© 2021 Ozan Berk Imir

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.

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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.

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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.

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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

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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

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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.

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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.

26

FIGURES AND TABLES

Figure 1. Thesis summary

27

Figure 2. Hypothesis and methods for studying PFAS impact on prostate cancer

proliferation

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.

36

Figure 11. Link between metabolic pathways and epigenetic marks.

37

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