YEAR OF FUNDING: 2022
Investigator: Georg T. Wondrak
Assigned to: Adaptive Response to Environmental Stress (RFG3)
Aim 1: Molecular characterization of the gastrointestinal epithelial response to chlorination stress using (i) oral gingival, (ii) oral mucosal, and (iii) colorectal organotypic human tissue reconstructs.
Aim 2: Impact of chlorinated drinking water administration on AOM/DSS-induced murine colorectal
Significance: The disinfection of drinking water by hypochlorous acid (HOCl)-dependent
chlorination may well be regarded as the most important public health milestone in human history 1,2. Among the sustainable development goals adopted by members of the United Nations in 2015 is goal 6, which aims to provide all people with equal access to safe and affordable drinking water, sanitation and hygiene as consistent with the 2010 proclamation of the general assembly that such encompasses a human right. Despite substantial progress, it is currently estimated that more than two billion people lack access to safely managed drinking water and basic hygiene, while nearly half of the human population lacks functional sanitation. Indeed, according to global population projections and climate change models, supply problems and water-related health disparities surrounding safe water will be of utmost importance for this century 1,2. Environmental exposure-relevant chlorination agents include (i) HOCl (and its corresponding anion) as well as (ii) diverse organic chloramines [trichloroisocyanuric acid (TCIC) as a predominant example], all of which are EPA-approved under FIFRA (Federal Insecticide, Fungicide, and Rodenticide Act) regulations used globally for drinking water and freshwater disinfection causing substantial exposure in human populations as reviewed by us recently. Chlorination stress-related pathophysiological outcomes are attributable to the molecular consequences of genotoxic, proteotoxic, inflammatory, and redox stress responses with modulation of crucial transcription factor systems including p53, Keap1/NRF2, HSF1, IKK/NFkB, and AP-1 as substantiated recently by us and others.
Relationship of the project to the SWEHSC theme and NIEHS: The data obtained from this pilot
grant will be used to finalize a competitive NIEHS-RO1 proposal for submission in spring/summer 2023 (or, preferably earlier, based on NIH-ACTS study section membership and continuous submission option). Our studies are relevant to the mission of SWEHSC, as they will comprehensively explore the impact of
environmental chlorination stress on human epithelial health, a topic largely understudied in striking contrast to the global use of chlorination agents for freshwater and drinking water disinfection. Importantly, all necessary data on cutaneous consequences of chlorination stress have already been generated and published by us, providing a strong premise/support for the anticipated R01-application that will benefit from additional GI-directed prototype data relevant to drinking water chlorination as generated in this SWEHSC pilot grant.
Investigator: Zelieann R. Craig
Assigned to: Adaptive Response to Environmental Stress (RFG3)
Aim 1: Establish the impact of phthalates on the neural control of ovulation in rodents.
Aim 2. Establish the impact of phthalates on ovarian ovulatory response capacity in rodents.
Significance: Exposure to endocrine-disrupting chemicals has been recognized
by the World Health Organization as a threat to female fertility. Phthalate exposure in women has been confirmed by detection of phthalate metabolites in urine and ovarian follicular fluid, and has been associated with early menopause, and decreased oocyte yield, clinical pregnancies and live births. Classical animal studies demonstrated that DEHP (di-2-ethylhexyl phthalate), its metabolite MEHP (mono-2-ethyhexyl phthalate), and DBP target the ovary and disrupt ovarian function. Phthalates, as in women, distribute to and are metabolized in the mouse ovary and disrupt folliculogenesis. In the proposed studies, we have chosen to focus on a mixture of three phthalates commonly detected in human samples (DEHP, DBP, and benzyl butyl phthalate, BBP). The exposure levels selected for these studies are based on human exposure estimate data for general population (DBP: 7-10μg/kg/day, DEHP: 0.7-18.9μg/kg/day19,23-24, and BBP: 2μg/kg/day) and occupationally-exposed individuals (DBP: 0.1-76μg/kg/day, DEHP: 0.5-170μg/kg/day, BBP: 286μg/kg/day) Our selected in vitro exposure levels are based on concentrations of phthalate metabolites found in human follicular fluid4-8 (1.48-2.63ng/mL for DBP and BBP metabolites, 5.96-14.1ng/mL for DEHP).
Relationship of the project to the SWEHSC theme and NIEHS: The burden of phthalates may be disproportionate in regions with a significant Mexican American population which are near the Mexico-US border. This work fits with growing RFG3 research on phthalates effects on female fertility and ovarian function, placental physiology, and developmental programming of metabolic and cardiovascular cell function.
YEAR OF FUNDING: 2021
Investigator: Francesca Polverino
Aim 1: Phenotypical characterization of the T cell immune microenvironment
in the emphysematous lung.
Aim 2: Transcriptomic characterization of the T cell immune compartment in
the emphysematous lung. Hypothesis:
Significance: Our proposal is novel and ground-breaking under several different aspects, both methodological and conceptual. In particular, we will: a) use a novel Chromium single cell Gene Expression & Immune Repertoire Profiling kit 24,25 (scRNAseq) to identify rare or unique T cell populations that would otherwise go undetected in analyses of pooled cells, define antigen specificities, determine the oligoclonality of TCRs and self-reactivity of T cells belonging to distinct transcriptional clusters 24,25; and b) perform novel in situ single-cell phenotyping using DSP to characterize
for the first time the complete lung T cell microenvironment in the emphysematous lung. Importantly, we will compare the nature of the T cell responses in different lung lobes with various degrees of emphysema within the same subjects and in different subjects. The pilot study proposed in this application will form the basis for a competitive NIEHS RO1 application. Successful completion of our T cell studies will pave the way to novel immunomodulatory and anti-inflammatory therapies which will target specific T lineage cells (monoclonal antibodies) and/or reset the threshold for T cell activation in a selected subset of subjects (likely emphysema) with off-targeted T cell responses.
Relationship of the project to the SWEHSC theme and NIEHS: The data obtained from this pilot grant will be used to develop a competitive RO1 proposal for submission to
NIEHS extramural funding within 6 months from the end of the SWEHSC pilot funding. Our studies are very relevant to the mission of SWEHSC, as they will define the nature of the adaptive immune responses in a clinical phenotype (likely emphysema) mainly caused by exposure to noxious particles and gases. Our ground-breaking data will pave the way to therapies targeting T cells or T cell products, leading to earlier and more personalized therapeutic intervention which could greatly alleviate the burden of first and second-hand smoke-induced emphysema.
Investigator: Lynn B. Gerald
Aim 1: Expand our relationships with the Diné community to include experts in and advocates for reducing household air pollution.
Aim 2: Locally develop and build the BFF air cleaner.
Aim 3: Evaluate the feasibility of the BFF air cleaners and use of PurpleAir sensors for air quality measurement to improve asthma in 10 Diné homes of children with asthma.
Significance: Better understanding and management of household wood stove smoke-induced asthma in Diné children can achieve better respiratory outcomes including reduced asthma prevalence, improved lifelong lung function, and reduction in pulmonary disability and disability-adjusted life years. These gains may be applicable in Native Americans and the three billion people globally where home smoke exposure is a health concern, including recent concerns that household air pollution may increase the risk of developing or dying from COVID-19 (17). Household smoke exposure reduction is an achievable objective that could have significant impact. This pilot study will establish the approach to effectively reduce household air pollution and improve outcomes for Navajo Nation children. If this intervention effectively reduces household air pollution, and is safe and acceptable for Diné families, then a clear next step would be a study to determine if this intervention improves respiratory outcomes for Diné children.
