor 2 gene, as predictors of gestational and preterm birth.
Description: How do we balance the potential hazards from new and inadequately characterized engineered nanomaterials with the potential and power of nanotechnology? It has become apparent, that potential environmental health effects are critical factors in the likely success or failure of new nanotechnologies. Furthermore, it is becoming apparent that the current nanotoxicology approach of evaluating the toxic properties of the large universe of existing and emerging ENMs at one material at the time, will place a significant financial burden to an emerging industry and may slow down significantly the development of the nanotechnology industry as a whole. Alternatively, a promising approach will be to examine families of engineered and rigorously characterized particles, and to study the role of such factors as particle size, composition, shape, charge and surface chemistry and their link to specific biological outcomes.
As part of this pilot project, a novel method was developed which is suitable for ENM in-vivo inhalation and in-vitro toxicological characterization studies. ENMs are produced continuously in the gas phase using industry relevant flame spray pyrolysis reactors, allowing their continuous transfer to inhalation chambers, without altering their state of agglomeration. Important properties of the generated aerosols (i.e. particle size, concentration, shape, state of agglomeration, surface chemistry) can be modified allowing for both in-vitro and in-vivo investigations of toxicity. The ability of the developed technique to generate a variety of industry relevant, property controlled exposure atmospheres for inhalation studies was systematically investigated in our recently established Particle Technology Laboratory. The suitability of the technique to characterize the toxicity of ENMs in both in-vitro cellular and intact animal models was also shown in pilot studies. We demonstrated both pulmonary and systemic toxicity using a variety of toxicological assays under acute exposure conditions. The future use of this novel method will improve our understanding in terms of the health and safety of ENMs and help us to investigate further our central hypothesis that physical and chemical characteristics of ENMs determine their bioavailability, redistribution, and toxicity in the lungs.
Resulting Publications: Demokritou, P. , Buchel R., Molina, R., DeLoid, G., Brain, J., and Pratsinis S. Development and characterization of a Versatile Engineered Nanomaterial Generation System (VENGES) suitable for toxicological studies. Inhalation Toxicology, 1-10, 2010
Georgios A. Sotiriou, Edgar Diaz, John Godleski, Joseph Brain, Sotiris. E. Pratsinis and Philip Demokritou. A novel technique for in-vivo toxicological characterization of engineered nanomaterials. Manuscript submitted to Nature Nanotechnology, 2010
Other Outcomes: Our aforementioned developed methods and systems as well as the other infrastructure of our recently established Particle Technology Laboratory (PTL Lab) have been made available to our HSPH graduate level students and faculty interested in studying the fate and transport of engineered nanomaterials in the environment and their biointeractions. The use of these systems along with the development of new in vitro screening methodologies and in vivo inhalation studies will help to determine which set of physico-chemical properties correlate best with bio-interactions.
A number of research collaborations were established both at University level but also with other research groups at leading academic institutions such as the Harvard Center for Nanoscale Systems at SEAS, ETH Zurich and Nanotechnology Center at University of Massachusetts, Lowell.
Description: There are very limited data on exposure to bisphenol A (BPA) among infants in the neonatal intensive care unit (NICU) and even less on the identification of specific sources of BPA exposure. Our objective is to determine if nutritional intake or medical devices are a source of BPA exposure among infants in the NICU. One nutrition sample and two urine samples were