THE EFFECTS OF PER- AND POLYFLUOROALKYL SUBSTANCES (PFAS) AND
HIGH FAT DIET ON METABOLISM, REPRODUCTIVE HEALTH &
PROSTATE CANCER PROGRESSION
BY
OZAN BERK IMIR
THESIS
Submitted in partial fulfillment of the requirements
for the degree of Master of Science in Nutritional Sciences
in the Graduate College of the
University of Illinois at Urbana-Champaign, 2021
Urbana, Illinois
Master’s Committee:
Professor Jodi A. Flaws, Chair
Associate Professor Zeynep Madak-Erdogan, Director of Research
Professor Joseph M. K. Irudayaraj
ii
ABSTRACT
Per- and polyfluoroalkyl substances (PFASs) are a group of synthetic chemicals that have
been utilized in various industries and settings. Their resistance to water, oil, grease, and extreme
temperatures enabled utilization of PFAS in adhesives, cleaning-products, paints, coatings,
packaging, building materials, and firefighting foams. Due to highly fluorinated carbon chains
ranging in length, PFAS are resistant to decomposition, and have increased longevity in media,
such as soil, water, dust, and air. Because of increased stability in the environment, many
communities in the US are exposed to high concentration of PFAS. Multiple studies reported
increased PFAS blood concentrations in fluorochemical factory workers, office workers, and
firefighters, all of which are exposed to these materials on a day-to-day basis. Epidemiological
studies linked PFAS exposures to increased risk of various pathological conditions including
non-alcoholic fatty liver disease, infertility, and cancer. Epidemiological studies suggested a link
between increased blood PFAS levels and prostate cancer incidence, but the mechanism of action
for such a phenomenon is unknown. In the present thesis research, we investigated how PFAS
impacts prostate cancer metabolism, progression, and prognosis. We tested the hypothesis that
metabolic alterations from high fat diets combined with PFAS exposures play a significant role
in prostate cancer initiation and tumor progression.
iii
ACKNOWLEDGEMENTS
I would like to offer my sincere appreciation to my mentor Dr. Zeynep Madak-Erdogan
for giving me all the time, effort, and dedication she has put into the culmination of my project
and my education. I am grateful for the guidance that she has given, and the helping hand she has
offered many times when I needed it most. I would also like to thank Dr. Jodi Flaws and Dr.
Joseph Irudayaraj for being in my committee, helping me develop this project and facilitating the
creation of this document.
To all current and former M-Lab members: Eylem Külköylüoglu-Çotul, Qianying Zuo,
Ashlie Santaliz-Casiano, Alicia Arredondo-Eve, Justina Zurauskiene, Ayca Nazli Mogol, Brandi
Smith, Shoham Band, Kinga Wrobel, Yiru Chen Zhao, Karen Chen, and Kadriye Hieronymi,
thank you so much for your support on this project and your friendship. I will dearly miss all the
great conversations and cherished moments that we have had together. It was a pleasure and a
privilege to work with you all. I would also like to thank our undergraduate students Alanna Zoe
Kaminsky, Sruthi Sridhar, Haihley Colette Connors, Matthew Chanho Jin, Nina Litvak, and
Saumya Agrawal. Despite the COVID-19 pandemic, you have exceeded all expectations for
which I am proud to have worked with you, and with the determination you have at hand and the
help of M-Lab, I have no doubt that you will not only achieve but also outperform your goals in
life!
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TABLE OF CONTENTS
CHAPTER 1: INTRODUCTION .................................................................................................1
CHAPTER 2: PERFLUOROALKYL AND POLYFLUOROALKYL SUBSTANCES AND
EFFECTS OF EXPOSURE ON METABOLIC AND REPRODUCTIVE HEALTH ...........5
2.1. LINK BETWEEN PFAS EXPOSURE AND PROSTATE CANCER RISK .........................5
2.2. MOLECULAR MECHANISMS OF METABOLIC DEREGULATION BY PFAS
EXPOSURE ....................................................................................................................................7
CHAPTER 3: PFAS EXPOSURE LEADS TO INCREASED RATE OF TUMOR
PROGRESSION .........................................................................................................................11
3.1. INTRODUCTION .................................................................................................................11
3.2. MATERIAL AND METHODS .............................................................................................12
3.3. RESULTS ..............................................................................................................................16
3.4. DISCUSSION ........................................................................................................................21
CHAPTER 4: CONCLUSIONS AND FUTURE DIRECTIONS ...........................................23
4.1. CONCLUSIONS ....................................................................................................................23
4.2. FUTURE DIRECTIONS .......................................................................................................25
FIGURES AND TABLES ..........................................................................................................26
REFERENCES ............................................................................................................................37
1
CHAPTER 1
INTRODUCTION
Per- and polyfluoroalkyl substances (PFAS) are fluorocarbons with a carbon backbone
flanked with fluorine atoms and capped with a carboxyl group. PFASs adhere to metal, plastic,
or other designated charged surfaces by virtue of their more electronegative carboxylic acid
group and polymerize with other PFAS compounds via the fluorine atoms on their long carbon
chains, forming a stain-repellant surface coat which renders the surface inaccessible (Behr,
Plinsch, Braeuning, & Buhrke, 2020; Domingo & Nadal, 2019; Ojo, Xia, Peng, & Ng, 2021;
Sunderland et al., 2019). PFAS compounds are frequently used in many industries as stain-
repellant coating material on food packaging and grime-free cooking equipment such as Teflon
pans as well as being coated into pipes to make them leak-proof (Sunderland et al., 2019).
Perfluoroalkyl substances, both within their manufacture and in their application onto
coating surfaces, pose a threat against human health. Soil, water, and air contamination; direct
contact to coated surfaces; and the consumption of food that has had contaminant exposure are
some of the ways by which one can get exposed to PFAS compounds (Legoff et al., 2021;
Mahinroosta & Senevirathna, 2020; Temkin, Hocevar, Andrews, Naidenko, & Kamendulis,
2020). Due to their low manufacturing cost and wide range of use-cases, PFASs come in many
forms. Perfluorooctane sulfonate (PFOS), perfluorobutane sulfonic acid (PFBS) and
perfluorooctanoic acid (PFOA) are among the most readily used and the most environmentally
persistent of these pollutants. Due to the health risks involved with the use of PFASs, many
environmental and public health institutions such as the EPA and NIH have issued prompt
advisories following the emergence of the scientific data (Agency, 2021; Sciences, 2021). Many
industries have since been trying to develop safer PFAS alternatives that could replace these
2
flagged chemicals. Hence, other frequently used types of PFAS compounds such as
perfluorononanoic acid (PFNA), perfluorohexane sulfonic acid (PFHxS) and perfluorodecanoic
acid (PFDeA) were developed to serve this purpose, but all of them have also been detected as
persistent environmental contaminants found mostly in drinking water and surface soil
(Mahinroosta & Senevirathna, 2020).
