Pilot: Center for Appalachian Research in Environmental Sciences: Adverse Reproductive and Metabolic Effects of Co-Exposure to Heat, Humidity and di(2-ethylhexyl) Phthalate

Grants and Contracts Details


Swanson, Hannon & Rashid Abstract Human exposures to high temperatures and high humidity (HTH) are predicted to dramatically increase as a consequence of climate change. While increases in mortality and decreases in birth rates are well documented adverse effects, the impact of HTH on underlying physiological/endocrine processes that contribute to poor health outcomes is not clear. Uncertainties also exist regarding the impact of co-exposure to HTH and ubiquitous environmental contaminants, like endocrine disruptors. The plasticizer di(2-ethylhexyl) phthalate (DEHP), is amongst the best characterized endocrine disruptor and is present in a plethora of consumer goods which contributes to its daily human exposure. Chronic exposures to DEHP adversely affect multiple tissues, including reproductive (i.e., testes and ovaries) and metabolic (i.e., liver) organs as well as incurring developmental effects in the offspring of exposed mothers. Studies on the effects of high heat indicate that DEHP-altered events, such as those involved in lipid, glucose and steroid metabolism as well as DEHP clearance (i.e. drug metabolizing enzymes) are impaired. Thus, we propose that exposures to HTH will contribute to endocrine dysfunction, thereby enhancing the adverse effects of endocrine disruptors and exacerbate their impact on reproductive and metabolic outcomes. Towards this end, we will utilize the well-studied DEHP to determine the impact of co-exposure to DEHP and HTH on reproductive outcomes (Aim #1), stress and endocrine homeostasis (Aim #2) and metabolism and clearance of DEHP (Aim #3). This highly collaborative project will leverage the three investigator’s expertise in environmental health, maternal health/reproduction, developmental programming, drug metabolism and diabetes/obesity. Successful completion of the proposal will serve as a foundation for assessing the human risk associated with co-exposures to endocrine disruptors and HTH and provide strong, preliminary data for future NIH applications. Swanson, Hannon & Rashid Research Description Significance. As climate change progresses, the global incidences of high temperature and humidity (HTH, typically assessed as heat indices) will dramatically increase.1; 2 By mid-century, global population exposure to heat indices in excess of 37.8°C (100 °F) will likely increase by 4-20 fold.2 The acute adverse health outcomes include increased mortality, heat stroke, heat exhaustion and exacerbation of chronic disease states such as cardiovascular and respiratory diseases and diabetes.3 Additional affected outcomes include impaired reproductive health which contributes to decreases in overall birth rates and increased risks of still births.4-6 Unfortunately, the most vulnerable populations (i.e., elderly, homeless, pregnant women, outdoor laborers) are again the most susceptible to the negative consequences associated with HTH, signifying the potential exacerbation of current health disparities. However, the extent to which HTH exposures accelerate the development and progression of chronic disease states and exacerbate the impact of environmental contaminants is as yet undetermined. Thus, the long-term goal of this proposal is to address this gap in knowledge by testing the hypothesis that exposures to HTH will contribute to endocrine dysfunction, thereby enhancing the adverse effects of endocrine disruptors and exacerbate their impact on reproductive health and metabolic diseases. How does exposure to endocrine disruptors affect female reproductive health? Di (2-ethylhexyl) phthalate (DEHP) is a plasticizer incorporated into a myriad of common consumer goods leading to daily human exposure.7; 8 DEHP, a prototypical endocrine-disrupting chemical (EDC) with antiandrogenic and antiestrogenic properties, is linked to reproductive and metabolic dysfunction.9 Many studies in humans show a positive association with DEHP exposure and impairments in glucose homeostasis. While DEHP exposure in males is linked to testicular dysfunction, much less is known about