Relationship of the project to the SWEHSC theme and NIEHS:
The proposed pilot project will provide our team with the necessary collaborations and preliminary data to
support our planned NIEHS R01 household intervention clinical trial for Diné children with asthma. Our research fits well within two themes of the SWEHSC including environmental exposures in underserved southwest populations and environmental lung diseases. We also plan to work with four of the SWEHSC cores. The preliminary work proposed in this pilot project will substantiate a competitive NIEHS R01 clinical trial application for the RFA on Interventions to Improve Native American Health (https://grants.nih.gov/grants/guide/pa-files/PAR-20-238.html).
Investigator: Frank von Hippel
Specific Aims: This pilot project has two specific aims directed at assessing exposures to pesticides (Aim 1)and toxic metal(loid)s (Aim 2) via breastmilk in neonates in the Lake Atitlán watershed in Guatemala. The fetus and neonate are the most vulnerable to contaminant exposure, but to maximize the opportunity to collect pilot data, this proposal focuses on the neonate and exposure through breastmilk. Information on specific pesticides (including many banned in the US) and metal(loid)s elevated in breastmilk is necessary to demonstrate exposure risk and to refine research aims for an RO1 application. An important research goal is to test for an association between contaminant exposure and stunting.
Significance: Lake Atitlán is the deepest and one of the most important lakes of Central
America; it is also home to a diverse group of Mayan cultures.1-4 Approximately 260,000 people live within the watershed.5 The majority of the population is Indigenous, relies upon subsistence agriculture within the basin, and uses the lake for drinking water, transportation, and as a protein source through fisheries.1,4,6,7 Guatemala has the highest prevalence of childhood stunting in the Americas and the sixth highest prevalence in the world.8 Indigenous, rural and poor populations such as those in the Atitlán watershed are disproportionately affected.9 In our study region, 66% of children <5 years old are stunted.9 In order to assess potential contamination of Lake Atitlán with agrochemicals, and resultant health effects, PI von Hippel began a pilot project in 2013 that focused on contamination of water and subsistence foods. Annual sampling trips were funded by the NGO Amigos del Lago de Atitlán, including the use of their field station.
Relationship of the project to the SWEHSC theme and NIEHS: The team will use the IHSFC as a
resource for human exposures and emerging contaminants. The Core will help to establish a statistical
methodology and database, and ensure that protocols are culturally sensitive and meet local needs. The team will also use the CEC to develop a culturally appropriate approach to report results back to the communities and to develop future intervention strategies. We anticipate that the RO1 will focus on the interaction between contaminant toxic effects and health outcomes in children. Depending upon the findings of this pilot study, these health outcomes may relate to endocrine disruption, nutrition, stunting, immune function, or cognitive development. A study of this nature would not be possible in the U.S. given access to food resources and lack of stunting, but a study of the interaction between contaminant burden and stunting in Guatemala could have broad implications in the developing world. Many agrochemicals in heavy use in Guatemala are banned in the U.S., but still commonly employed in low- and middle-income countries. Therefore, depending upon the prevalent chemicals in breastmilk, our aims may address chemical exposures that are especially relevant to developing countries. The Atitlán population is primarily Indigenous and poor, and hence our aims will relate to health disparities associated with some of the most vulnerable communities in the Western Hemisphere. The goals of the RO1 will be to characterize exposures and health effects in order to develop interventions to reduce exposures and improve health outcomes for children in the watershed. Many of our goals are priorities of the NIEHS strategic plan26, such as “Evidence-based prevention and intervention”, “Environmental health disparities and environmental justice”, and “Training and capacity building in global health.”
Investigator: Bernard Futscher
Assigned to: Environmental Lung Disease (RFG2)
Aim 1: Determine if the epigenetic clock is accelerated in an established murine model of
environmental toxicant-induced lung pathology.
Aim 2: Discover new DNA methylation biomarkers of accelerated/decelerated aging.
Significance: Significant deviations from the normal healthy ticking of the epigenetic clock have been detected in murine models and human populations of disease, suggesting a potential utility for such clocks across multiple basic and translational research disciplines [1-4]. A variety of age-related pathologies display strong associations between disease state and an accelerated epigenetic clock, including cancer, the impaired immunocompetence often seen in older individuals, frailty and menopause, and neurodegenerative disorders. The concept that epigenetic rejuvenation can help reverse the aging process is suggested by the observation that during the process of induced pluripotency, differentiated cells reverse their DNA methylation state from a differentiated to a stem cell state. Taken together, the observed DNA methylation changes may reflect conserved processes of mammalian aging and within them their history of environmental exposures.
Relationship of the project to the SWEHSC theme and NIEHS: This research project proposes innovative collaborative research to develop and use DNA methylation biomarkers to quantitate the process of mammalian aging and the role environmental exposures can have on this process. If our overarching hypothesis is supported by the experimental results obtained in this pilot project, then a variety of paths forward are seen that integrate into SWEHSC themes and objectives. With experimental success, this project would initiate a potential productive collaboration between the Futscher & Ding labs that can model environmental exposures, assess their impact on epigenetic aging, and develop projects that interrogate potential mechanisms responsible for accelerated aging. Multiple NIH grant proposals
(R21 or R01) are expected to result from this innovative collaborative effort. The ultimate goal of this research is to translate these fundamental research findings into practical application. With the development of a validated human epigenetic clock, multiple SWEHSC investigators with interest in human cohorts from arid environments would be sought as collaborators on sun exposure (Dr. Wondrak), first responders (Dr. Burgess), and environmental arsenic exposure (Superfund).
Investigator: Daniela C Zarnescu
Aim 1: Evaluate the effects of chronic arsenic exposure on the function of developing and aging neuronal circuits involved in locomotor control, sleep, and memory in Drosophila.
Aim 2: Identify arsenic induced alterations in glycolytic output in neurons, in vivo.
Aim 3: Identify arsenic induced changes in the neuronal translatome using ribosomal tagging in vivo.
ALS/FTD is a progressive neurodegenerative spectrum disorder that affects
motor neurons (MNs) in ALS and the frontal cortex in FTD. Existing therapies are palliative at best. Although both ALS and FTD have a well defined genetic component, >70% of ALS and a large proportion of FTD cases have unknown etiology 11. A significant, yet poorly understood contributing factor to ALS/FTD is the environment. Here we will address this important gap of knowledge by studying the effect of low level, chronic arsenite exposure to neuronal homeostasis in Drosophila models of ALS/FTD based on TDP-43, an RNA binding protein found in cytoplasmic inclusions in 97% of ALS and 45% of FTD cases. Drosophila is a powerful genetic system that has been successfully used to model neurodegeneration 12-15. Our laboratory developed fly models of ALS/FTD whereby expression of hTDP-43 in fly neurons recapitulates ALS phenotypes including locomotor dysfunction and reduced survival 8,9. Using these fly models we identified abnormalities in the expression of Futsch/MAP1B, a microtubule stabilizing protein, the Hsc70-4/HSPA8 chaperone and the glypican Dlp/GPC6, that we subsequently confirmed in ALS spinal cords and iPSC-MNs.