PFAS exposure was found to cause various cellular and systemic metabolic alterations. A
2016 study by researchers from Nanjing University in China revealed that the liver metabolism
of mice that were exposed to PFAS was altered such that the amino acid and TCA cycle
dependent energy metabolism were statistically significantly shifted (Costello & Franklin, 2006;
Yu et al., 2016). In addition, a 2012 study published in Environmental Science & Technology
used a proteomic label quantification technique called iTRAQ to visualize biomarkers of toxicity
when mice were exposed to PFOS, which led to the discovery of both novel toxicity biomarkers
and revealed that PFOS exposure significantly upregulated peroxisomal 𝛽-oxidation controlling
enzymes (F. Tan et al., 2012). With increased dose of PFOS concentrations (5.0 mg/kg BW),
higher peroxisome (23.5-fold, p-val<0.05) endoplasmic reticulum (10.1-fold, p-val<0.05),
mitochondria (3.9-fold, p-val<0.05) and membrane protein (1.6-fold, p-val<0.05) concentrations
were observed (Domazet, Grøntved, Timmermann, Nielsen, & Jensen, 2016). Furthermore,
PFAS exposure was found to dysregulate multiple lipid metabolism pathways (glycosphingolipid
metabolism, fatty acid metabolism, de novo lipogenesis, and linoleic acid metabolism), as well
as amino acid metabolic pathways (aspartate and asparagine, tyrosine, and arginine and proline
metabolism) (Alderete et al., 2019; Qu et al., 2020). PFAS can, therefore, impact lipid
metabolism and alter cellular energetics which may have detrimental health outcomes.
3
In addition to the cellular shifts observed in in vitro and in vivo settings, PFAS was
shown to trigger systemic changes in patient metabolisms as well. GWAS studies in exposed
patients have historically shown a positive correlation between PFAS and metabolites such as
fatty acid and glycerophospholipids (Thomas et al., 2008). Additionally, a Metabolomics journal
publication from 2019 featuring a HILIC-coupled LC/MS metabolomics analysis of 114 children
showed that children with low to moderate PFOA, PFOS, PFNA and PFHxS concentrations in
their serum exhibited altered arginine, proline, aspartate, asparagine, butanoate, glycine, serine,
alanine, and threonine metabolism (Kingsley et al., 2019).
Lifestyle factors and environmental exposures impact prostate cancer risk. Although
chronic exposures to PFAS are associated with elevated the prostate cancer incidence and/or
mortality, the cellular targets and potential mechanisms are unknown. Using a combination of
human prostate and relevant human prostate cancer models, spatial and molecular data, and
sophisticated analytical tools, my thesis provided some key mechanistic insights into prostate
carcinogenesis as a function of PFAS plus high fat diet exposures: metabolic changes in benign
or tumor cells that lead to epigenomic reprogramming and altered signaling, which ultimately
increase tumorigenic risk and tumor aggressiveness.
In my thesis project, the effects of PFAS on prostate cancer proliferation, growth
progression and metabolic activity were explored. To understand how the metabolic landscape is
shifted upon PFAS exposure, a metabolomics approach was employed. An animal study focused
on the growth rate of tumors in animals that were exposed to PFOS, a type of PFAS compound,
and high fat diet that may affect liver health in combination with PFAS. This research will help
us gain preliminary knowledge on the potential dangers of PFAS exposure and enhance our
understanding on how it may impact reproductive cancer characteristics.
4
The overall objective in the present thesis project was to characterize the actions of PFAS
and its interaction with high fat diets that together drive prostate carcinogenesis (Figure 1). As
such, we hypothesized that PFAS exposures together with high fat diets drive metabolic changes
that reprogram prostate cells through epigenetic modifications that, independently or combined,
initiate prostate cancer and promote tumor progression. Our studies unraveled new and clinically
relevant insights regarding novel metabolic and epigenetic states that provide a rational
framework for future development of effective preventative, and therapeutic strategies for PFAS
induced prostate cancers.
5
CHAPTER 2
PERFLUOROALKYL AND POLYFLUOROALKYL SUBSTANCES AND EFFECTS OF
EXPOSURE ON METABOLIC AND REPRODUCTIVE HEALTH
2.1. LINK BETWEEN PFAS EXPOSURE AND PROSTATE CANCER RISK
2.1.1. Per- and polyfluoroalkyl substances:
PFAS are a family of chemicals containing long hydrophobic (8-carbon) chains fully
saturated with fluorine atoms (i.e., perfluoroalkyl chains) and a hydrophilic polar functional
group (Prevedouros, Cousins, Buck, & Korzeniowski, 2006). These chemicals are thermally
stable and lipid- and water-repelling, prompting their extensive use since the 1970s in household
products such as carpets, Gortex products, and non-stick cookware. Such widespread usage has
led to accumulation of these persistent organic pollutants in the environment (Fraser et al., 2013;
Karásková et al., 2016; Oakes et al., 2010; Rappazzo, Coffman, & Hines, 2017). Two PFAS,
perflurooctanoate acid (PFOA) and perfluorooctanesulfonic acid (PFOS), are the most abundant
in the environment and are a major source of exposure to humans through food and drinking
water. Unfortunately, these and newer PFAS substitutes (e.g., GenEx) do not readily degrade and
are commonly referred to as “forever chemicals”. An estimated 99% of Americans are exposed
to and/or have these chemicals in their body (Calafat et al., 2019; Kato, Wong, Jia, Kuklenyik, &
Calafat, 2011). Wastewater treatment plants are a major sink for PFAS by receiving PFAS-
impacted wastewater. Landfill leachate, wastewater effluent, biosolids application, and
groundwater recharge using surface runoff re-introduces PFAS to water sources including
drinking water. Crucially, PFAS exposures exert negative effects on health.