its impact on female reproductive health. In animal studies, DEHP exposure disrupts ovarian function by depleting the ovarian follicular reserve, altering estrous cyclicity, inhibiting follicle growth, increasing follicle death, decreasing the levels of sex steroid hormones, and inhibiting ovulation.10-13 In women, increased levels of DEHP metabolites are associated with increased risk of being infertile, decreased follicle counts, altered steroid hormone levels, and poor in vitro fertilization outcomes (decreased oocytes, embryonic quality, pregnancies, and live births).14-18 How does exposure to HTH alter development metabolic syndrome? The rise in global temperatures are thought to contribute to an increase in diabetes and glucose intolerance19 due in part from reduced brown fat activity.20 Epidemiological studies link higher ambient temperatures to increased HbA1c levels.21 Long term exposure to high relative humidity environments (>80%) are associated with an increased risk of metabolic syndrome but reduced HDL cholesterol levels in women.22 In male mice, chronic exposures to high ambient temperatures increase serum levels of TC, TG, LDL-C and alter expression of genes regulating thermogenesis and fatty acid and drug metabolism in multiple tissues, particularly in brown adipose tissue.23 How does exposure to HTH impact body burdens of EDCs like DEHP? The available literature indicates that HTH exposures likely increase body burdens of EDCs and/or enhance formation of their toxic chemical entities. Studies performed using a variety of xenobiotics have consistently shown that in heat exposed rats and mice, less compound is required to achieve a toxic effect.24 Metabolism and clearance of EDCs typically involve phase I (i.e., cytochrome P450) and phase II (e.g., glucuronosyltransferases) drug metabolism as well as enzymes involved in antioxidant activity.25-27 In rats28; 29 and mice23 exposed to high temperature environments, the hepatic expression levels of drug metabolizing genes decrease while those involved in apoptosis and oxidative stress are enhanced. It is thus likely that HTH exposure will compromise liver function and drug metabolism thereby enhancing the adverse effects of EDCs. In this proposal, we will use DEHP as our model EDC. Oral exposure to DEHP leads to high accumulation of its metabolites in the liver.7; 8 Here, DEHP is rapidly converted by hydrolysis to its monoester and putative toxic form, MEHP, which is further metabolized by CYP4A (and other cytochrome P450s) and alcohol/aldehyde dehydrogenases. These secondary oxidation products are then glucuronidated and excreted primarily in the urine and typically serve as measures of human exposures.30 Innovation. HTH will continue to increase in prevalence and severity as the climate crisis worsens. The proposed studies are forward-looking as they establish a preclinical model of HTH for elucidating the underlying mechanisms by which HTH mediate and/or exacerbate adverse phenotypes. Moreover, the studies address an emergent knowledge gap related to how the physiological effects of EDCs are affected by climate change- Swanson, Hannon & Rashid induced HTH. In addition to this conceptual innovation, our experimental approach is innovative in several ways. 1) Innovative technology: Use of rodent incubators (Powersscientific) allow for precise control of temperature (up to 50°C) and humidity. Controlling both temperature and humidity requires specialized equipment and has limited study of HTH’s effects. 2) Innovative Approach: Many rodent studies of HT use temperatures that are within the thermoneutral temperature of mice, which can be as high as 32°C. However, by using temperatures above the upper critical temperature, we will reproduce physiological responses to heat stress observed in humans, thereby enhancing the relevance of our model to human HTH exposures. In addition, HTH co-exposure with an EDC is innovative in that we will be among the first investigators to explore physiologically, the intersectionality of global warming and ubiquitous chemical exposure. Using this newly developed model, we will identify the extent to which HTH may alter reproduction, metabolism, and disposition of EDCs using the well-studied DEHP as