Perfluorooctanoic Acid Activates Multiple Nuclear Receptor Pathways and Skews Expression

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bioRxiv preprint doi: https://doi.org/10.1101/2020.01.30.926642; this version posted January 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Perfluorooctanoic acid activates multiple nuclear receptor pathways and skews expression

of genes regulating cholesterol homeostasis in liver of humanized PPARα mice fed an

American diet

Schlezinger, JJ, Puckett, H, Oliver, J, Nielsen, G, Heiger-Bernays, W, Webster, TF

Department of Environmental Health, Boston University School of Public Health, Boston, MA,

02118, USA

Corresponding author: Jennifer J. Schlezinger, Ph.D. ORCiD: 0000-0001-6834-4369 Boston University School of Public Health Dept. of Environmental Health 715 Albany Street, R-405 Boston, MA 02118 Phone: 617-638-6497 Fax: 617-638-6463 Email: [email protected]

Author contributions Jennifer Schlezinger, Wendy Heiger-Bernays and Thomas Webster contributed to the study conception and design. Material preparation and data collection were performed by Jennifer Schlezinger and Hannah Puckett. Data analysis and interpretation were performed by all authors. The first draft of the manuscript was written by Jennifer Schlezinger, Greylin Nielsen and Wendy Heiger-Bernays, and all authors commented the manuscript. All authors read and approved the final manuscript.

Acknowledgements This work was supported by the National Institute of Environmental Health Sciences Superfund Research Program P42 ES007381 (Jennifer Schlezinger) and R01 ES027813 (Thomas Webster). Greylin Nielsen and Jennifer Oliver are supported by training grant T32 ES01456. The authors thank Mr. Nathan Burritt for his excellent technical assistance and Dr. Juliet Gentile (Research Diets, Inc.) for her expert assistance in designing the diet.

Abstract

Humans are exposed to per- and polyfluoroalkyl substances (PFAS) in their drinking water,

food, air, dust in their homes, and by direct use of consumer products. Increased concentrations

of serum total cholesterol and low density lipoprotein cholesterol are among the endpoints best

supported by epidemiology. The objectives of this study were to generate a new model for

examining PFAS-induced dyslipidemia and to conduct molecular studies to better define

mechanism(s) of action. We tested the hypothesis that PFOA exposure at a human-relevant level

dysregulates expression of genes controlling cholesterol homeostasis in livers of mice expressing

human PPARα (hPPARα). Female and male hPPARα and PPARα null mice were fed a diet

based on the “What we eat in America” analysis and exposed to perfluorooctanoic acid (PFOA)

in drinking water (8 µM) for 6 weeks. This resulted in a serum PFOA concentration of 48 μg/ml.

PFOA increased liver mass, which was associated with histologically-evident lipid accumulation.

PFOA induced PPARα and constitutive androstane receptor target gene expression in liver.

Expression of genes in four pathways regulating cholesterol homeostasis were also measured.

PFOA decreased expression of Hmgcr in a PPARα-dependent manner. PFOA decreased

expression of Ldlr and Cyp7a1 in a PPARα-independent manner. Apob expression was not

changed. Gene expression in females appeared to be more sensitive to PFOA exposure than in

males. This novel study design (hPPARα mice, American diet, long term exposure) generated

new insight on the effects of PFOA on cholesterol regulation in the liver and the role of

hPPARα.

Keywords: Perfluorooctanoic acid, peroxisome proliferator activated receptor α, constitutive

androstane receptor, cholesterol

Introduction

Per- and polyfluoroalkyl substances (PFAS) are pervasive in the environment because of their

persistence and extensive use in consumer products and fire-fighting foam. Daily human

exposures occur via PFAS contaminated food, drinking water, dust and air (EFSA 2018; Kim et

al. 2019; Makey et al. 2017). Multiple adverse health outcomes have been associated with PFAS

exposure including birth outcomes, immunologic effects, and metabolic disruption. Among the

best supported and most sensitive endpoints in both cross-sectional and longitudinal

epidemiology studies are lipid-disrupting effects (Frisbee et al. 2010; Geiger et al. 2014; Graber

et al. 2018; He et al. 2018; Nelson et al. 2010; Steenland et al. 2009). The European Food Safety

Authority has proposed dyslipidemia, an abnormal amount of lipids (triglycerides, cholesterol

and/or fat phospholipids) in the blood, as a critical effect (EFSA 2018).

Mechanisms by which PFAS may cause lipid-disrupting effects are not well understood.

Fatty acids are endogenous ligands for peroxisome proliferator activated receptor α (PPARα), a

transcription factor that regulates lipid homeostasis. Since many PFAS are structurally similar to

fatty acids, PPARα binding and activation is a logical molecular initiating event for a lipid-

disrupting pathway. As such, PPARα was found to account for 80-90% of perfluorooctanoic acid

(PFOA) regulated genes (Rosen et al. 2017). There are well known species differences in the

function of mouse PPARα (mPPARα) and human PPARα (hPPARα) (Gonzalez and Shah 2008).