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2.1.2. Prostate cancer and increased risks from environmental factors
Prostate cancer is the most commonly diagnosed noncutaneous cancer in American men
and the second leading cause of cancer-related deaths (Siegel, Miller, & Jemal, 2020), although
its etiology remains elusive. Our collaborators at UIC, previously determined that exposure to
two well-established endocrine disruptors/environmental toxicants, bisphenol A (BPA) and
arsenic, increases prostate cancer risk and identified molecular mechanisms that underpin these
activities, including prostate stem-progenitor cell (SPC) reprogramming through epigenetic
modifications (Ho et al., 2015; Xie et al., 2020). In addition to environmental and occupational
exposures, lifestyle factors including diet and body weight dictate overall increased prostate
cancer risk (Freedland & Aronson, 2004; Narita et al., 2019). Relevant to this thesis,
epidemiology studies show that prostate cancer risk and mortality increase with PFAS exposure
(Barry, Winquist, & Steenland, 2013; Kirsten T. Eriksen et al., 2009; Gilliland & Mandel, 1993)
as well as with obesity (Hardell et al., 2014; Vidal et al., 2020). Despite this evidence,
mechanistic data on the molecular underpinnings of PFAS chemicals in the prostate are
limited/nonexistent. It is well-established that metabolic adaptations in prostate cancer alter the
epigenetic landscape, in part due to changes in substrate availability for epigenetic enzymes
(detailed below). Further, epigenetic marks dictate the activity of PPARα, a critical transcription
factor in PFAS-associated carcinogenesis(Stanifer et al., 2018; Wolf et al., 2008) that is liganded
by both PFAS (Evans, 2020.) and metabolites associated with HFDs (David Patsouris, Reddy,
Muller, & Kersten, 2006).
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2.2 MOLECULAR MECHANISMS OF METABOLIC DEREGULATION BY PFAS
EXPOSURE
PFAS are not thought to be genotoxic (Eriksen et al., 2010; Freire et al., 2008; Lindeman
et al., 2012), suggesting that PFAS elicit effects without causing direct DNA mutations. One
plausible means through which PFAS may exert effects is epigenetic and transcriptomic
alterations. Associations between PFAS exposures and altered methylation, either genome‐wide
or at specific histone loci, are described from other laboratories and our collaborators (Rashid,
Ramakrishnan, Fields, & Irudayaraj, 2020; Tian et al., 2012; Wan et al., 2010; Wen, Mirji, &
Irudayaraj, 2020). PFAS exposure is also associated with lower global DNA methylation in
neonates (Guerrero-Preston et al., 2010; Kobayashi et al., 2017; Watkins et al., 2014).
2.2.1 Link between systemic metabolism and hormone-dependent cancers
The metabolic status of cancer cells determines phenotypic characteristics and drug
responses of hormone-dependent cancers (Cotul et al., 2020; Kulkoyluoglu-Cotul et al., 2019;
Madak-Erdogan et al., 2019; Madak-Erdogan et al., 2013). Of note, a recent study showed that
high fat diets (HFDs) induce changes in one-carbon metabolism, impact histone methylation
marks, and increase prostate cancer progression in an in vivo model of MYC-induced prostate
cancer (Labbé et al., 2019). Previously, using a unique multi-omics approach and serum samples
from obese and non-obese individuals, our laboratory identified obesity-associated factors that
increase breast carcinogenesis (Madak-Erdogan et al., 2019). We showed that free fatty acids
work through PPARα to rewire breast cancer metabolism and affect ERα cistromes that alter
transcriptomes. Structurally, PFAS resemble free fatty acids and bind to the same sites on serum
proteins. Several studies found that PFAS chemicals work through PPARα to impact metabolism
and the immune system (DeWitt et al., 2009; Stanifer et al., 2018; Wolf et al., 2008).
8
Studies have shown that multiple types of perfluoroalkyl substances have activated
variants of peroxisome proliferator-activated receptors. Researchers from the German Federal
Institute for Risk Assessment investigated the impact of various PFASs on the activation of
PPARα through their dependent target genes, as well as its effect on eight other human nuclear
receptors by using luciferase-based reporter gene assays (Kunath et al., 2015). The study
observed activation of PPARα when exposed to PFOA, PFOS, PMOH (“Gen X”), PFHxS,
PFBA, PMPP, and PFHxA, but PFOA and PMOH exhibited the strongest impact of activation.
PPARγ was the only other human nuclear receptor found to be activated by PFASs (PMOH and
PMPP) of those that were tested. This study provides additional research on the impact of PFASs
on PPAR pathways (Behr et al., 2020). Another study was done by researchers from the Chinese
Academy of Science that examined the structural impact of 16 PFAS binding affinity and
activation potency on PPARγ using fluorescence polarization based competitive binding assays
and Hep G2 cells. The experiments showed a high expression and activation of PPARγ
correlated with medium-sized PFC chain lengths (PFOA, PFDA). This study demonstrated
another case of PFC activation of PPARγ (Zhang, Ren, Wan, & Guo, 2014). Researchers from
the University of Minnesota Medical School sought to determine the activation of PPARα in
primary rat and human liver cell cultures by exposing them to 25μM of PFAS for 24 hours. They
found relative activation potencies by many PFASs with the carboxylic acids induced a greater
response compared to sulfonates as follows: perfluorononanoic acid ≥ PFOA > PFDA >
perfluorohexanoic acid > PFBA and perfluorohexane sulfonate > PFOS > PFBS. Activation of
mouse PPARα showed to be statistically greater than the human PPARα (Bjork & Wallace,
2009). In conclusion, these studies contribute evidence towards the influence of PFOS and
PFOA on the activation of PPARα.
9
PFOA and PFOS have been found to activate human and mouse peroxisome proliferator-
activated receptor alpha. A study done by researchers from the United States Environmental
Protection Agency determined the ability of PFOS and PFOA to activate PPARs using transient
transfection cell assays. COS-1 cells that were cultured with mouse or human PPARα, β/δ, and γ
reporter plasmids and exposed to PFOS and PFOA for 24 hours displayed an increase in PFOA
transactivity compared to PFOS in both human and mouse receptors (Takacs & Abbott, 2007). In
addition to these findings, the study reported that PFOA activated both human (p < 0.05; 30 and
40μM) and mouse (p < 0.05; 10, 20, 30, and 40 μM) PPARα in a dose-dependent manner,
whereas PFOS activation of PPARα only occurred in the mouse construct (p < 0.01; 120μM).