a prototype. Thus, this work will serve as preliminary data and a foundation for future NIH applications designed to assess the human risk associated with co-exposures to EDCs and HTH. Approach Overall Strategy. The goal of the current study is to establish an in vivo model at UK to be used for elucidating the adverse impact of HTH on human health; in particular, that pertaining to endocrine function. Towards this end, we aim to 1) develop a murine model of maternal exposure that mirrors human exposures to high heat indices and align with our ongoing maternal exposure paradigms and previously reported laboratory- based outcomes; 2) expand on previous studies focused on the adverse effects of elevated ambient temperatures by incorporating exposure to DEHP, a prototypical EDC, and by also integrating elevated humidity in this model and 3) determine the extent to which co-exposures to HTH and DEHP may alter reproductive health in females (Aim 1), metabolic homeostasis (Aim 2), and pharmacokinetics (Aim 3). Experimental Design. Ten week old C57Bl mice (80 males and 120 females) will be acclimated for 7 days prior to HTH and DEHP exposure. Mice will then be exposed to: 1) room temperature (RT), 2) high temperature alone (HT), 3) HT+DEHP, 4) HTH alone (HTH), or 5) HTH+DEHP. HT and HTH mice will be placed in climate chambers at 34°C/30-70% humidity and 34°C/85-90% humidity, respectively for 12 h per day during the dark cycle when mice are active.31; 32 The 20 mg/kg/day dose of DEHP (150ppm DEHP in diet equates to roughly 20 mg/kg/day based on typical food consumption) was selected because it has been shown to disrupt ovarian follicle development, steroidogenesis, and estrous cyclicity in mice.13; 33 Mice will be placed in the following 3 paradigms with analyses described under each aim: 2-week exposure (n=8/treatment group; 40 total females, 40 total males), 5-week exposure (n=8/group; 40 females, 40 males), female only breeding trial following 2-week exposure (n=8/group, 40 females). Aim #1. Test the hypothesis that exposure to HTH and DEHP impairs reproductive outcomes. Exposure to elevated temperatures and DEHP have separately been shown to disrupt female reproductive health, but the combined exposure of HT and DEHP, as well as the addition of high humidity, is unknown. Assessment of reproductive health. During the 2- and 5-week exposure paradigms, estrous cyclicity will be monitored daily via vaginal PBS smears, and percent of time in each stage and average cycle length will be compared across treatment groups. Following 2- and 5-week exposure, mice will be euthanized in estrus, and serum and reproductive tracts will be collected. Serum will be used to measure E2, P4, T, FSH, and LH levels. One ovary/mouse will be collected for histological evaluation of folliculogenesis, and percent of follicles per stage, total follicle counts, corpora lutea counts, size of follicles per stage, and atretic (dead) follicles will be compared across treatment groups. The other ovary will be used for mRNA (n=4) and protein (n=4) measurements of factors involved in regulating cyclicity (FSHR, LHCGR, E2 receptors, P4 receptors, androgen receptor), steroidogenesis (STAR, CYP11A1, HSD3B1, CYP17A1, HSD17B1, CYP19A1), and folliculogenesis (AMH, PI3K factors, apoptosis factors, and others pending the follicle count outcomes). One uterine horn will be collected for histology (measurements of morphology, gland number and thickness), and the other will be collected for mRNA analysis (estradiol and progesterone receptors). Assessment of reproductive outcomes. In the breeding paradigm, 2-week exposed females will be mated with unexposed, proven male breeders. Copulation will be monitored by the presence of a vaginal plug, and time to pregnancy will be compared across treatment groups. During the course of gestation, maternal weight gain and length of pregnancy will be assessed. Following parturition, total number of pups, male:female pup ratio, number of dead pups, and average weight of live pups will be compared across treatment groups. Swanson, Hannon & Rashid Additional metrics of fertility to be compared across groups include: fertility index (number of pregnant females/number of females with vaginal plug), gestational