Activation of mPPARα results in peroxisome proliferation and dysregulation of cell cycle genes,

which does not occur in humans (Morimura et al. 2006). However, both mPPARα and hPPARα

efficaciously regulate multiple biological pathways involved in lipid homeostasis, acute phase

response and inflammation (Rakhshandehroo et al. 2009). mPPARα and hPPARα share 91%

amino acid identity (Sher et al. 1993), and important differences in the ligand binding domains

have been identified (Keller et al. 1997; Oswal et al. 2014). As a result, there are differences in

ligand specificity and gene expression patterns (Keller et al. 1997; Oswal et al. 2014;

Rakhshandehroo et al. 2009). However, both mPPARα and hPPARα have been shown to

contribute to regulation of cholesterol homeostasis (Bouchard-Mercier et al. 2011; Flavell et al.

2000; Peters et al. 1997; Robitaille et al. 2004; Sparso et al. 2007; Tanaka et al. 2007; Vohl et al.

2000).

Studies using reporter assays show that hPPARα is activated by PFAS. hPPARα

activation increases with perfluoroalkyl acid chain length up to 8 carbons, yet all carbon chain

lengths tested (i.e., C4 to C12) significantly activate PPARα (Behr et al. 2019; Maloney and

Waxman 1999; Rosenmai et al. 2018; Takacs and Abbott 2007; Vanden Heuvel et al. 2006).

hPPARα target gene expression is induced by PFAS in human hepatocyte models, including

primary hepatocytes (Behr et al. 2019; Bjork et al. 2011; Buhrke et al. 2013; Buhrke et al. 2015;

Peng et al. 2013; Rosen et al. 2013; Wolf et al. 2012; Wolf et al. 2008). However, in human and

rodent hepatocyte models, transcriptome profiling shows that PFAS also regulate target gene

expression of other nuclear receptors (Abe et al. 2017; Bjork et al. 2011; Buhrke et al. 2015;

Scharmach et al. 2012).

Studies of the effects of PFOA on serum lipids and cholesterol in animal models have

produced contradictory results. Mice exposed to PFOA and fed standard rodent chow (low in fat

with negligible cholesterol) show decreased serum cholesterol levels (reviewed in (Rebholz et al.

2016)), in contrast to the increase shown in human epidemiology. But diet also influences serum

cholesterol levels in mice (Dietschy et al. 1993), and when mice are fed a cholesterol and fat-

containing diet, PFOA does induce hypercholesterolemia (Rebholz et al. 2016). Strain and sex

also modify PFAS-induced effects on serum lipids (Pouwer et al. 2019; Rebholz et al. 2016).

Last, in mice expressing human PPARα in liver, PFOA increased serum cholesterol (Nakamura

et al. 2009). The goals of this study were to generate a new model for examining PFAS-induced

dyslipidemia and to conduct molecular studies to better define the mechanism of action by which

this occurs. We tested the hypothesis that PFOA exposure dysregulates genes controlling

cholesterol homeostasis in livers of mice expressing human PPARα (hPPARα) and fed an

American diet, the first time this combination has been examined. We focused on liver because it

is an essential site of regulation of multiple aspects of cholesterol homeostasis (Dietschy et al.

1993). The role of hPPARα was determined through comparison with effects in PPARα null

mice. The data document important sex differences and identification of molecular pathways

important for PFAS-induced dyslipidemia.

Materials and Methods

Materials

Perfluorooctanoic acid (cat. # 171468, 95% pure) was from Sigma-Aldrich (St. Louis, MO). All

other reagents were from Thermo Fisher Scientific (Waltham, MA), unless noted.

In vivo exposure

All animal studies were approved by the Institutional Animal Care and Use Committee at Boston

University and performed in an American Association for the Accreditation of Laboratory

Animal Care accredited facility (Animal Welfare Assurance Number: A3316-01). Male and

female, humanized PPARα mice (hPPARα) were generated from mouse PPARα null, human

PPARα heterozygous breeding pairs (generously provided by Dr. Frank Gonzalez, NCI)(Yang et

al. 2008). Experiments were carried out using 11 cohorts of mice generated from four breeding

pairs (Table S1). Genotyping for mouse and human PPARα was carried out by Transnetyx

(Cordova, TN). The expression level of hPPARα in liver was confirmed by RT-qPCR.

At weaning, mice were provided a custom diet based on the "What we eat in America

(NHANES 2013/2014)" analysis for what 2-19 year old children and adolescents eat (Research

Diets, New Brunswick, NJ)(USDA 2018). The diet contains 51.8% carbohydrate, 33.5% fat, and

14.7% protein, as a % energy intake (Table S2). Fats are in the form of soybean oil, lard and

butter, with cholesterol at 224 mg/1884 kcal. Vehicle (Vh) and treatment water were prepared

from NERL High Purity water (23-249-589, Thermo Fisher Scientific), which is prepared using

the most efficacious methods to remove PFAS (i.e., reverse osmosis and carbon

filtering)(Appleman et al. 2014). A concentrated stock solution of PFOA (1x10-2 M) was

prepared in NERL water and then diluted in NERL water containing 0.5% sucrose. Mice were

administered vehicle (0.5% sucrose) drinking water or PFOA (8 μM) drinking water ad libitum

for 6-7 weeks. Food and water consumption were determined on a per cage basis each week.