Relationship of the project to the SWEHSC theme and NIEHS: Our proposal is related to the RFG3
Adaptive Response to Environmental Stress theme. Neuronal degeneration is intimately linked to cellular
stress including the formation of SGs, reduced protein synthesis, metabolic alterations and free radicals that we are already studying in the lab. Expanding our research focus to environmental stressors such as arsenite is a natural progression of our work and is of utmost importance in the field. For example, 90% of ALS cases are sporadic and although a genetic component has been identified, the vast majority have unknown etiology. Although it is known that ALS and FTD have an environmental component, this aspect of disease mechanism remains poorly understood. Our studies are timely and poised to make a significant impact on understanding the role of environmental factors in neurodegeneration. We look forward to interact with, and receive guidance from SWEHSC experts on this project to ultimately generate results to support an RO1 application to NIEHS. Dr. Scott Boitano has kindly agreed to advise us as we enter this new area of research.
Investigator: Chi Zhou
Assigned to: Adaptive Response to Environmental Stress (RFG3)
Aim 1: Determine the effect of DBP exposure on endothelial cell function in HUVECs from lean (LN) and obese (OB) pregnancies. We will also determine the endothelial function markers, lipid disposition, and DBP metabolites in human fetoplacental tissues from LN and OB pregnancies.
Aim 2: Determine the effect of DBP exposure on high-fat high sugar diet (western diet)-induced maternal obesity and fetal vascular/endothelial dysfunction as well as placenta transcriptomic profiles using a mouse model.
Significance: Results from this proposed research will provide important information on how
phthalates exposure affects placental function as well as the fetal-sex specific programming of fetal vascular/endothelial development and function in lean and obese pregnancies, which will lay the ground for future studies on phthalates exposure related endothelial/vascular dysfunction in the offspring.
Relationship of the project to the SWEHSC theme and NIEHS: Women at reproductive age have a significant burden of DBP exposure. Consumer products and medications are a major source of DBP exposure in humans, but manufacturers are not required to disclose it as an individual fragrance ingredient. This proposed research will provide important information on how phthalates exposure affects pregnancy outcomes and fetal vascular/endothelial development and function. Data generated from this study will provide valuable information for the regulation of phthalates usage. This is especially relevant to the local population in the State of Arizona as Hispanic and non-Hispanic black populations have disproportionally higher phthalates exposure and obesity occurrence.
YEAR OF FUNDING: 2020
Our overall hypothesis is co-exposure differentially alters the hypothalamic gene network in male and female rats, which determines the severity of its metabolic consequences.
Specific aims: Aim 1a will identify the common hypothalamic gene network that mediates co-exposure induced weight gain in male and female rats. After co-exposure, we will conduct RNA-seq in the hypothalamus from males and females and validate DEGs with Nanostring. Aim 1b will identify the hypothalamic gene network conferring higher susceptibility to co-exposure induced weight gain in female rats using weighted gene co-expression network analysis (WGCNA) to determine the gene networks, modules (clusters of genes), sub-networks, and key genes within sub-networks (hub genes) associated with weight gain and sex.
Significance: The incidence of noise pollution, insufficient sleep, and obesity highlights the severity of co-exposure. Despite sex differences in obesity susceptibility and SD, mechanisms explaining these differences remain unknown. Identifying cellular mechanisms is key to providing a personalized approach to disease prevention, management, and improving the limited success of current therapies.
Relationship of the project to the SWEHSC theme and NIEHS: This project addresses the ‘adaptive response to environmental stress’ SWEHSC theme, would add an animal model of co-exposure to the SWEHSC portfolio and seeks to establish co-exposure to noise pollution and Western diet as an “obesogenmimic” that elicits negative health effects due to stress on hypothalamic cellular metabolism. This project addresses 1) the first theme (Advancing Environmental Health Sciences) in the NIEHS 2018-2023 strategic plan, which aims to explain how our interactions with the built environment and factors including diet affect biological systems and health with metabolic disorders stated as a major research areas of interest and 2) 3 goals within the first theme (#2 Individual Susceptibility, #5 Co-Exposure and #7 Data Science and Big Data). Last, I identified 4 program directors in NIEHS who funded basic science research on links between noise, circadian rhythms, and health, which shows interest by NIEHS in noise pollution research.
Ergothioneine (ET), a thiourea derivative of histidine, is a rare and naturally occurring amino acid. ET, which is only produced by certain plants and vegetables, is a widely used nutritional supplement. ET is a stable antioxidant that detoxifies free radicals and oxidants, increases intracellular thiol levels, controls nuclear factor-κB activation, and inhibits inflammatory gene expression. Moreover, ET treatment was able to reduce the acute lung injury and inflammation in cytokine insufflated rats.
ET has several potentially beneficial roles in vivo, including protecting against UV and γ-radiation, ameliorating inflammation, and preventing neurodegenerative conditions and cardiovascular diseases. Moreover, ET has great potential as cancer chemopreventive agent through scavenging free radicals, chelation of divalent metallic cations, activation of glutathione peroxidase (Se-GPx) and Mn superoxide dismutase (SOD) and inhibition of superoxide-generating enzymes such as NADPH-cytochrome c reductase, and affecting the oxidation of various hemoproteins such as hemoglobin and myoglobin8. Given that, ET has been the subject of a number of clinical trials as a chemoprotective agent against various diseases. However, ET is a polar and hydrophilic compound, which makes it very difficult to diffuse across the cell membrane. It has to be transported by the organic cation/carnitine transporter 1 (ETT or OCTN1) for intracellular delivery. However, the cellular uptake of ET is saturable due to the saturation of the ETT and efflux, and is Na+ and pH-dependent; additionally, ET absorption is very poor following administration, resulting in limited therapeutic effects in vitro and in vivo.
Nanomedicine has been emerging as a powerful paradigm for disease therapy and toxicity reduction because of its ability to enhance intracellular delivery, improve pharmacokinetics and bioavailability, as well as to preferentially target drugs to diseased tissues (e.g. lung injury) while decreasing nonspecific systemic toxicities. Among the existing nanocarriers for drug delivery, liposome has been the most successful, due to its biocompatible and biodegradable lipid compositions, and in vivo stability. A number of liposomal nanoformulations have been approved by FDA (Doxil®, Myocet®, Daunoxome®, Depocyt®, Mepact®, Marqibo® and OnivydeTM).
Liposome is composed of a phospholipid bilayer; water-soluble drugs can be incorporated to its spacious hydrophilic interior core, whereas hydrophobic drugs are anchored in the lipophilic bilayer. To date, there has been no liposomal ET reported. This proposal will develop the first ever nano-ET delivery system to address its poor cellular uptake and inadequate in vivo bioavailability, with a near-term goal of testing the nano-ET delivery system in animal models of environmental diseases, and eventually in chemoprevention trials in people. We hypothesize that liposome-delivered ET can enhance the antioxidant potential of ET through improvements in its delivery efficiency and bioavailability.
Aim 1: To establish an optimal liposomal ET nano-delivery system (Lipo-ET).
Aim 2: To determine the intracellular delivery and antioxidant efficiency of Lipo-ET in vitro.
Aim 3: To investigate the pharmacokinetics (PK) and biodistribution (BD) of Lipo-ET in mice.
Relationship of the project to the theme of the SWEHSC and NIEHS:
This proposal is pertinent to the SWEHSC theme and will establish an innovative nano-ET platform for preventing lung toxicity, particularly in aging populations of the Southwest who are exposed to elevated levels of various toxicants in the agricultural, construction and environmental dust, tobacco smoke, and/or mine tailing pollution, and are predisposed to toxicity due to advanced age and medication-related adverse effects, as well as the Ozone-triggered asthma.