index (number of females giving birth/number of pregnant females), and birth index (number of females giving birth/number of total females). Expected Results, Potential Pitfalls and Alternative Approaches. This aim will provide, to our knowledge, the first evidence linking combined exposure to HT, HTH, and a prototypical EDC to impaired female reproductive outcomes. We expect that combined exposure to HT+DEHP and HTH+DEHP will lead to worse reproductive outcomes than HT and HTH alone. We also expect that the addition of high humidity will lead to greater defects in reproductive function than HT alone. More specifically, we expect that HT+DEHP and HTH+DEHP (versus HT and HTH alone) and HTH (versus HT alone) will have more disruptions in estrous cyclicity, and alterations in steroid and gonadotropin hormones levels, in follicle/corpora lutea/atretic counts, and in the uterine and ovarian mRNA and protein levels of factors that mediate cyclicity, steroidogenesis, and folliculogenesis. Thus, we also expect that females in the breeding trial will have increased time to pregnancy and decreased pup numbers and other fertility indexes. While not all of these altered outcomes may be observed, any changes in these measurements could negatively impair female reproductive health and fertility, and will be the focus of future proposals. In total, these experiments are straightforward and have been extensively utilized by Dr. Hannon.13; 34; 35 Aim #2. Test the hypothesis that exposure to HTH, alone or in combination with DEHP, increases adiposity and disrupts glucose homeostasis. The effect of HTH on obesity will be determined by measuring body weight on a weekly basis. After 4 weeks, each of the 5 treatment groups will undergo body composition analysis via EchMRI. For evaluation of glucose homeostasis, we will perform glucose tolerance tests (GTT) and insulin tolerance tests (ITT) at 2 weeks and 5 weeks after exposure. We will quantify fasting glucose, insulin and triglyceride levels at 4 weeks of age. After 5 weeks, mice will be euthanized and plasma and tissues harvested. From terminal plasma, we will measure adiponectin (positively associated with insulin sensitivity36), alanine transaminase (ALT: associated with liver damage37 and insulin resistance38). From the liver, we will measure gene expression related to glucose and lipid regulation (GCK1, PCK1, G6PC, GLUT2, ACC1, FASN, and PPARa). Expected Results, Potential Pitfalls and Alternative Approaches. Consistent with observations in humans, we anticipate that HT will impair glucose homeostasis.39 Elevated humidity diminishes evaporative water loss causing core body temperature to increase, which will further impair glucose homeostasis. Because DEHP decreases insulin sensitivity and increased ambient temperature generally augments acute toxicity of xenotiotics40, we anticipate that co-exposure to DEHP will further exacerbate the impaired glucose homeostasis. Compared to RT controls, we also anticipate that all treatments will cause adiposity due to decreased metabolic rate. Adipose tissue will be collected for future analyses. One potential pitfall is that although DEHP contributes to obesity,41 it also causes hypothermia42 which may mitigate the metabolic disruption caused by HTH. Aim #3. Test the hypothesis that HTH impairs metabolism and clearance of DEHP. The liver, a primary DEHP target organ, rapidly forms MEHP and other metabolic products thereby allowing subsequent systemic exposure to reproductive tissues.43 Activation of hepatic nuclear receptors, such as the PPARs, impact a plethora of metabolic processes. In these experiments, the livers, pancreas, and small intestines will be collected from mice subjected to the 2- and 5-week exposures (Aim #1). DEHP Clearance. Urine will be collected for analyses of DEHP metabolites 24 hr prior to sacrifice as previously described.44 Body burdens of DEHP and MEHP in collected livers will also be quantitated.45; 46 HPLC analyses of urine and liver samples will be performed by UK-CARES Analytical Core. DEHP Metabolic Capacity. Cytosolic and microsomal fractions of livers, pancreas and small intestines will be prepared and the activity of DEHP metabolizing enzymes (ADH, ALDH, Lipase, CYP4A and UGT) will