Body weight was measured weekly. Mice were analyzed for body composition (total body fat

mass, lean mass, water) using an EchoMRI700 (EchoMRI LLC, Houston, TX), fasted for 6

hours and then euthanized. Livers were collected from each mouse and weighed. Aliquots of

liver for gene expression were flash frozen in liquid nitrogen and stored at -80°C and for

histology were fixed in 10% neutral buffered formalin.

PFAS analysis

PFAS concentrations were determined in pooled water samples. Aliquots were taken each time

drinking water was made for a cohort and combined in a single sample; drinking water from five

cohorts was analyzed. Individual serum samples were analyzed. PFOA concentrations were

determined by LC-MS/MS according to methods MLA-110 (EPA Method 537 Modified) for

water and MLA-042 for serum (SGS AXYS Analytical Services Ltd., Sidney, British Columbia,

CA).

Gene expression analyses

Total RNA was extracted and genomic DNA was removed using the Direct-zol RNA Miniprep

Kit (Zymo Research, Orange, CA). cDNA was synthesized from total RNA using the iScript™

Reverse Transcription System (BioRad, Hercules, CA). All qPCR reactions were performed

using the PowerUp™ SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA). The

qPCR reactions were performed using a StepOnePlus Real-Time PCR System (Applied

Biosystems, Carlsbad, CA): UDG activation (50°C for 2 min), polymerase activation (95°C for 2

min), 40 cycles of denaturation (95°C for 15 sec) and annealing (various temperatures for 15

sec), extension (72°C for 60 sec). The primer sequences and annealing temperatures are provided

in Table S3. Relative gene expression was determined using the Pfaffl method to account for

differential primer efficiencies (Pfaffl, 2001), using the geometric mean of the Cq values for

beta-2-microglobulin (B2m), GAPDH (Gapdh), and 18sRNA (R18s). The average Cq value from

two livers from female C57/BL6J mice was used as the reference point. Data are reported as

“Relative Expression.”

Histological analyses

5µm liver sections were stained with hematoxylin and eosin. Micrographs (20x) were visualized

on a Nikon Eclipse TE2000 microscope (Nikon Corporation; Tokyo, Japan).

Statistical analyses

Data are presented as data points from individual mice or as means ± standard error (SE). Mice

were considered hPPARα positive if they were either homozygous or heterozygous. Information

on outliers is presented in Table S1. In the gene expression analyses, values more than four

standard deviations different than the mean were excluded from the analyses. Overall, six values

were excluded in five of the twenty genes analyzed. Within sex and genotype, statistical

significance was determined by unpaired, two-tailed t-test (Prism 6, GraphPad Software Inc., La

Jolla, CA). Regression analyses were performed using Microsoft R Open version 3.6.1. To

investigate the interactions between treatment, sex and genotype in modifying phenotype and

gene expression, we used multiple linear regression modeling (MLR). Each outcome was

assessed using a MLR model with predictors including sex and an interaction term for genotype

and treatment. Models were also stratified by sex, allowing effect estimates to vary between

males and females. Statistical significance was evaluated at an α= 0.05 for all analyses.

Results

Drinking water concentrations of PFOA averaged 3509 ± 138 μg/L. Based on average daily

water consumption (0.21 ml/g mouse/day), the daily exposure was approximately 0.7 mg/kg/day.

This resulted in serum concentrations of 47 ± 8 μg/ml in females and 48 ± 10 μg/ml in males (N

= 4 for each sex). Assuming a 20 day half-life (Lou et al. 2009), and that at 6 weeks of exposure

77% of steady state was reached, the steady-state Cs/Cdw ≈ 18.

Daily exposure to PFOA for 6 weeks did not significantly impact weight gain in hPPARα

mice of either sex (Fig. 1a and 1c). However, PFOA treatment significantly reduced weight gain

in male PPARα null mice (Fig. 1c, Table 1). A similar trend was observed in female PPARα null

mice, but the effect was not significant (Fig. 1a, Table 1). Body composition was not affected by

PFOA in either genotype or sex (Fig. 1b and 1d). No differences in water or food consumption

were observed (Fig. S1).

PFOA significantly increased liver to body weight ratios in both sexes and both

genotypes (Fig. 2). In females, PFOA induced a significantly greater effect on liver to body

weight ratios in PPARα null mice than in hPPARα mice (Fig. 2a, Table 1); this effect was not

observed in males (Fig. 2c, Table 1). Histological analyses showed significant microvesicular

lipid accumulation (steatosis) in PFOA-treated hPPARα mice of both sexes (Fig. 2b and 2d).

Microvesicular steatosis also was evident in Vh-treated PPARα null mice of both sexes (Fig. 2b

and 2d). Macrovesicular steatosis was present in PFOA-treated PPARα mice, with the largest

lipid droplets observed in male PPARα null mice (Fig. 2b and 2d).