Specific Aims and Hypothesis: Di-ethyl-hexyl-phthalate (DEHP), a phthalate diester, is the most common plasticizer additive in various cosmetics, perfumes, shampoo, polyvinyl chloride (PVC) plastics, and many other everyday products. Recent studies have demonstrated that the metabolites of DEHP are present in significantly higher amounts in women with preterm birth and intrauterine growth restricted fetuses (IUGR). Specifically, the DEHP metabolites MEHP and MECPP were strongly associated with preterm birth and IUGR. Additionally, studies have demonstrated that maternal phthalate exposure can also affect fetal health and lead to adverse health outcomes later in offspring life. The effects of phthalate exposure on preterm birth, IUGR, and health problems in offspring demonstrate that DEHP metabolites not only affect placental function (e.g. steroidogenesis or nutrient transport) but can cross the placenta to affect fetal growth. However, the impact of maternal phthalate metabolites on placental growth and function are not well studied. Moreover, the specifics of maternal phthalate exposure and its transport across the placental barrier is not known. It is also not known if the DEHP metabolites undergo accumulation in an organ-specific manner in the fetus, making it more susceptible to adverse effects. Thus, we hypothesize that maternal DEHP metabolites are readily transferred across the placenta and accumulate in various fetal tissues. Furthermore, the DEHP metabolites promote abnormal gene expression in placental and fetal tissues complicating the pregnancy outcome.
Specific Aim 1: Determine transplacental fluxes of DEHP metabolites under steady-state conditions in vivo in maternal and fetal sheep.
Specific Aim 2: Determine the effect of DEHP metabolites on placental gene expression. Also, we will evaluate the impact of DEHP metabolites on the gene expression in the fetal brain hippocampus and frontal cortex region. Overall, the two specific aims of this pilot project will provide substantial background data that will allow for further investigation on the role of phthalates in placental function and “fetal programming”. These new findings will elucidate molecular pathways and gene networks, which can lead to placental dysfunction and increase the offspring risk for “fetal programming” of adult diseases.
Relationship to SWEHSC and NIEHS: Phthalates have become a major environmental contaminant in the past few decades. Recent studies have demonstrated a widespread presence of phthalates in human blood. However, the effect of phthalates on placental physiology and fetal development is not known. The proposed research aligns with the goal of the SWEHCS and NIEHS to investigate the effects of environmental agents on human health and disease. The data collected from the proposed research will provide important information for pregnant mothers regarding the proportion of phthalates that reach the blood of their unborn baby. In the future, the proposed research may lead to the formation of regulations to limit the use of plasticizers, which can adversely affect the placental function and accumulate in fetal tissues.
Specific Aims and Hypotheses:
We have established toxicology and Alternaria-induced asthma research programs at the University of Arizona that are largely focused on cellular signaling pathways following airway epithelial exposures. We have successfully used the xCELLigence real time cellular analysis (RTCA) impedance-detecting device to monitor physiological changes associated with nanoparticle or arsenic (i.e., toxic environmental exposures) or activation of protease-activated receptor-2 (PAR2), a primary GPCR target for Alternaria in allergic asthma. Despite our highly successful initial screening with RTCA, mechanistic understanding of environmental exposure and/or receptor activation relies on individual cellular signaling pathways, and potential “biased signaling events”. We currently screen these signaling pathways with digital imaging microscopy, traditional Immunoblotting or In-Cell-Westerns of immortalized human bronchial epithelial cells and/or immunoblotting of primary airway cell cultures. An interesting outcome of our Alternaria exposure model is the identification of biased signaling at PAR2 where activation of PAR2-Ca2+ signaling in vivo is associated with beneficial physiology (e.g., bronchorelaxation) and the activation of PAR2-b-arrestin/MAPK signaling is associated with detrimental physiology (e.g., mucus overproduction, inflammation). While successful, our progress in evaluating environmental exposures has been slowed by the limited capacity we have in our in vitro approaches and the lack of use of well-differentiated airway epithelial cells in our high capacity assays. In 2019 we began a successful partnership with the Functional Genomics Core to improve screening methods and capacity. We are now in a position to advance these techniques to develop high capacity screening methods to be used for grants aimed at understanding airway epithelial environmental exposure and/or receptor activation.
To accomplish these goals, we propose the following Specific Aims:
Aim 1) Develop a high capacity, biased cellular signaling assay airway epithelial cells.
Aim 1a) Use immortalized human airway epithelial cells.
Rationale/Approach: In our Alternaria-exposure experiments we have shown that activation of PAR2 in airway epithelium can lead to both barrestin/ MAPK and Ca2+ cellular signaling and asthma indicators with independent outcomes. We have obtained unique probes from Montana Molecular (Bozeman, MT) that have allowed for direct measurement of b-arrestin and Ca2+ signaling pathways that we have adopted for use in an immortalized human airway epithelial cell line (16HBE14o-). Using the 96-well imaging microscope in the Functional Genomics Core (Perkin-Elmer Operetta) and PAR2 ligands developed in our laboratory, we will optimize transfection protocols to best assay full and biased ligand activation of PAR2. We will use the licensed Perkin-Elmer Harmony-Software and open source Cell Profiler software to quantify fluorescence data. We will next modify the protocol for the rapid readout of a multi-plate reader to increase capacity. These experiments will result in the first cell signaling protocol that can be used to evaluate barrestin/ MAPK and Ca2+ signaling simultaneously. Significantly, the probes are not receptor-specific and would thus allow for exposure-induced cellular signaling independent of PAR2 as we have proposed.
Aim 1a) Use well-differentiated primary cultured mouse tracheal airway epithelial cells for high capacity measurements.
Rationale/Approach: Environmental exposures or require high capacity screening that is most easily accomplished using immortalized cell lines. However, immortalized cell lines can lack qualities of cells in vivo that may specifically alter cellular signaling. Primary cultured, well-differentiated mouse tracheal epithelial (MTE) cells grown at air liquid interface on filter supports in our and other laboratories retain many of the characteristics of in vivo airway epithelium and thus, present a better model for ultimate targets for the environmental exposure. Our preliminary data demonstrates the ability to image these cells on filters in a 24-well format using the Operetta. Additionally, we have been able to transfect MTE cells with the b-arrestin and Ca2+ signaling probes described in Aim 1a. We will adapt procedures described in Aim 1a to transfer our protocol to the well-differentiated cells in the current 24-well format using the Operetta. Corning has recently introduced a 96-well format transwell filter plate and the next target will be to move our protocol to a 96-well format. A second improvement to add specificity will be to introduce the newly designed PAR2-tdTomato mouse developed with our collaborators at UT-Dallas.
Combined with a humanized luciferase reporter, we could advance our currently used b-arrestin BRET assay in transfected CHO cells to better represent physiologically intact, well-differentiated airway epithelial cells. While straight-forward in description, implementation of high capacity screening in well-differentiated cells is a large leap in technique that could prove highly valuable tools for evaluation of environmental exposure screening. Anticipated Outcome: The PI (Scott Boitano) and Lead Technician (Joy Prisco) have combined for over 55 years of tissue culture and cellular/molecular biology experience, with 25 years exclusively focused on in vitro screening and thus have the extensive insight needed to develop these high-capacity protocol. Our recent
experience in the Functional Genome Core, strong preliminary data and the availability of unique and specific PAR2 probes made in our laboratory suggest the successful development of a high capacity protocol for simultaneous multiple cellular signaling assays. Notably, the probes we have obtained from Montana Molecular measure signaling are neither cell-type nor PAR2-specific, and thus, these protocols could be quickly adapted for use in other cell types and/or other receptors in environmental exposure/cell signaling research at the SWEHSC or University of Arizona. Successful execution in the 96-well format will fundamentally change our ability to screen environmental exposures or receptor ligands and provide members of the SWEHSC/Functional Genomics Core users with valuable protocols that could be easily adapted. Should we be able to complete our Aim, we would expand to include the use of normal human bronchial epithelial (NHBE) airway cells with our collaborators Drs. Kraft and Ledford (NIH/NHLBI pending award, R41HL152942).