be analyzed in vitro.44; 47 Assessment of Relevant Drug Metabolism Regulatory Pathways. We will also assess the mRNA levels of phthalate-metabolizing genes (Lpl, Aldh1a1, Adh1, Ugt1a6a and Cyp1B1)46 and PPAR dependent (Cyp4a14, Acat2) and PPAR independent (CAR-Cyp2c29, Cyp3a11; PXR-Cyp3A11; Nrf2-Cyp8b1, Gstm4) gene pathways.48; 49 Expected Results, Potential Pitfalls and Alternative Approaches. This aim will provide, to our knowledge, the first evidence linking combined exposure to HT, HTH, and a prototypical EDC to impaired xenobiotic metabolism/clearance. While exposure to heat is linked to impaired clearance of several drugs,24 its impact (alone and combined with high humidity) may be drug/xenobiotic dependent. We expect that HTH will decrease DEHP clearance, increase hepatic levels of MEHP, and impair the upregulation and activities of the involved drug metabolizing enzymes but may enhance oxidative stress/ Nrf2 pathways. Use of [14C]DEHP to assess DEHP clearance and DEHP/MEHP in the in vitro analyses may be required to improve accuracy. As time and resources allow, we will perform transcriptomic (RNA-seq) and metabolomics analyses. OMB No. 0925-0001 and 0925-0002 (Rev. 10/2021 Approved Through 09/30/2024) BIOGRAPHICAL SKETCH Provide the following information for the Senior/key personnel and other significant contributors. Follow this format for each person. DO NOT EXCEED FIVE PAGES. NAME: Swanson, Hollie Isabel eRA COMMONS USER NAME (credential, e.g., agency login): hswanson POSITION TITLE: Professor of Pharmacology and Nutritional Sciences EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable. Add/delete rows as necessary.) INSTITUTION AND LOCATION DEGREE Completion FIELD OF STUDY (if Date applicable) MM/YYYY South Dakota State University, Brookings, SD B.S. 1985 Chemistry Oregon State University, Corvallis, OR M.S. 1988 Food Science/ Toxicology Purdue University, West Lafayette, IN Ph.D. 1991 Food Science/ 1991-1992 Toxicology Michigan State University Postdoc Northwestern University Postdoc Biochemistry 1992-1995 Pharmacology A. Personal Statement The overall goal of this proposal is to determine the impact of co-exposure to high heat, humidity and the endocrine disruptor, di(2-ethylhexyl) phthalate. This project will be accomplished as a collaborative, multiple principal investigator team and I will serve as the contact PI. I have substantial expertise and leadership to successfully serve as in this role as given my ongoing contributions to the UK Superfund and UK-CARES centers. Within UK-CARES, I currently serve as the Integrated Health Sciences Facility Core (IHSFC) translational research hub in Molecular Toxicology. My training encompasses the fields of Biochemistry, Cancer Research, Molecular Biology, Pharmacology and Toxicology and my expertise has focused on nuclear receptors and drug metabolism. My research efforts during the past 24 years have focused on various aspects of the AHR (aryl hydrocarbon receptor) and other nuclear receptors (e.g., estrogen receptor (ER), Nrf2, NFκB) and their roles in regulation of drug metabolizing enzymes, mediators of inflammation and cell fate decisions. My work has involved a variety of cell types including those of the liver, lung, colon, breast, skin and oral mucosa. I have been particularly interested in how nuclear receptors are activated by a variety of ligands and how they engage in crosstalk. For example, the AHR and ER pathways exhibit considerable crosstalk and share many common ligands. In collaboration with Dr. Carolyn Klinge, we discovered that the AHR participates in protein-protein interactions with ER-containing complexes that impact the expression of ER-regulated genes. In collaboration with Dr. Kyung-Bo Kim, a synthetic chemist, we developed novel ligands for both the AHR and ER which targeted either the AHR or ER and initiated their proteolytic degradation. The proposed work of UK-CARES and the IHSFC is highly complementary to these published studies as it will employ similar approaches and methodologies. With respect to cell fate decisions, my published work has
Effective start/end date11/22/223/31/23


  • National Institute of Environmental Health Sciences


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