Activation of hPPARα was evident in livers of PFOA-treated, humanized mice. Human

PPARA mRNA was highly expressed in transgenic mice of both sexes, and lack of PPARα

expression was confirmed in the PPARα null mice; expression of PPARA was not influenced by

PFOA treatment (Fig. 3, Table 2). Expression of the PPARα target genes Acox (Acyl-CoA

oxidase 1 is the first enzyme of the fatty acid beta-oxidation pathway), Adrp (Perlipin 2 coats

intracellular lipid storage droplets), Mogat1 (Monoacylglycerol O-acyltransferase 1 catalyzes the

synthesis of diacylglycerols), and Vnn1 (Vanin 1 biotransforms pantetheine in cysteamine and

pantothenic acid, a precursor of coenzyme A) were upregulated by PFOA exposure in hPPARα

mice but not in PPARα null mice in both sexes (Fig. 3, Table 2). Pdk4 (Pyruvate dehydrogenase

kinase 4 inhibits the pyruvate dehydrogenase complex) was upregulated by PFOA exposure in

hPPARα mice but downregulated in female PPARα null mice (Fig. 3, Table 2). Sex-dependent

differences in expression were evident for Mogat1 and Pdk4 (Table 2). Mogat1 was upregulated

to a greater extent by PFOA in male hPPARα mice. Pdk4 was downregulated to a greater extent

by PFOA in female hPPARα null mice.

In addition to PPARα, evidence suggests that at least PPARγ and CAR also are molecular

targets of PFAS (Abe et al. 2017; Buhrke et al. 2015; Vanden Heuvel et al. 2006). Expression of

PPARγ mRNA (Nr1c3) along with its target genes Fabp4 (Fatty acid binding protein 4 binds and

transports long chain fatty acids) and Cd36 (CD36 is a fatty acid translocase) were upregulated

in PFOA treated mice of both sexes (Fig. 4a). There was a small but significant decrease in

induction of Nr1c3 expression and a trend toward a decrease in induction in Fabp4 expression in

male PPARα null mice (Table 3). Induction of Cd36 was nearly completely abrogated in PPARα

null mice of both sexes (Fig. 4a). In contrast, expression of CAR mRNA (Nr1i3) was modestly

induced by PFOA in only male hPPARα mice (Fig. 4b). Expression of the CAR target genes

Cyp2b10 (Enzymes in the CYP2B family oxidatively metabolize a broad range endogenous and

exogenous substrates) and Gstm3 (GSTM3 is a glutathione s-transferase of the mu class) was

highly upregulated in both sexes and in both genotypes (Fig. 4b). CAR target gene expression

was induced to a greater extent in PPARα null mice than in hPPARα mice (Fig. 4b, Table 3).

Prior to experimentation, we hypothesized that activation of PPARα by PFOA may

influence serum cholesterol homeostasis through four potential pathways in liver: increased de

novo cholesterol synthesis, increased cholesterol export into the blood, decreased hepatic uptake

of LDL-C from blood, and/or decreased cholesterol turnover to bile acids (Fig. 5). Expression of

Hmgcr, the rate limiting step in cholesterol synthesis, was decreased by PFOA exposure in

hPPARα but not PPARα null mice (Fig. 5a, Table 4). Expression of Apob, the apolipoprotein

associated with VLDL-C and LDL-C, was not changed by PFOA exposure in either genotype or

sex (Fig. 5b). Expression of Ldlr, which is responsible for hepatic uptake of LDL-C, was

decreased by PFOA exposure in both hPPARα and PPARα null mice (Fig. 5c). Lastly,

expression of Cyp7a1, the rate limiting step in conversion of cholesterol to bile acids and thus

cholesterol efflux from the body, was down regulated by PFOA in both hPPARα and PPARα

null mice but more so in hPPARα mice than PPARα null mice (Fig. 5d). Interestingly, PFOA’s

effect on expression of cholesterol homeostasis genes, particularly Cyp7a1, was greater in female

than male mice (Table 4).

While PPARα is known to regulate cholesterol homeostasis (Bouchard-Mercier et al.

2011; Flavell et al. 2000; Robitaille et al. 2004; Sparso et al. 2007; Tanaka et al. 2007; Vohl et al.

2000), there are no studies showing direct interactions of PPARα with the promoters of the genes

involved in cholesterol homeostasis. Rather, Hmgcr is regulated by SREBP2 (Srebf2; (Brown

and Goldstein 1997; Sharpe and Brown 2013)). Apob is regulated by C/EBPα and HNF4α

(Metzger et al. 1993). Ldlr is regulated by SREBP1 (Srebf1), SREBP2 and estrogen receptor

(Brown and Goldstein 1997; Parini et al. 1997; Yokoyama et al. 1993). Cyp7a1 is regulated by

HNF4α, LXR and FXR (Gupta et al. 2002; Kir et al. 2012). PFOA treatment did not regulate

C/EBPα, HNF4α, SREBP1 or SREBP2 at the transcriptional level (Fig. 6), although regulation

at the post-translational level is still a possibility. Interestingly, Srebf1 was significantly

downregulated in PPARα null mice compared to hPPARα mice (Fig. 6, Table S4)