PPAR β/δ was activated by PFOA and PFOS in the mouse construct only (p < 0.05; p < 0.05) for
both PFASs. PFOA and PFOS did not activate the mouse or human PPARγ (Takacs & Abbott,
2007). A study by researchers from Boston University focused on determining the impact of
activation of PFOSAs containing PFOS on mouse and human PPARα using COS-1 cells cell-
based luciferase reporter trans-activation assays. PFOS activated both mouse and human PPARα
in a dose-dependent fashion (p < 0.05), with a maximum activation from 4-6-fold. PFOS was
also tested on FAO, a rat hepatoma cell line, which showed a dose-dependent induction of PTL
protein expression (p < 0.05), a common PPARα-dependent response (Shipley et al., 2004).
Another study conducted by researchers from the United States Environmental Protection
Agency examined the effects of PFOA on PPARα, β, and γ expression and PPAR-regulated
genes by orally dosing pregnant CD-1 mice. PPARα mRNA was found to be expressed at higher
levels than PPARβ or PPARγ (p <.001) when exposed to PFOA. PPARα was dominant with
PFOS exposure in the following types of tissues: liver, heart, kidney, thymus, adrenal, and
intestines (Abbott et al., 2012). These studies provide statistically significant data that support
10
the activation of PPARα by PFOS and PFOA. Examining the specific types of PFAS activation
on PPAR can provide essential insight on what to avoid for the usage of PFAS in firefighting
foams. Overall, these studies support and suggest that PFAS exposure increase the risk of
prostate cancer via activation of PPARs.
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CHAPTER 3
PFAS EXPOSURE LEADS TO INCREASED RATE OF TUMOR PROGRESSION
3.1. INTRODUCTION
It is well-documented that PFASs, mainly PFOA and PFOS, may act as an endocrine
disruptors, and lead to abnormalities in pathways regulating steroidogenesis, cholesterol
homeostasis, pregnancy, weight maintenance, and molecular toxic waste disposal (Blake et al.,
2020; Dennis et al., 2020; Poteser, Hutter, Moshammer, & Weitensfelder, 2020). The effects of
PFAS on reproductive cancers, however, was the main focus of this study.
To discern any possible correlation between PFAS and cancer, it is essential to outline
the molecular and clinical characteristics of male reproductive cancers. Male reproductive
cancers may present themselves as either testicular cancers or prostate cancers, and prostate
cancer is the most common type of male cancer in the United States, with 248,530 new cases and
34,130 deaths in 2021 so far, amounting to a prevalence of 1 in every 8 men having the risk of
being diagnosed with prostate cancer throughout their lives (team, 2021). Contrary to testicular
cancer, which presents itself frequently between 20 to 35 years of age, prostate cancer risk tends
to increase in the later stages of life, with around 60% of the cases being older than 65
(Grossman et al., 2018). This is likely due to the dysregulation of testosterone production with
increasing age. Interestingly, PFAS exposure tends to have a similar effect on physiological
testosterone metabolism (Bandak et al., 2018; Gann, Hennekens, Ma, Longcope, & Stampfer,
1996; Kokontis et al., 2005; Miura et al., 2020; Watts et al., 2018). Male mouse pups exposed to
PFAS in utero can have an approximately 50% reduction in their serum testosterone levels, and
genes associated with apoptosis and cell-to-cell junctions may get either inhibited or
12
transcriptionally dysregulated, which can create a tumorigenic microenvironment that may
facilitate the development of reproductive cancers (Zhong et al., 2016).
As PFAS exposure was found to be associated with liver metabolism dysfunction and
development of NAFLD, the association between a high fat diet consumption that can create an
imbalance in cholesterol metabolism (Lysne et al., 2016; X. Tan et al., 2013; Venkateswaran &
Klotz, 2010) was introduced in combination with PFAS to an immunocompromised mouse
model to test the detrimental effects of PFAS-high fat diet combination on prostate cancer
progression. To facilitate human prostate tumor survival in this mouse model, we implanted a
testosterone tube dorsomedially to increase the serum testosterone levels of mice. With
testosterone added to the in vivo model, we used dihydroxytestosterone (DHT) as a control in all
subsequent in vitro studies. In preliminary negative tests, the proliferative characteristic that
PFAS seemed to promote in prostate cancer cells was not observed in the absence of DHT, so
DHT control proved to be an essential component to the prostate cancer-PFAS paradigm. We
hypothesized that our PFAS treated subjects, would have accelerated tumor progression and
coincident in vitro models with added DHT would have a more proliferative prostate cancer
profile upon PFAS introduction due to a shift in PPAR𝛼-mediated epigenetic and metabolic
alterations.
3.2. MATERIAL AND METHODS
Cell Culture
RWPE1, RWPE-Kras (RWPE2) cells were a gift from Dr. Gail Prins from University of
Illinois, Chicago. Cells were maintained in Gibco Keratinocyte SFM 1X growth media with
glutamine (Gibco 17005042, Fisher Scientific, Waltham, MA 02451).
WST-1 In vitro Cell Viability Assay
13
RWPE-kRAS and RWPE1 prostate cancer cells were maintained as described above. The
day before the treatments, cells were seeded at a density of 5000 cells/well in a 96-well plate.
Next day, cells were treated with varying concentrations (10-5 M, 10-6 M, 10-7 M, 10-8 M, 10-9 M
10-10 M) of PFOS or PFBS with or without 1 nM dihydrotestosterone (DHT) (2 biological
replicates, 6 technical replicates). Treatments were repeated again after two days. Effects of
PFAS on cell viability was quantitated by using the WST-1 reagent treatment, followed by an
incubation period of 45 minutes. Absorbance readings were measured at 450nm using Cytation5
plate reader (BioTek,Winooski, VT, USA), and statistical analyses were performed using
Graphpad© Prism8 software (GraphPad Software Inc., La Jolla, CA, USA, www.graphpad.com).
In vivo Prostate Cancer Xenograft Models
Animal experiments and protocols were approved by the University of Illinois at Urbana-
Champaign and the National Institutes of Health standards for the use and care of animals
(IACUC Protocol #20159) were followed. RWPE-1 and RWPE-KR as prostate cancer epithelial
cell lines were used for the tumor xenograft study. 4-week-old athymic nude male mice
(RRID:RGD_5508395) were ordered from Jackson laboratory (stock no. 007850). After a week
of acclimatization, to test synergy between PFAS and HFD, we compared carcinogenesis in mice
fed a high fat diet vs control diet. Because standard diets contain isoflavones with estrogenic
activity that interferes with metabolic effects, we used purified (AIN93M) diets. Purified HFDs
for our studies (Harlan TD.88137), aka the “western diet”, are high in butterfat (~42% Kcal from
fat) and sucrose, with a modest level of cholesterol (0.2%). This diet is commonly used for
metabolic syndrome studies.