Specific Aim 2) Develop high capacity cell trafficking assays. Rationale/Approach: In environmental exposures or ligand/receptor activation, mechanistic understanding can be gleaned from exposure/ligand/receptor trafficking. Similar to cell screening assays, high capacity measurements are lacking. We have used the Operetta 96-well microscope to initiate high capacity tracking (Fig. 3) and propose to optimize the procedure to better screen exposure tracking. We will use our Alternaria exposure protocol and labeled PAR2 ligands from our laboratory (Dr. Vagner) for optimization of procedures. We currently have Cy5- and phrodo- (pH sensitive dye) labeled compounds. We have optimized ligand activation/trafficking experiments and will focus on developing methods for fluorescence quantification. As above, we will be using Harmony and Cell Tracker software for data extraction and transfer before adapting our protocol for multi-plate
readers. Also as above, we will be able to employ primary cultured PAR2-tdTomato MTE to improve the assay. Notably, we will be able to track the ligand and receptor independently. The understanding of ligand/receptor position during agonism/antagonism may provide important mechanistic understanding to biased signaling (We have in hand antagonists that block both b-arrestin/MAPK and Ca2+ signaling as well as those that block only one of the two major pathways and will be able to directly test this novel hypothesis).
Anticipated Outcome: Similar to above, experience with these techniques and strong preliminary data are supportive of successful completion of the experimental protocols. Also similar to above, these protocols can be adapted to provide high capacity screens for a variety of exposures and tissues.
Relationship of the project to the theme of the SWEHSC and NIEHS: The work described herein is meant to complement our current in vitro physiological RTCA assay with advanced high capacity in vitro protocols to measure subsequent cellular signaling. An immediate benefit to our laboratory is to best position ourselves to extend our currently funded PAR2 drug discovery programs for environmental-exposure asthma (current NIH/NIAID R21AI140257; pending NIH/NHLBI R41HL152942 – priority score 22). We will apply for a full NIH R01 with the environmental asthma award within the year and the techniques described herein will greatly advance our chances of success. NIEHS has shown considerable interest in environmental exposures (e.g.,
Alternaria) and disease (e.g., asthma). While we have been funded by NIAID in the past, we have noted that Alternaria and Alternaria-induced asthma is significantly up-regulated after environmental disasters and should be considered for NIEHS funding. Importantly, successful completion of this grant will fit with the long-standing efforts of NIH/NIEHS to reduce animal experiments by providing novel in vitro approaches to evaluate environmental exposures. Since the protocols can be quickly adapted to several cell types, SWEHSC members will directly benefit from novel means of high capacity cell signaling screening.
Environmental mold (fungal) exposure has long been recognized as a significant risk factor for asthma and asthma exacerbation. According to NIEHS and CDC, the most common indoor molds are: Alternaria (Alt), Aspergillus (Asp), Cladosporium (Cla), Penicillum (Pen) as well as more toxic black mold (Stachybotrys chartarum). Correspondingly, “Severe Asthma with Fungal Sensitization” (SAFS) has been specially coined for severe asthma with the sensitization to one or more of these major molds. Using Alt as a model, we have established the first mouse model of fungal asthma mimicking natural exposure to live spores. We discovered previously unknown immunogenic RNAs (or iRNAs) as a novel fungus associated molecular pattern to activate type I interferon (IFN-I) response. IFN-I receptor (IFNAR) blockade exacerbated asthmatic phenotypes in mice, suggesting that IFN-I pathway was protective against asthma. Indeed, genetic polymorphisms that affect downstream signaling molecules (e.g. IRF1, STAT1) of IFN-I pathway were linked to asthma. Additionally, we and others reported that fungal secretions potently repressed IFN-I signaling, further supporting the protective role of IFN-I in anti-fungal immunity. Most recently, a dsRNA-like molecules were discovered in the house dust mite (another type of asthma-causing exposure), suggesting that RNA sensing might be a significant, but rarely studied, modulator in asthma. Therefore, we hypothesize that innate sensing of fungal iRNA activates protective IFN-I pathway, and the impairment of this protection leads to asthma. We propose to elucidate the protective function of IFN-I in fungal asthma model (Specific Aim 1). We have built a strong collaborative team for this project: Immunology (Chen, Schenten), animal models (Chen, Daines and Ledford), mycology (Pryor), clinical asthma (Daines) and single-cell sequencing (Bhattacharya). These data will support highly competitive R01 grant proposals to NIEHS as well as to other NIH institutes.
Relationship of the project to the theme of the SWEHSC and NIEHS
NIEHS: our proposal about mold (fungal) exposure falls in two themes of NIEHS strategic plan: #1 Advancing Environment Health Sciences, #2 Promoting translation. For theme #1, our study will advance the understanding of mechanistic basis of mold induced airway diseases (Goal #1: basic biology). As SNPs in IFN pathway have been linked to the susceptibility of fungal asthma, this study will also address the Goal #2: Individual Susceptibility. In the planned proposal to NIH, we propose to study co-exposures of all four common fungi found in indoor molds. Thus, this project addresses Goal #5: Co-exposure. For theme #2, we will address Goal #3: Evidence-Based Prevention and Intervention by establishing a foundation for the development of IFN-I based immunotherapy to treat fungal asthma.
SWEHSC: our proposal about mold (fungal) asthma addresses the second major theme of SWEHSC-Environmental Lung Diseases. Mold exposure has been prevalent in Southwestern US. Importantly, Alt, a major mold species, has first been found to cause asthma by researchers in the Arizona Respiratory Center (now Asthma and Airway Disease Research Center), of which PI Chen and Co-investigators-Daines, Ledford are the members.
YEAR OF FUNDING: 2019
Asthma, the most common chronic disease of childhood, imposes a societal burden that is higher than that of tuberculosis and HIV/AIDS combined. Currently, asthma can be treated but not cured. Therefore, strategies to understand the mechanisms promoting onset and therapies to prevent asthma are urgently needed. As a prototypical complex disease, childhood asthma pathogenesis results from gene-environment interactions at sensitive developmental windows. These interactions have made it challenging for researchers to identify single genes with large effects in populations. Our hypothesis is that we can map asthma protection genes by leveraging the asthma-protective effects of Amish farm dust exposure and the power of systems genetics. The aim of this pilot study is to assess whether genetic variation drives differences in asthma-protective responses to Amish farm dust measured as inhibition of allergen-induced lung eosinophilia in mice.
Relevance to SWEHSC Our long-term goal is to leverage the HMDP panel to map the genetic pathways underpinning the striking inhibition of allergen-induced lung eosinophilia induced by inhaled Amish farm dust, an exposure that is associated with essentially universal asthma protection in the relevant human population. Identifying the host’s pathways involved in asthma protection would open the way to targeted, effective asthma-preventive strategies.