Discussion

Increased serum total cholesterol and LDL-C are strongly associated with PFAS exposure in

humans. However, studies of the effects of PFOA on serum lipids and cholesterol in animal

models have produced contradictory results. New models are needed to investigate the

mechanism(s) of action through which PFAS could interfere with cholesterol homeostasis. Here,

we tested the hypothesis that PPARα activation is a critical molecular initiating event in the

adverse outcome pathway linking human-relevant PFOA exposure with dyslipidemia in a novel

model, mice expressing hPPARα fed an American diet. Over all, PFOA modulates at least the

PPARα, PPARγ and CAR pathways in hPPARα mice, as well as multiple genes involved in

cholesterol metabolism and homeostasis. Not all effects were PPARα-dependent. Our results

show that the hepatic response to PFOA exposure is sexually dimorphic.

The PFOA exposure in this study was designed to recapitulate serum PFOA

concentrations observed in some epidemiological studies. The PFOA’s toxicokinetic parameters

differ significantly between rodents and humans with rodents having increased renal clearance

capacity and a substantially shorter half-life compared with humans (Harada et al. 2007; Lou et

al. 2009). Thus, we used a higher than typical exposure dose (≈ 0.7 mg/kg/day) to generate a

serum PFOA concentration (≈ 48 μg/ml) similar to that found in fluorochemical workers in the

US (≈ 22 μg/ml (Steenland et al. 2010)). We estimated a steady-state serum (Cs) to drinking

water (Cdw) ratio of ≈ 18 in the mice in this study, which is similar to the ratio previously

determined in CD1 mice (Cs/Cdw ≈ 12 (White et al. 2011)). As expected from the differences in

half life, in humans the Cs/Cdw ratio was estimated to be on the order of 114 and 141 (Hoffman

et al. 2011).

Our results demonstrate that both hPPARα and PPARα null mice exposed to PFOA and

fed an American diet develop hepatosteatosis. PFOA induced a significant increase in liver:body

weight ratio associated with histologically evident increases in hepatocyte lipid accumulation.

PFOA-induced hepatosteatosis has been observed previously in mice expressing wildtype

PPARα (mPPARα), hPPARα mice and PPARα null mice (Das et al. 2017; Minata et al. 2010;

Nakagawa et al. 2012; Nakamura et al. 2009; Tan et al. 2013). While hepatosteatosis is induced

in PFOA-exposed mice fed standard composition rodent diets, the severity is increased when

mice are co-exposed to PFOA and a high fat diet (Tan et al., 2013). Importantly, in an exposure

scenario that generated an approximately steady state body burden, hPPARα mice were more

susceptible to hepatic steatosis than mPPARα mice (Nakagawa et al. 2012), underscoring the

importance of using a humanized mouse model to investigate PFOA-induced hepatic endpoints.

PFOA activated hPPARα. However, upregulation of PPARγ and CAR target genes

indicate that PFOA exerts biological effects through multiple pathways. The ability of PFOA to

upregulate the transcription of PPARγ has been documented (Rosen et al. 2008). Upregulation of

Fabp4 and Cd36, genes classically thought to be regulated by PPARγ was abrogated in PPARα

null mice. This is not necessarily a surprise, as there can be a significant overlap in regulation of

genes by PPARs. While regulation of fatty acid transporter genes is largely controlled by PPARγ

in adipose, their expression is PPARα-dependent in liver (Motojima et al. 1998). CAR

transcriptional activity was strongly upregulated by PFOA in both hPPARα and PPARα null

mice. Studies with PXR, CAR and FXR null mice show that CAR is the most significant

contributor to PFAS-induced changes in gene expression, after PPARα (Abe et al. 2017; Cheng

and Klaassen 2008). Interestingly, our findings show that PFOA induces CAR target gene

expression to a higher degree in PPARα null mice than hPPARα mice indicating a potential

interaction between the two nuclear receptors. Increased CAR activation in the absence of

PPARα has been identified previously and may be due to antagonistic effects of these two

nuclear receptors (Corton et al. 2014; Rosen et al. 2017).

We hypothesized that PFOA alters lipid homeostasis through one of four mechanisms in

the liver: increased de novo cholesterol synthesis, increased cholesterol export into the blood,

decreased hepatic uptake of LDL-C from blood, and/or decreased cholesterol turnover to bile

acids. We observed a significant reduction in the expression of Hmgcr, Ldlr, and Cyp7a1 in

female mice. Apob expression was unchanged in both sexes. These changes in expression also

were observed in C57BL/6J female mice (Rebholz et al. 2016). While the decrease in Hmgcr

expression would be expected to decrease serum cholesterol, reductions in Cyp7a1 and Ldlr

expression would be expected in increase serum cholesterol. The downregulation of Cyp7a1

occurs more broadly across models, as we observed Cyp7a1 downregulation in both male and

female SV129 mice and Rebholz et al., 2016 observed its downregulation in both sexes in both