After 10 days, 2 x 106 RWPE-Kras cells were injected to the left and right flanks of the
mice. In addition, testosterone sialistic tubes were implanted to provide continuous testosterone,
14
which is needed for xenograft establishment and growth. Animals were administered PFOS by
oral gavage seven days per week at 10 µg/kg/mice. Food consumption and animal weights were
monitored twice weekly. Tumor size measurements were performed three times a week using a
digital caliper. Animals were sacrificed five weeks after the initial cancer cell line injection.
Tumors, livers, prostates, and blood were harvested and were flash frozen or were fixed in
formalin for future staining.
OMICS-Based Metabolic Profiling
RWPE-Kras cells were seeded in growth media. The next day, they were treated with
Veh (cell growth media), 5mL of 10-8 M PFOS, 5mL of 10-8 M PFBS with or without 10-9 M
DHT. Cellular metabolites were extracted using a 1:2:1 mixture of
Acetonitrile:Isopropanol:Water. Extracts were sent to University of Illinois at Urbana-
Champaign’s Metabolomics Core Facility to detect and quantify metabolites using Gas
chromatography mass spectroscopy (GC/MS) analysis. Metabolic profiles were obtained from
Agilent GC-MS system (Agilent 7890 gas chromatograph, an Agilent 5975 MSD, and an HP
7683B autosampler, Lexington, MA, USA). The spectra of all chromatogram peaks were
evaluated using the AMDIS 2.71 and a custom-built database with 460 unique metabolites. All
known artificial peaks were identified and removed prior to data mining. Individual metabolomic
data sets for each treatment were separated and grouped into files to make comparisons between
treatment conditions using web based Metaboanalyst software. The sample class annotations
consisted of Veh vs. PFOS, Veh vs. PFBS, Veh vs. DHT, DHT vs. DHT + PFOS, and DHT vs.
DHT + PFBS. These files were uploaded to the Enrichment Analysis tool of MetaboAnalyst
software version 5.0 (RRID:SCR_015539). The data was not normalized, transformed, or scaled,
but it was compared to the SMPDB reference metabolome which represents metabolite values
15
from normal metabolic human pathways. The top 25 enriched metabolic pathways and
associated metabolites were retrieved along with their p-values and enrichment ratios. Heatmaps
were developed for each treatment group based on class averages using the default settings for
clustering, and the data was restricted to the top 25 metabolites using PLS-DA VIP.
For sequencing-based transcriptome/RNA analysis, RWPE-kRas cells were treated with
Veh or 10 nM PFAS with and without 1 nM DHT for 24 hours. cDNA libraries were be prepared
and sequencing reactions evaluated at the UIUC Sequencing core. Processing of data and
analysis was be performed as previously described (Madak-Erdogan et al., 2019; Madak-
Erdogan et al., 2013; Madak-Erdogan et al., 2016; Madak-Erdogan, Lupien, Stossi, Brown, &
Katzenellenbogen, 2011; Madak-Erdogan, Ventrella, Petry, & Katzenellenbogen, 2014; Zhao &
Madak Erdogan, 2016). GSEA analysis was performed to identified enriched gene set grouping
as previously described (Madak-Erdogan et al., 2013; Madak-Erdogan et al., 2016; Zhao &
Madak Erdogan, 2016).
Plate-Based Pyruvate and Acetyl CoA Assays
We prepared metabolite extracts from aforementioned in vitro cell models and xenograft
tumors to validate changes in pyruvate and acetyl-CoA levels using fluorescence based plate
assays (Sigma, #MAK071 and #MAK039). RWPE-Kras cells were seeded in 10 cm plates at a
density of 500,000 cells/plate and were treated with Veh (cell growth media), 5mL of 10-8 M
PFOS or 5mL of 10-8 M PFBS with or without 10-9 M DHT for 24 hours. The pyruvate
concentrations in the cells were detected by Cytation 5 plate reader upon formation of
fluorescent metabolite as pyruvate underwent oxidation by pyruvate oxidase. Pyruvate
concentrations were in nmol/uL.
16
For Acetyl CoA measurement, the fluorescence produced from NADH and probe
reaction that is coupled to conversion of Acetyl-CoA to CoA was detected using Cytation 5 plate
reader. Each experiment was repeated twice, with three technical replicates.
Western Blotting for Epigenetic Markers’ Assessment
RWPE-Kras cells were seeded at a density of 500,000 cells/plate on 10 cm plates. Cells
were treated with Veh (cell growth media), 5mL of 10-8 M PFOS or 5mL of 10-8 M PFBS with or
without 10-9 M DHT for 24 hours. Cell lysates were collected in lysis buffer (0.5 M EDTA, 1 M
TrisHCl pH 8.1, 10% SDS, 10% Empigen, ddH2O) with 1X Complete Protease Inhibitor
(Roche) and 1X Phosphatase Inhibitor (Thermo Scientific, Waltham, MA, USA). Cell lysates
were sonicated and protein concentrations were determined by BCA assay (Thermo Scientific).
Samples were boiled in SDS-containing loading buffer and each sample was run in 10% precast
gels (BioRad) and transferred to nitrocellulose membrane. The membranes were blocked in
Blocking Buffer (Odyssey®, Li-Cor, Lincoln, NE, USA) and target proteins were probed with
Acetyl Histone Antibody Sampler Kit (#9933, Cell Signaling) (RRID:AB_10699455), Tri-
Methyl Histone Antibody Sampler Kit (#9783, Cell Signaling) antibodies in 1:1000 dilution and
𝛽-actin (Sigma SAB1305546) (RRID:AB_2541177) antibody in 1:10000 dilution. The
secondary antibodies obtained from Odyssey were used at 1:10000 dilution. The membranes
were visualized by using Licor Odyssey CLx infrared imaging device and software.
3.3. RESULTS
3.3.1. Exposure to PFAS increases rates of cell proliferation in malignant prostate cancer
cell lines in vitro.
Epidemiology studies suggest an increase in prostate cancer incidence and/or mortality
with increasing years of chronic occupational PFAS or living in regional PFAS hotspots (Chang
17
et al., 2014; K. T. Eriksen et al., 2009; Lundin, Alexander, Olsen, & Church, 2009; Vieira et al.,
2013), particularly in men with familial prostate cancer risk, suggesting a gene–environment
interaction (Hardell et al., 2014). Whether PFAS exposures initiate carcinogenesis or promote
progression of latent or later stage prostate cancer is unknown for which we hypothesized that
PFAS exposure would increase the cell proliferation rate of the prostate cancer cells compared to
a non-treated control group (Figure 2). To test the impact of PFAS exposure on prostate cancer
initiation, we performed cell viability assays using a benign human prostate cell line, RWPE-1,
and a derivative cancerous cell line, RWPE-kRAS (Figure 3A), which showed that PFAS
enhances cell viability in an environmentally relevant dose range in both cell types (Figure 3B).