SPECIFIC AIMS AND HYPOTHESIS
Obesity rates have risen dramatically over the last four decades, highlighting that this is likely not due to genetics alone, but rather an interaction between genetics and the environment. Along this same time frame, the use of pesticides and herbicides in the United States has increased dramatically, with RoundUp being one of the most widely used herbicides in the agriculture industry since 1980. Originally developed to target the shikimate pathway which is only found in plants (and bacteria), it was assumed that RoundUp and its chemically active component, glyphosate, would have no physiological effect on mammals, however glyphosate is now linked to cancer. Although other herbicides and pesticides are associated with metabolic dysfunctions often observed during obesity, no study has directly assessed whether RoundUp promotes the development of obesity. Despite mammalian cells lacking the shikimate pathway, glyphosate could impact the bacteria that inhabit the gastrointestinal tract, which play a major role in host physiology. Obesity is associated with altered gut microbiota profiles, and early-life gut microbiota developmental issues increase propensity for later life metabolic disease. Several studies have shown that glyphosate potentially alters the gut microbiota profile in rodents, with observed shifts in increased pathogenic and decreased beneficial bacteria. Given these data, we hypothesize that glyphosate alters the gut microbiota leading to the development of metabolic dysfunction. This hypothesis will be tested in the following aims:
Specific Aim 1: Determine if glyphosate promotes metabolic dysfunction in adult mice. Adult mice will be subject to a dose response of glyphosate, the main active ingredient in RoundUp, to determine changes in energy homeostasis and intestinal dysbiosis.
Specific Aim 2: Determine if early-life exposure of glyphosate increases susceptibility to development of obesity. This aim will be identical to Aim 1 except weaned mice will be used to determine if early life exposure to glyphosate is detrimental to adult metabolic phenotype.
Specific Aim 3: Determine if glyphosate alters the gut microbiota to promote metabolic dysfunction. Intestinal microbial contents from groups in Aims 1 and 2 will be analyzed and compared to determine how glyphosate alters the gut microbiota, and germ-free mice will be inoculated with this gut microbiota to determine causality.
RELATIONSHIP TO SWEHSC
Examining the effect of environmental exposure to a common herbicide (glyphosate) on host metabolic phenotype and gut bacteria fits well with RG#3 in SWEHCS. Also, this proposal will benefit from the insight provided by the members, who are experts in examining effects of environmental stressors on genetics, epigenetics, and metabolism. Furthermore, the work generated from this proposal would provide significant data for a competitive funding opportunity from NIEHS, which has a main goal to: “Foster research on environmental triggers of disease”.
Investigator: Jon Chorover
Assigned to: Environmental Exposures in Underserved Southwest Populations (RFG1)
Specific Aim. Develop and validate a novel, predictive toxicological assay that better represents in vivo conditions than current leaching tests by progressively aligning the products of abiotic gastric simulant in vitro bioassays (IVBA) with germ-free and intact microbiome mouse model in vivo ingestion. (The mouse model approach was recommended in a prior Pilot Project review.) Assays will be evaluated for samples of fine particulate matter (PM2.5 and PM10) from mine tailings and specimen natural and synthetic minerals. For both IVBA and mouse-derived particle suspensions, we will analyze arsenic speciation in the fluids (IVBA, cecum, urine) by HPLC-ICP-MS and HPLC-ESI-MS, and the solids by synchrotron X-ray absorption spectroscopy. This will enable us to tune the IVBA to obtain products consistent with in vivo results.
Our overarching hypotheses are that (i) contaminant molecular speciation, host particle crystallinity, and particle size (i.e., specific surface area) control bioaccessibility, and (ii) bio-fluid simulant leaching experiments commonly used to mimic in vivo metabolism do not accurately predict arsenic bioaccessibility because they do not account for changes in arsenic speciation due to in vivo variation in redox status and metabolic activity.
Background. Bioaccessibility is controlled by dissolution reactions that release toxic elements into biofluid.
Metalliferous mine tailings are a significant health risk to proximal populations. In (semi)arid regions, tailings and their associated contaminants are prone to windborne dispersion as fugitive dusts. These problems are extensive and persistent because impacted sites lack normal soil stabilization and plant cover that can mitigate aerosolized PMs. Although metal(loid) speciation is known to control bioavailability and toxicity, little is known about in vivo reaction trajectories and changes in particle-scale metal speciation for inhaled and ingested tailings particles through the GI tract, owing largely to the complexity of monitoring physiologically relevant analogs. Pharmacokinetic interactions of toxic dusts and target organs are modeled via IVBA, where IVBA bioaccessibility is defined as the physiological solubility of toxins available for absorption. As such, IVBA has been proposed by EPA as a low-cost and expeditious toxicological predictive tool to examine bioavailability of metal(loid)s 1. Use of IVBA is most informative when validated with animal models, and IVBA in vivo correlation (IVIVC) studies can evaluate (and validate) biological toxin uptake and metabolic transformation with laboratory release. As requested by reviewers, the IVIVC specifics are more fully described in this proposal revision. Results will lead to predictive risk assessment tools and targeted remediation strategies for stakeholders, many of which are in the SW US.
Significance. Risks to human health of atmospheric PM are becoming increasingly clear. This proposed project builds on our prior work indicating that mine tailings in (semi-)arid climates may pose greater human health risk than in humid environments. The Agency for Toxic Substances and Disease Registry Priority List of Hazardous Substances shows that 4 of the top 10 hazardous substances are metal(loid)s commonly found in tailings, including arsenic (#1); lead (#2); mercury (#3); and cadmium (#7) (e.g. ATSDR, 1997-2017). The primary transport mechanisms of these substances are mediated by wind and water. With the associated likelihood of off-site dust transport, direct inhalation and ingestion of PM can lead to toxic exposures with biological effects that are controlled by the rate of dissolution in vivo.
Evidence is mounting that the microbiome of higher organisms can influence speciation of toxic metal(loid)s. A review of microbial arsenic metabolism in human and rat intestines indicated that reduction and methylation of arsenic is common, as is formation of thiolated arsenic species. Microorganisms in the distal intestine of swine were responsible for increasing release of arsenic from soil particles. Dissimilatory arsenate reductase activity and arsenate-respiring bacteria have been demonstrated in bovine rumen fluid, hamster feces, and the termite hindgut. Conversely, IVBA extraction techniques are commonly carried out under oxic conditions, and in the absence of active microbiota, which inaccurately mimics the bioactive reducing conditions of the distal GI tract. There is thus an urgent need to compare arsenic bioaccessibility in PM under appropriate redox conditions with appropriate comparative mouse models to assess metabolic influences on bioavailability. This pilot project would provide essential preliminary data to support a larger NIEHS proposal to address this need; results would solidly align with the all three theses of the NIEHS 2018-2023 Strategic Plan (e.g. theme 1 goal 6; theme 2 goal 4; theme 3 goal 3).
Relationship to the theme of SWEHSC and NIEHS. The proposed project is aligned with the SWEHSC goal of facilitating and implementing innovative research to understand mechanisms underlying modulation of human disease risk due to environmental exposures in arid environments. Specifically, geodust and airborne particulate matter (especially that deriving from industrial mining sites and containing high concentrations of toxic metals) represents an environmental exposure that is particularly prevalent and widely dispersed in the arid SW US. Whereas the PI (Chorover) and one Co-PI (Root) have primary expertise in environmental biogeochemistry, and records of research pertaining to the geochemical weathering of mine tailings systems, they have a strong interest in redirecting their research team’s focus to environmental health aspects of geochemistry. Co-PI Kiela, a professor of Pediatrics and a specialist in gastroenterology and nutrition, has primary expertise in the use of mouse models to elucidate the physiology of absorption and secretion in the gut, and the impact of the gut microbiome. The current project takes a novel approach to integrating geochemistry with pediatric health by implementing in vitro bioassays and in vivo mouse models to assess bioavailability of arsenic in airborne particulate matter deriving from mine tailings environments. If funded, the results would provide preliminary data needed for a larger proposal to be submitted NIEHS, with specific synergies: 1) animal and cell-based studies to understand how timing of environmental exposures effects health outcomes; 2) addressing the relationship between environmental exposures and biological response; 3) developing approaches to address exposure–pathway–disease relationships; 4) investigating endocrine disruptors and exposure biology (including validation of additional metals like cadmium and mercury).