C57Bl/6J and Balb/C mice. Importantly, PFOA also downregulates Cyp7a1 expression in human

hepatocytes (Behr et al. 2020). Cholesterol is converted to bile acids, a major mechanism for

removal of cholesterol from the body, through two primary molecular pathways with CYP7A1

being the rate limiting enzyme in the primary pathway (Dietschy and Turley 2002). In humans,

the CYP7A1-mediated primary pathway accounts for 90% of bile acid production whereas it

only accounts for 60% in mice (Phelps et al. 2019). Further, CYP7A1 is a major factor regulating

lipoprotein synthesis and assembly (Wang et al. 1997). A reduction of more than 50% in Cyp7a1

expression was associated with increased plasma cholesterol levels in mice (Rebholz et al.,

2016). Expression of Ldlr plays a substantial role in regulating serum LDL-C, with serum

concentrations increasing an order of magnitude in Ldlr null mice (Osono et al. 1995). It is likely

then that the PFOA-induced decrease in Ldlr expression has a biologically significant effect on

serum cholesterol. Intriguingly, PFOA substantially upregulated Vnn1, overexpression of which

is associated with increased liver lipid content, serum triglycerides and LDL-C, decreased HDL-

C and enhanced atherosclerotic plaque formation (Hu et al. 2016). It remains to be determined if

any or all of these changes in gene expression contribute to serum lipid dysregulation of PFOA.

Only the repression of Hmgcr expression by PFOA appeared to be PPARα dependent.

Hmgcr expression was also decreased in PPARα null mice. Previous studies have shown that

GW7647, a PPARα ligand, increases the occupancy of PPARα at the Hmgcr promoter, in

concert with SREBPs (van der Meer et al. 2010); however this resulted in an increased

expression of Hmgcr. Thus PFOA-liganded PPARα appears to be acting distinctly from

GW7647-liganded PPARα. On the other hand, repression of Ldlr and Cyp7a1 expression by

PFOA occurred in both hPPARα and PPARα null mice. PPARα overexpression or treatment

with a PPARα ligand has been shown to suppress HNF4α protein expression, thereby reducing

its interaction with the Cyp7a1 promoter (Marrapodi and Chiang 2000). However, we did not

observe a decrease in Hnf4a mRNA in PFOA-treated animals. No studies have reported

regulation of Ldlr by PPARα.

A single previous study has investigated the effects of PFAS on lipid homeostasis in

female mice (Rebholz et al. 2016). The results presented here corroborate the observation of sex-

dependent effects of PFOA on liver physiology. The data show differences at the macro level

(liver:body weight) and gene expression level. Most interesting are the differences in effect of

PFOA on expression of genes involved in cholesterol homeostasis. It is well known that

cholesterol homeostasis differs in mouse strains and sexes (Bruell et al. 1962). However, before

the current study, only Rebholz et al., 2016 investigated the influence of strain and sex on the

response to PFOA and showed that C57Bl/6J mice were more sensitive to modulation of

cholesterol homeostasis by PFOA than Balb/C mice. They also showed that female C57BL/6

mice, the mice with the greatest increase in serum cholesterol, were the only mice to have

significant changes in expression of multiple genes involved in cholesterol homeostasis (Rebholz

et al. 2016). We observed significant changes due to PFOA in these same genes (Hmgcr, Ldlr,

and Cyp7a1) in female hPPARα mice. It is critical that future studies take into account the

complexity of the genetics that contribute to cholesterol homeostasis when investigating PFOA-

induced effects.

Conclusions

PFOA activates human PPARα and CAR at human relevant serum concentrations in vivo.

Multiple genes involved in cholesterol homeostasis are modified by PFOA by both PPARα-

dependent and independent mechanisms. Investigation of the effects of PFOA and their

dependence on PPARα beyond the four biomarker genes analyzed here is necessary. The

essential role of hPPARα in basal cholesterol homeostasis, as well as fatty acid homeostasis, and

known species differences in ligand binding gene batteries support the conclusion that our model

is an important new tool in dissecting the multiple, interacting mechanisms of PFOA action on

cholesterol homeostasis. Importantly, PFOA-induced effects appear to be stronger in females

than in males. Regulation of cholesterol homeostasis is complex, is modified by diet, with

multiple pathways able to compensate for deficiencies (Dietschy and Turley 2002; Dietschy et al.

1993). Thus, further research is needed to delineate the biologically significant effects of PFAS

on multiple aspects of cholesterol homeostasis.

Funding

This work was supported by the National Institute of Environmental Health Sciences Superfund

Research Program P42 ES007381 to JJS and R01 ES027813 to TW. GN and JO are supported by

training grant T32 ES01456.

Conflict of Interest

The authors declare that they have no conflict of interest.

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Tables Table 1: Effect estimates (β) and standard errors (SE) for phenotypic outcomes. Regression models were fit to evaluate associations of phenotypic outcomes with treatment and genotype, including a treatment-genotype interaction term. The left hand column adjusts for sex. The two right columns stratify by sex, allowing results to differ between males and females. Statistical significance was evaluated at α = 0.05 for all analyses.