When prostate cancer cells were exposed to varying concentrations of PFAS (10-12 M – 10-4 M),
they exhibited a consequent degree of variance in their rates of proliferation with the first peak in
cell proliferation increase observed in 10-8 M PFAS, both for PFBS and PFOS. RWPE-kRAS
cells showed a more consistent cell proliferation response, with the 10-8 M peak being retained.
The more aggressive RWPE-kRAS cells exhibited a significant 3.1-fold increase in the rate of
cell proliferation when exposed to PFOS (𝜇=0.3729, 95% CI: 0.1155-0.6303, p-val=0.0048,
*=p<0.05,**=p<0.01) and a significant 2.9-fold increase in the rate of cell proliferation when
exposed to PFBS (𝜇=0.3484, 95% CI: 0.09105-0.6058, p-val=0.0078, *=p<0.05,**=p<0.01)
compared to a DHT-treated control group. There was no significant difference between the rates
of proliferation of either PFAS compound exposed groups and the DHT-treated control group.
3.3.2. Exposure to PFAS increases the rate of RWPE-kRAS xenograft tumor growth in vivo
both in the presence and absence of HF diet.
Compelling evidence from human prostate cell lines and transgenic murine prostate
cancer models indicates that a high fat diet contributes to prostate cancer progression by shifting
18
the prostate metabolome to a pro-cancerous state (Labbé et al., 2019; Priolo et al., 2014). Of
note, these actions are mediated through PPARα, the receptor targeted by PFAS, providing
potential for synergistic tumor promotion. To test the effects of PFAS exposure, we generated a
xenograft tumor growth model on nude immunocompromised mice and injected RWPE cells that
have kRAS overexpression ectopically to be able to measure tumor size growth (Figure 4A). At
the end of 40 days post-injection, we observed that ectopic tumor volume increased with the
fastest rate of growth when PFAS exposure was combined with consumption of HF diet (Figure
4B). This suggests that PFAS exposure in combination with high fat diet consumption likely
increases tumor growth more than normal cancer progression. In addition, the rate of growth of
the tumors exposed to just PFAS increased compared to the control treated tumor growth rate,
which supports the hypothesis that PFAS is detrimental to the prostate cancer progression rate.
Finally, we observed that PFAS treatment increased prostate cancer growth rate more than HF
diet consumption. As the animals have received slow-release testosterone tube implants, it is
likely that the combined PFAS and DHT treatment had a synergistic effect on the tumor growth
and a similar effect was also observed in the cell proliferation assays.
3.3.3. Prostate cancer cells that are exposed to PFAS in vitro exhibited increased building
block synthesis and altered energy production-adjacent pathways when DHT is present.
Metabolic plasticity is a hallmark of cancer (Hanahan & Weinberg, 2011; Pavlova &
Thompson, 2016). Cancer cells respond to their environments by rewiring their metabolic
pathways to sustain biological functions. Previous work using transgenic mouse models for Myc-
induced prostate cancer found that HFDs increase one-carbon metabolism and associated
changes in histone methylation marks, further increasing Myc activity in prostate tumors (Labbé
et al., 2019). However, the impact of environmental exposures on prostate cancer cell metabolic
19
wiring is unknown. Since we observed a synergy between PFOS exposure and high fat diet to
increase RWPE-kRAS tumor burden (Figure 4 ), and an increase in RWPE-kRAS cell viability
with PFOS treatment(Figure 3), we performed various –OMICS analysis to study metabolic
changes (Figure 5). Prior to our metabolomics, we knew that our prostate cancer cells would
proliferate more given a PFAS treatment in the presence of DHT, so we hypothesized that the
cell-proliferative energetics pathways would be upregulated in the prostate cancer metabolome.
To test this hypothesis, we analyzed metabolites that change in response to PFOS treatment
using GC/MS analysis of RWPE-kRAS cell extracts. This analysis showed that PFOS treatment
increased the metabolites associated with increased glucose metabolism through Warburg effect,
transfer of acetyl groups into mitochondria and citric acid cycle (Figure 6A), particularly
pyruvate (Figure 6B). To determine whether the increase in pyruvate production was due to
increased expression of enzymes in glycolytic pathway, we performed RNA-seq using tumors
from Figure 4. GSEA of the gene expression data identified pyruvate metabolism and glycolysis
pathways as significantly upregulated by PFOS exposure in tumors from HFD-fed animals
(Figure 6C). Particularly, components of pyruvate dehydrogenase complex (PDC), which is
responsible for acetyl-CoA production from pyruvate were increased with PFOS treatment in
tumor from high fat diet fed mice (Figure 6D). Consistent with these results, acetyl-CoA level
was increased in RWPE-kRAS cells in the conditions where we observed an increase in cell
viability (Figure 6E). These results from prostate transformed cell lines show that PFAS
exposure increases pyruvate and acetyl-CoA production.
3.3.4. Prostate cancer cells that are exposed to PFAS exhibited increased mitochondrial
dependence (citric acid cycle, PPP), increased epigenetic activation (H3K27Ac), acetyl-coA
metabolism; and altered amino acid metabolism (serine & lysine) in the absence of DHT.
20
Like the prior round of GC/MS analysis, the same conditions were tested without the
presence of DHT either in the control or the treatment group to see the effects of DHT on the
PFAS-prostate cancer paradigm (Figure 7A). As a result, we observed that PFAS treatment
upregulated the ‘Threonine & 2-Oxobutanoate Degradation’ (4.757-fold, n=3, p-val=0.000903),
‘Phosphatidylethanolamine Biosynthesis’ (4.057-fold, n=3, p-val=0.0143), ‘Homocysteine
Degradation’ (4.036-fold, n=3, p-val=0.0144), ‘Lysine Degradation’ (3.403-fold, p-val=0.0165),
‘ – all pathways that are involved in mitochondrial dependence, citric acid cycle regulation and
the pentose phosphate pathway (Chen et al., 2021; Leav et al., 2010). Interestingly, biotin
metabolism was also significantly upregulated (4.039-fold, n=3, p-val=0.0148), which plays an
important role in acetyl-coA carboxylase function as a prosthetic group . These data further
reinforced our initial hypothesis that acetyl-coA metabolism is affected with PFAS treatment
(Figure 7B, Figure 8).