The overarching goal of this research is to understand how inhaled environmental exposures influence reproductive health. The proposed pilot project will focus on measuring AMH as a proxy measure of ovarian reserve and timing of menopause. Specifically we will measure AMH levels in a highly exposed group of women firefighters and compare them to non-firefighting women. AMH will be collected via dried blood spots (DBS). We hypothesize that firefighters will have lower AMH levels compared to non-firefighting women. Among firefighters, we hypothesize that there will be an inverse dose-response relationship between number of years in the fire service, AMH levels, and rate of decline in AMH. Specifically we will aim to:
Aim 1: Investigate whether AMH levels are lower in female firefighters compared to comparison women, taking into account age and other potential confounding factors.
Aim 2: Among female firefighters, investigate whether there is a dose-dependent relationship between years of firefighting and AMH levels.
Aim 3: Among a smaller cohort of female firefighters with previously collected serum, investigate whether there is a relationship between: a) AMH with previously measured PFAS levels and epigenetic (blood microRNA and DNA methylation) markers, and b) rate of decline in AMH over a two year period with interim measures of exposure to fires.
RELATIONSHIP OF PROJECT TO THE THEME OF SWEHSC AND NIEHS
The proposed project is a time- and cost- effective way to investigate an emerging biomarker among women highly exposed to environmental contaminants. In line with the mission of SWEHSC, this project will utilize the Integrated Health Sciences Facility Core to implement innovative research aimed at understanding the mechanisms underlying human disease risk due to environmental exposures. The proposed aims will display feasibility and generate pilot data for an R01-level grant submission to NIEHS. NIEHS has expressed interest in understanding environmental influences on reproductive health and long-term health (PA-17-091) and currently is funding 12 R01-level grants focused on environmental exposures and women’s reproductive health in line with their 2018 strategic plan. Specifically, this proposal will show feasibility and generate pilot data for collecting AMH from DBS on a large-scale population level, with a comparison group recruited via social media. Additionally, the data generated in Aim 3, utilizing previously collected epigenetic and PFAS data will generate preliminary mechanistic data for future grant submissions. Additionally, we will collect residential information that can be used as preliminary data related to particulate matter and AMH levels. Future environmental health research supported by this pilot could focus on the mechanism of association related to inhaled environmental exposures and could expand to other environmental respiratory exposures such as wildfires and air pollution.
We propose that iAs exposure leads to altered glyoxalase function and increased MGO, promoting diabetes. We will test this hypothesis by utilizing both in vitro and in vivo models for iAs toxicity:
Specific Aim 1: Determine the impact of As on the glyoxalase pathway. The effects of As on the cellular levels of DHAP, MGO, GSH, and LGSH will be evaluated in WT, GLO1-/-, GLO2-/-, and GLO1/2-/- HEK293 cells. As-GSH adducts will be quantified. Activity assays will be performed using recombinant GLO1 and GLO2 to determine the impact of As on enzymatic activity. As-modified GLO1 and GLO2 will be quantified in cells exposed to NaAsO2 using reactivity-based protein profiling with a Cys-reactive probe. Proteomics will be conducted in the University of Arizona Analytical and Biological Mass Spectrometry Facility (MSF) to map As-sensitive sites of modification on GLO1 and GLO2.
Specific Aim 2: Determine the role of MGO in iAs diabetogenicity using a murine model chronically exposed to iAs. WT, Glo1+/-, and Glo1-/- mice will be fed a high-fat high-sucrose diet for a period of 8 weeks in the presence or absence of 250 ppb NaAsO2 in the drinking water. The following parameters will be assayed to evaluate the severity of diabetes: fasting blood glucose, glomerular filtration rate, serum creatinine, Hb1Ac, and serum carbonyl content (e.g. MGO). Standard biochemical parameters (e.g. western blotting, immunohistochemistry) will be performed on liver and kidneys isolated from each treatment group. Glo1 and Glo2 activity will be performed. MGO-derived PTMs will be quantified using a targeted LC-MS/MS assay and immunohistochemistry. Lastly, As-modified proteins will be identified and quantified using in-gel fluorescence and proteomics using a Cys-reactive probe and click chemistry.
Although previous studies have shown that arsenic exposure can modulate SLC transporters (i.e., glucose transporters, excitatory amino acid transporters) in the brain, its effects on Oatp1a4 expression and activity at the BBB is unknown.
Our hypothesis is that arsenic exposure modulates Oatp1a4 expression and activity at the BBB, leading to significant effects on statin drug delivery to the brain in the setting of stroke. Such changes in statin drug delivery will impact the neuroprotective efficacy of these therapeutics. Therefore, we propose two specific aims:
Specific Aim 1: Investigate, in vivo, BBB localization and functional expression of Oatp1a4 in experimental animals exposed to arsenic and subjected to focal cerebral ischemia. Effects of experimental stroke on Oatp1a4 functional expression at the BBB will be studied using the intraluminal suture tMCAO approach. Male CD-1 mice will be exposed to inorganic arsenic via drinking water for 24 weeks. For this pilot study, we will not examine female CD-1 mice due to confounding effects of estrogens on BBB integrity and stroke pathophysiology. We will measure localization and molecular expression of Oatp1a4 using confocal microscopy, quantitative PCR and western blot analysis. Using our in situ brain perfusion methodology, we will investigate Oatp-mediated transport of statins in animals subjected to tMCAO and/or exposed to arsenic.
Specific Aim 2: Investigate, in vivo, effects of arsenic on neuroprotective drug efficacy following focal cerebral ischemia. Arsenic effects on neuroprotective drug efficacy will be studied in our tMCAO model by measuring specific biomarkers associated with apoptosis and oxidative stress as previously described. We will also examine arsenic effects on functional outcomes post-focal cerebral ischemia using behavioral methodologies currently in place in our laboratory (i.e., Morris water maze, Rotorod performance test).
Investigator: Paul Sheppard
Assigned to: Environmental Exposures in Underserved Southwest Populations (RFG1)
Specific Aims And Hypothesis: Aim 1: Assess temporal validity of dendro-metal concentrations by evaluating whether annual concentrations of metals in tree rings predict historic EPA-measured ambient annual metals, over a 25-year time period. Hypothesis: Annual metals concentrations in tree rings will be strongly predictive of EPA-measured annual PM2.5 levels, and will be useful as a historic exposure measure in epidemiology studies.