ALL MALE FEMALE Test β (SE) P value β (SE) P value β (SE) P value Weight Gain, Week 6 (%) PFOA treatment -24.62 (13.82) 0.08 -33.89 (22.10) 0.14 -15.43 (17.25) 0.38 hPPARα Genotype -32.29 (12.65) 0.01 -47.58 (19.23) 0.02 -15.15 (16.63) 0.37 Treatment*Genotype 26.79 (18.11) 0.15 43.54 (28.00) 0.13 7.99 (23.51) 0.74 Male Sex 25.71 (8.97) 0.006 - - - - Body Fat (%) PFOA treatment -2.87 (2.33) 0.22 -4.58 (3.76) 0.23 -1.28 (2.94) 0.67 hPPARα Genotype -0.08 (2.13) 0.97 -1.64 (3.27) 0.62 1.64 (2.83) 0.57 Treatment*Genotype 2.37 (3.05) 0.44 4.96 (4.77) 0.31 -0.34 (4.00) 0.93 Male Sex 0.89 (1.51) 0.56 - - - - Liver to Body Weight (%) PFOA treatment 4.75 (0.39) <0.0001 4.99 (0.63) <0.0001 4.49 (0.44) <0.0001 hPPARα Genotype -0.41 (0.36) 0.25 -0.19 (0.54) 0.72 -0.60 (0.43) 0.17 Treatment*Genotype -1.17 (0.51) 0.03 -0.95 (0.79) 0.24 -1.50 (0.60) 0.02 Male Sex 0.43 (0.25) 0.10 - - - -

Table 2: Effect estimates (β) and standard errors (SE) for relative expression of PPARA and its target genes. Regression models were fit to evaluate associations of gene expression outcomes with treatment and genotype, including a treatment-genotype interaction term. The left hand column adjusts for sex. The two right columns stratify by sex, allowing results to differ between males and females. Statistical significance was evaluated at α = 0.05 for all analyses.

ALL MALE FEMALE Test β (SE) P value β (SE) P value β (SE) P value PPARA PFOA treatment 0.20 (34.91) 0.99 3.21 (45.13) 0.94 2.04 (50.01) 0.97 hPPARα Genotype 378.16 (31.96) <0.0001 314.37 (39.28) <0.0001 452.67 (48.19) <0.0001 Treatment * Genotype 9.96 (45.74) 0.82 48.64 (57.20) 0.40 -45.49 (68.15) 0.51 Male Sex -50.55 (22.67) 0.03 - - - - Acox PFOA treatment 0.69 (0.31) 0.03 0.67 (0.39) 0.09 0.67 (0.44) 0.14 hPPARα Genotype 0.28 (0.28) 0.32 0.28 (0.34) 0.42 0.33 (0.42) 0.45 Treatment*Genotype 3.07 (0.40) <0.0001 3.56 (0.50) <0.0001 2.46 (0.61) 0.0005 Male Sex 0.49 (0.20) 0.02 - - - - Adrp PFOA treatment 0.49 (0.37) 0.19 0.73 (0.51) 0.16 0.23 (0.53) 0.67 hPPARα Genotype 0.01 (0.34) 0.97 0.32 (0.44) 0.47 -0.29 (0.51) 0.57 Treatment*Genotype 2.34 (0.48) <0.0001 2.39 (0.64) 0.001 2.22 (0.73) 0.006 Male Sex 0.19 (0.24) 0.44 - - - - Mogat1 PFOA treatment 3.38 (8.87) 0.70 4.67 (13.76) 0.74 1.01 (3.73) 0.79 hPPARα Genotype -3.31 (7.55) 0.66 0.08 (10.71) 0.99 0.33 (3.59) 0.93 Treatment*Genotype 66.47 (10.71) <0.0001 78.59 (15.70) <0.0001 34.39 (5.09) <0.0001 Male Sex 15.73 (5.34) 0.004 - - - - Vnn1 PFOA treatment 0.38 (0.55) 0.49 0.27 (0.56) 0.63 0.46 (0.97) 0.64 hPPARα Genotype 0.12 (0.51) 0.81 0.03 (0.49) 0.95 0.26 (0.94) 0.78 Treatment*Genotype 6.20 (0.73) <0.0001 6.86 (0.71) <0.0001 5.42 (1.33) 0.0005 Male Sex 0.18 (0.36) 0.61 - - - - Pdk4 PFOA treatment -3.31 (1.99) 0.10 -0.88 (3.23) 0.79 -5.71 (2.21) 0.02 hPPARα Genotype -2.84 (1.83) 0.13 -0.70 (2.81) 0.81 -4.96 (2.14) 0.03 Treatment*Genotype 12.64 (2.64) <0.0001 11.62 (4.09) 0.009 12.91 (3.08) 0.0004 Male Sex -1.03 (1.31) 0.44 - - - -

Table 3: Effect estimates (β) and standard errors (SE) for relative expression of Nr1c3 (PPARγ) and Nr1i3 (CAR) and their target genes. Regression models were fit to evaluate associations of gene expression outcomes with treatment and genotype, including a treatment-genotype interaction term. The left hand column adjusts for sex. The two right columns stratify by sex, allowing results to differ between males and females. Statistical significance was evaluated at α = 0.05 for all analyses.