3.3.5. PFAS treatment increase PPAR signaling and histone acetylation in prostate cancer
cells
Based on previously published studies and our data, we hypothesized that in these
prostate cells, PFAS converge with a high fat diet to activate PPARα, alter the cell metabolome,
and shift carcinogenic risk in normal cells while driving progression in prostate cancer cells.
PPARs are transcription factors involved in the regulation of metabolic processes, and HFDs are
known to impact hepatic cells through PPARα activation (D. Patsouris, Reddy, Müller, &
Kersten, 2006). Our laboratory previously showed that metabolites associated with obesity
activate PPARα signaling to modulate ERα activity in breast cancer cells (Madak-Erdogan et al.,
2019).It is noteworthy that PFAS chemicals also activate PPARα and, to a lesser extent, PPARβ,
to affect metabolism and the immune system (DeWitt et al., 2009). Structurally, PFAS resemble
21
free fatty acids and bind to the same sites on serum proteins (Evans, 2020.). Published studies
show a central role for PPARα signaling in PFOA/PFOS-induced liver and kidney
carcinogenesis (Stanifer et al., 2018; Wolf et al., 2008). However, this pathway has not been
interrogated for prostate cancer (Figure 9). We hypothesized an upregulation of global gene
activity both through epigenetic markers and within other transcriptional factors affect global
gene activity. To interrogate transcriptional changes in prostate tumors upon PFAS exposure, we
further analyzed our RNASeq analysis. Our data from RWPE-kRAS xenografts show that PFAS
exposure combined with high fat diet increases tumor growth and induces transcriptomic
changes in PPARα-target genes and genes involved in chromatin organization that in turn
regulate transcription (Figure 10A and 10B). In a transgenic MYC model of prostate
carcinogenesis, a high fat diet induced changes in H4K20 methylation and impacted expression
of MYC-target genes (Labbé et al., 2019). Consistent with these results and earlier changes
identified in pyruvate and acetyl-CoA synthesis-related metabolites, we examined the state of a
range of histone acetylation and methylation marks in RWPE-kRAS cells exposed to PFOS in
HLE and identified significant increases in H3K9 and H3K27 acetylation (Figure 10C). These
data support a fundamental role of PPAR signaling and epigenetic changes in prostate cancer
xenograft response to PFAS. This suggests that in the presence of PFAS, cellular energetics has
shifted from a more proliferative mode to a more mitochondrially dependent mode when
testosterone is absent, but further experimentation and repeats should be performed to confirm
this emergent pattern.
3.4. DISCUSSION
In the present study, we characterized the effects of PFAS using cell proliferation,
metabolomics, metabolite profiling, and western blot assays as well as in vivo xenograft models.
22
Our cell proliferation and in vivo xenograft model experimental results indicate that prostate
cancer cell proliferation rate in vitro was increased nearly 3-fold and the tumor growth rate was
faster in the presence of PFAS. According to our metabolomics and metabolite profiling assays,
PFAS shifts the cellular energetics of prostate cancer cells, which then increases the rate of
proliferation if there is DHT present, but moves the cells to a more energetically efficient and
mitochondria dependent state by enhancing oxidative phosphorylation, upregulating pentose
phosphate pathway and citric acid cycle.
Combining what has been observed in the metabolomics assay with the fact that
testosterone deregulation has increased incidence rates of prostate cancer in men really puts in
perspective the critical role that testosterone plays in male reproductive health. Add on top of
that the fact that rats treated with testosterone propionate experience a reduction in their prostate
cancer tumor size, indicating that proper testosterone concentration maintenance can facilitate
tumor prevention in the prostate microenvironment (Umekita, Hiipakka, Kokontis, & Liao,
1996). PFAS exposure seems to interact with the prostate in a similar manner to a testosterone
deficient prostate in that it modulates metabolic activity and reduces serum testosterone
concentrations, which incidentally a tumor-prone prostate landscape. Further research should be
performed to understand whether bioaccumulation of PFAS may lead to similar physiological
conditions, and how the liver metabolism deregulation interplays with the aforementioned
prostate microenvironment shifts.
23
CHAPTER 4
CONCLUSIONS AND FUTURE DIRECTIONS
4.1. CONCLUSIONS
In this thesis project, we studied the impact of PFAS exposure and high fat diets on
prostate carcinogenesis. We found that, PFAS + high fat diet exposure synergized to increase
prostate cancer xenograft growth, and PFAS treatment increased glucose metabolism and
pyruvate production in prostate cancer cells. Metabolic adaptations in prostate cancer change the
epigenetic landscape, in part due to changes in the availability of substrates for epigenetic
enzymes. Further, epigenetic marks dictate activity of critical transcription factors for prostate
carcinogenesis and progression including PPARα. Despite epidemiological evidence for an
association of PFAS exposure with prostate cancer, no mechanistic studies have established the
molecular underpinnings whereby PFAS exposures combine with high fat diet to increase
prostate carcinogenic risk and promote a more aggressive prostate cancer phenotype.
Epidemiology studies show that prostate cancer risk and mortality increase with PFAS
exposure (Barry et al., 2013; Kirsten T. Eriksen et al., 2009; Gilliland & Mandel, 1993) as well
as with obesity (Hardell et al., 2014; Vidal et al., 2020). Despite this evidence, mechanistic data
on the molecular underpinnings of PFAS chemicals in the prostate are limited/nonexisitent. In
preliminary studies, we found that PFAS exposure expands the prostate epithelial SPC
population, whereas a HFD plus PFAS exposure synergize to increase prostate cancer xenograft
growth in mice. Additionally, our results show that PFAS treatment increases glucose
metabolism and pyruvate production in prostate cancer cells. It is well-established that metabolic
adaptations in prostate cancer alter the epigenetic landscape, in part due to changes in substrate
availability for epigenetic enzymes. Further, epigenetic marks dictate the activity of PPARα, a
24
critical transcription factor in PFAS-associated carcinogenesis(Stanifer et al., 2018; Wolf et al.,
2008) that is liganded by both PFAS (Evans, 2020.) and metabolites associated with HFDs
(David Patsouris et al., 2006).