Approach Aim 1: Several EPA air monitors throughout the US have been in operation for almost 30 years with daily records of airborne metal concentrations. We will collect samples from trees within 30 meters of EPA metals air monitors7 at 4 different EPA sites (10 trees per site), and analyze up to 25 years of metals (annual data) from each tree, including As, Ba, Mn, Cd, Pb, Ni. Site selection will be determined with assistance from Google Earth, to ensure sites have a sufficient number of trees of suitable species (preferentially we will select pines, then maple, cottonwood, and oak, in listed order), within a defined distance to the respective EPA monitor. Trees will be ideally of similar age and circumference. We will monitor site characteristics for each tree, including measuring soil pH, tree circumference, tree wounds, tree species, leaf-drop patterns, cambial age, rainfall/water, climate, humidity, the heartwood-sapwood boundary, characteristics of the leaf, and the metal species. Before going into the field, we will work with the HPER core of SWEHSC to develop sampling SOPs. At the conclusion of this pilot study, we will have measurements for 40 trees (4 sites and 10 trees per site), and each tree will have up to 25 values that correspond to 25 years of metals concentrations (40x25 = 1,000 tree ring-year concentrations). Dr. Sheppard will cross-date tree rings against weather parameters for accuracy. Data Analysis: Dr. Edward Bedrick is an expert in longitudinal mixed models and will oversee model.
The objective of this pilot study is to obtain preliminary data on how As exposure affects the liver acetylome and its regulation in preparation for a new NIEHS grant proposal (R21 or R01, depending on the data obtained) focused on potential epigenetic mechanisms underlying the pathological effects of As on metabolism. Drs. Xinxin Ding and James Galligan will serve as collaborators on this pilot study. The central hypothesis is that As effects on the hepatic expression or activity of KATs and deacetylases drive changes in the acetylation of both histones and non-histone proteins. The objective will be met through two aims using livers from mice exposed to sodium arsenite in drinking water.
Specific Aim 1: Determine whether As exposure changes the expression or activity of KATs and deacetylases. We will assess the mRNA and protein expression of KATs and deacetylases with established in vivo activity and measure their activity through in vitro assays. Metabolites that contribute to regulation of acetylation (e.g. acetyl-CoA, and NAD+) will also be measured. The outcome will guide specific aim design for the planned NIEHS proposal through identification of enzymes impacted by As exposure and the nature of its impact (metabolites, expression, or activity).
Specific Aim 2: Assess the impact of As exposure on the liver acetylome. A targeted approach assaying acetylated candidate proteins (e.g. histones) using standard biochemical techniques will be combined with unbiased identification and quantification of acetylated hepatic proteins using proteomics mass spectrometry in the UA Analytical and Biological Mass Spectrometry Facility. The outcome will provide critical preliminary data that potentially links altered acetylation to As-induced pathologic changes, which will be validated experimentally in the planned NIEHS proposal.
YEAR OF FUNDING: 2018
Investigator: Boris Reiss
Assigned to: Environmental Lung Disease (RFG2)
Specific Aims and Hypothesis
Ambient air pollution and antibiotic resistance are among the world’s most urgent public health problems. Although animal models present emerging evidence that particulate matter concentration – a key component of ambient air pollution – contributes to antibiotic resistance, ecologic studies of this association do not exist. To address this research gap, we will calibrate a new low-cost air monitor (PurpleAir PA-II) to measure particulate matter 2.5 (PM2.5) and deploy monitors in communities where we collect antibiotic resistance data. In Aim 1, we propose calibration of PA-II monitors with federally certified ambient air analyzers in distinctly different air concentrations to accurately measure PM2.5. In Aim 2, we will determine the association between the prevalence of antibiotic resistance in select communities and regional PM2.5 concentrations. We will obtain antibiotic resistance data from microbiology labs and build mixed model regression models to test our central hypothesis that PM2.5 levels drive antibiotic resistance. Execution of these aims will generate pilot data through preliminary studies that will demonstrate feasibility, experience, and collaboration necessary for competitive extramural funding applications.
Investigator: Shane Snyder
Assigned to: Environmental Exposures in Underserved Southwest Populations (RFG1)
Background and Significance: Firefighters and associated personnel at a fire scene are exposed to a variety of contaminants from combustion, of which many have known toxic effects. In fact, numerous studies have concluded a greater cancer incidence and mortality in firefighters compared to the general population. Polycyclic aromatic hydrocarbons (PAHs) and their derivatives are one contaminant class in particular that is formed as a result of incomplete combustion of organic material and this class includes chemicals known to be mutagenic and/or carcinogenic. Researchers have quantified a select set of hydroxylated PAHs as metabolites in the urine of firefighters before and after fires as biomarkers of exposure. In addition to observing increased concentrations of hydroxylated PAHs in post-fire urine samples of firefighters, our research group has demonstrated an increased in vitro bioassay response using the PAH CALUX® bioassay. This bioassay provides insight into the toxicity of a set of compounds that are biologically active through interaction with the aryl hydrocarbon receptor (AhR). An investigation into the potency of 14 different hydroxylated PAHs was conducted by our research group to identify what proportion of the bioactivity was due to the current set of hydroxylated PAHs being routinely monitored. This revealed that less than 1% of the bioassay response in the post-fire urine was due to the hydroxylated PAHs being quantified, which suggests that the majority of the bioassay response from the urine extracts is from unknown AhR agonists. Our research group has also tested extracts of soot collected from the helmets of the firefighters using an Agilent gas chromatograph-quadrupole time of flight instrument (GC-QTOF), which showed numerous PAH derivatives in addition to the parent PAHs.
Within the compounds found on the firefighters’ helmets, one class of specific interest is that of alkylated PAHs, which are a sub class of PAHs that has been shown to be an important group to analyze in order to not underestimate the overall load of PAH contamination in environmental samples. Specifically, methylated PAHs have been shown to have greater potency in terms of the AhR response than parent PAHs and have been shown to be mutagenic and carcinogenic. Alkylated PAHs or their metabolites have not yet been identified in urine of firefighters, but are known to be created from combustion reactions and through bioalkylation substitution of parent PAHs within the body. Being able to identify some of the compounds within the urine extracts that are primarily responsible for the observed bioassay response will provide critical biomarkers of exposure that better correlate to the bioactivity observed.
Specific Aims and Hypothesis: The specific aim of the proposed study is to identify compounds present in the urine of firefighters that are responsible for the majority of the PAH bioassay response observed post-fire.
We propose in this study to identify the most important environmental media associated with urinary arsenic, serum CC16 concentrations, and negative respiratory outcomes in our Yaqui pediatric cohort. This study will allow us to build upon previous work conducted by both U of A and our collaborators in Sonora, Mexico to obtain preliminary data to support larger grant applications to assess arsenic toxicity from early age exposures via multiple environmental media on both sides of the US-Mexico Border. The preliminary data we would will obtain from this project are key to helping us start to determinate the level of combined arsenic exposures in both study sites, and whether is these combined exposures are sufficient to result in adverse health effects on the respiratory system of children, and whether soil and dust exposure contribute as much to arsenic toxicity as drinking water. Data from this study will help us further our innovative hypotheses that challenge the current paradigm of the relative importance of dust and soil exposure pathways on arsenic-related respiratory toxicity and will provide preliminary data for future collaborative applications. The information collected from this current proposal will also be used to plan and evaluate interventions in future studies to improve children’s health and reduce the burden of environmental-related diseases.
Investigator: Marti Lindsey
Assigned to: Community Engagement Core
Specific Aims and Hypothesis
Hypothesis: There is a high incidence of childhood asthma and air pollution in a Native American Community
Measure Asthma prevalence among Native American children ages 4-16, in collaboration with the Health and Environment and Education Departments, by distributing, completing and analyzing the International Study of Asthma and Allergies in Childhood (ISAAC) survey tool to 300 children.
Conduct Air monitoring activities with tribal members and students, high school, community college and university, to collect pilot data for more in-depth studies.
Build Citizen Science capacity among tribal members and students by developing, providing, and evaluating culturally appropriate training methods.