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Description
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
Status | Finished |
---|---|
Effective start/end date | 11/22/22 → 3/31/23 |
Funding
- National Institute of Environmental Health Sciences
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Projects
- 1 Finished
-
Center for Appalachian Research in Environmental Sciences: Administrative Core
Hahn, E. (PI), Haynes, E. (CoI), Pearson, K. (CoI), D'Orazio, J. (Former CoI), Fondufe-Mittendorf, Y. (Former CoI), Fowlkes, J. (Former CoI), Giannone, P. (Former CoI), Morris, P. (Former CoI), Smyth, S. (Former CoI), Stanley, S. (Former CoI) & Swanson, H. (Former CoI)
National Institute of Environmental Health Sciences
5/1/17 → 5/31/23
Project: Research project