We showed that metabolic alterations from high fat diets combined with PFAS exposures
play a significant role in prostate cancer initiation and tumor progression. Published studies
clearly demonstrate that metabolic changes impact epigenetic marks during tumor progression
(Deblois et al., 2020; Makohon-Moore et al., 2017; McDonald et al., 2017). Metabolic alterations
in cancer cells result in epigenetic reprogramming due to changes in the availability of substrates
for epigenetic enzymes (Boukouris, Zervopoulos, & Michelakis, 2016; Faubert, Solmonson, &
DeBerardinis, 2020; Pascual, Domínguez, & Benitah, 2018). Local acetyl-CoA production, via
recruitment of metabolic enzymes to chromatin, enables coordination of environmental cues with
histone acetylation and gene transcription, which increases fitness and survival of cancer cells.
Acetyl-CoA can be synthesized in the nucleus from pyruvate by the pyruvate dehydrogenase
complex (PDC) (Matsuda et al., 2016; Sutendra et al., 2014), from acetate by acetyl-CoA
synthetase 2 (ACSS2) (Li, Qian, & Lu, 2017; Li, Yu, et al., 2017), and from citrate by ATP-
citrate lyase (ACLY) (Wellen et al., 2009) (Figure 11).
Overall, our studies suggest that PFAS exposure through oral ingestion can lead to an
increased risk of prostate cancer incidence. This increased incidence risk of prostate cancer
through PFAS exposure prompted the investigation of various nuclear receptors and mechanisms
of induction. PPARα, a nuclear receptor that can induce the carcinogenesis and proliferation of
prostate cancer, was found to be activated by various types of PFAS. PFOS and PFOA were two
specific chains that showed a higher level of activation of PPARα compared to the other types
that researched in this investigation. In addition, the deregulation of liver metabolism through
25
PFAS exposure seems to have potential to develop non-alcoholic fatty liver disease or other
metabolic disorders. Finally, the interactions between PFAS and testosterone metabolism can
lead to a reduction in serum testosterone concentration which can increase chance of prostate
cancer development.
4.2. FUTURE DIRECTIONS
Additional research on the other mechanisms and receptors that induce prostate cancer
through PFAS exposure must be conducted to help pinpoint the specific substances that can be
used as an alternative to PFAS in various high use frequency avenues such as firefighting foams,
grime-resistant Teflon pans and water pipes to avoid triggering these oncogenic pathways.
Research in this field is necessary to help protect the health and wellbeing of populations who
are exposed to high levels of PFAS, such as firefighters. Researchers and manufacturers must
work together to develop substances that replicate the purpose of PFAS in firefighting foams, but
that do not pose a substantial risk to firefighters.
Further investigation of the specific types of cancers associated with PPAR activation
should be conducted to determine the link between specific PFASs on types of cancers with
PPAR being a factor of correlation. Understanding individual types of PFAS activation on
PPARα, β/δ, and γ can provide insight for selecting the proper PFAS to be used in firefighting
foams that can prevent specific types of cancers.
28
Figure 3. PFAS treatments increase viability of prostate benign (RWPE-1) and cancerous
(RWPE-kRAS) cells. A. Experimental design. B. Cells were treated with indicated doses of
PFAS for 1 week. Cell viability was measured using WST1 assay.
29
Figure 4. PFAS and HFDs synergize to increase prostate cancer xenograft growth. A.
Experimental design. B. 106 RWPE-kRAS cells were injected subcutaneously in 4 week old
athymic nude male mice. Mice were fed a high fat diet (HDF) and treated with 10 µg/kg PFOS
for 5days/week orally. Tumor volume was measured using an electronic caliper three
times/week. C. For the duration of the 40-day treatment regimen, animal weight steadily
increased, but did not vary significantly between different treatment groups. Body weight was
measured every 3 days. D. Food consumption between different groups did not significantly
vary. Both high fat and Western control diets were varied in terms of how much each group has
consumed throughout the week, depending on how many days elapsed since the last day of
measurement, but the amount of consumption did not vary between different treatment groups.
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80
20
25
30
35
Animal weight
Time (days)
An
imal w
eig
ht
(g)
CONTROL
HIGH FAT
PFOS
HIGH FAT + PFOS
0 5 10 15 20 25 300
20
40
60
Time (days)
Fo
od
weig
ht
(g)
Food consumption
CONTROL
HIGH FAT
PFOS
HIGH FAT + PFOS
C D
30
Figure 5. Mechanistic studies to understand molecular basis of PFAS induces prostate
carcinogenesis. Hypothesis and methods used are highlighted.
31
Figure 6. PFAS treatment increases pyruvate and acetyl-CoA levels in RWPE-kRAS cells.
A. PFOS-induced metabolites in RWPE-kRAS cells identified by GC/MS analysis. B. Pyruvate
levels from A. C. GSEA of PFOS+HFD induced genes in RWPE-kRAS xenografts identified by
RNA-Seq analysis. D. mRNA expression of PDHB and PDHX, components of PDC, were
increased with PFOS and high fat diet in RWPE-kRAS xenografts. E. Acetyl-CoA levels in
PFOS-treated RWPE-kRAS cells (10 nM PFOS ± DHT, 24 hr) using a fluorescence based assay.
*p<0.05, **p<0.01.
32
Figure 7. PFAS treatment increases betaine metabolism, which can be associated as a
metabolic side-effect of PFAS exposure in RWPE-kRAS cells. A. PFOS-induced metabolites
in RWPE-kRAS cells identified by GC/MS analysis. Individual metabolic pathways that were in
the top 25 of the upregulated pathways were analyzed for significance and enrichment ratio. The
only significantly upregulated metabolism was betaine metabolism ((p-val=0.00819, ER=4.097)
and the rest of pathways were upregulated, but were not statistically significant. B. The top 25
metabolic pathways that were upregulated are shown with varying degrees of significance (p-val
range: 0.008-0.3)
A
B
33
Figure 8. PFAS exposure upregulates mitochondrial dependence (citric acid cycle, PPP),
increased epigenetic activation (H3K27Ac), acetyl-coA metabolism; and altered amino acid
metabolism (serine & lysine) in the absence of DHT. Metabolic pathways that were
upregulated in the PFAS-treated group compared to a non-treated control group were cross
referenced and pathways that were upregulated in the PFAS group were listed based on
enrichment ratio.
34
Figure 9. Outline of studies to understand the impact of PFAS on epigenetic regulation in
prostate cancer cells.
35
Figure 10. PFAS treatment increases A PPAR signaling, B epigenetic regulation of
transcription-associated genes in RWPE-kRAS xenografts. C PFOS exposure increased histone
acetyl markers in RWPE-kRAS cells.
37
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