ALL MALE FEMALE Test β (SE) P value β (SE) P value β (SE) P value Nr1c3 PFOA treatment 3.09 (0.64) <0.0001 3.00 (0.98) 0.005 3.16 (0.82) 0.0009 hPPARα Genotype -0.89 (0.57) 0.13 -0.93 (0.85) 0.29 -0.79 (0.75) 0.31 Treatment*Genotype 2.55 (0.83) 0.003 3.26 (1.24) 0.01 1.67 (1.09) 0.14 Male Sex -0.12 (0.41) 0.77 - - - - Fabp4 PFOA treatment 0.39 (0.24) 0.11 0.28 (0.31) 0.38 0.48 (0.38) 0.22 hPPARα Genotype 0.54 (0.22) 0.02 0.57 (0.27) 0.047 0.49 (0.36) 0.19 Treatment*Genotype 0.55 (0.31) 0.09 0.68 (0.39) 0.09 0.42 (0.52) 0.42 Male Sex 0.08 (0.16) 0.59 - - - - Cd36 PFOA treatment 1.50 (1.12) 0.19 1.46 (1.62) 0.38 1.55 (1.65) 0.36 hPPARα Genotype 0.76 (1.00) 0.45 0.64 (1.41) 0.65 0.91 (1.51) 0.55 Treatment*Genotype 11.55 (1.45) <0.0001 11.99 (2.05) <0.0001 10.99 (2.19) <0.0001 Male Sex -0.11 (0.72) 0.88 - - - - Nr1i3 PFOA treatment -0.19 (0.14) 0.20 -0.28 (0.18) 0.13 -0.09 (0.21) 0.66 hPPARα Genotype -0.07 (0.13) 0.60 -0.25 (0.16) 0.12 0.15 (0.21) 0.48 Treatment * Genotype 0.29 (0.19) 0.13 0.62 (0.23) 0.01 -0.11 (0.29) 0.71 Male Sex -0.14 (0.09) 0.15 - - - - Cyp2b10 PFOA treatment 54.00 (3.52) <0.0001 43.51 (4.77) <0.0001 63.09 (4.25) <0.0001 hPPARα Genotype 0.35 (3.23) 0.91 0.05 (4.15) 0.99 0.16 (4.09) 0.97 Treatment*Genotype -27.13 (4.52) <0.0001 -16.83 (6.04) 0.01 -35.97 (5.79) <0.001 Male Sex -4.09 (2.29) 0.08 - - - - Gstm3 PFOA treatment 33.66 (3.67) <0.0001 42.39 (5.94) <0.0001 25.98 (3.74) <0.0001 hPPARα Genotype 0.27 (3.35) 0.93 0.86 (5.17) 0.87 0.21 (3.60) 0.95 Treatment* Genotype -22.12 (4.79) <0.0001 -29.29 (7.53) 0.0007 -16.46 (5.09) 0.004 Male Sex 4.84 (2.38) 0.046 - - - -

Table 4: Effect estimates (β) and standard errors (SE) for relative expression of genes contributing cholesterol homeostasis Regression models were fit to evaluate associations of gene expression outcomes with treatment and genotype, including a treatment-genotype interaction term. The left hand column adjusts for sex. The two right columns stratify by sex, allowing results to differ between males and females. Statistical significance was evaluated at α = 0.05 for all analyses.

ALL MALE FEMALE Test β (SE) P value β (SE) P value β (SE) P value Hmgcr PFOA treatment 0.04 (0.10) 0.68 0.11 (0.16) 0.50 -0.01 (0.14) 0.93 hPPARα Genotype 0.21 (0.10) 0.04 0.17 (0.14) 0.21 0.26 (0.14) 0.08 Treatment*Genotype -0.32 (0.14) 0.03 -0.29 (0.20) 0.16 -0.38 (0.20) 0.07 Male Sex -0.03 (0.07) 0.61 - - - - Apob PFOA treatment 0.20 (0.16) 0.19 -0.04 (0.20) 0.84 0.42 (0.24) 0.09 hPPARα Genotype 0.13 (0.15) 0.40 0.14 (0.18) 0.44 0.08 (0.24) 0.73 Treatment*Genotype -0.08 (0.21) 0.70 0.07 (0.26) 0.78 -0.18 (0.33) 0.60 Male Sex 0.10 (0.10) 0.34 - - - - Ldlr PFOA treatment -0.11 (0.05) 0.03 -0.03 (0.08) 0.73 -0.19 (0.05) 0.0004 hPPARα Genotype -0.04 (0.04) 0.31 0.005 (0.07) 0.95 -0.09 (0.04) 0.047 Treatment*Genotype 0.03 (0.06) 0.64 -0.005 (0.10) 0.96 0.05 (0.06) 0.46 Male Sex -0.02 (0.03) 0.49 - - - - Cyp7a1 PFOA treatment -0.63 (0.20) 0.003 -0.55 (0.31) 0.09 -0.68 (0.21) 0.004 hPPARα Genotype 0.69 (0.19) 0.0005 0.34 (0.28) 0.23 1.08 (0.20) <0.0001 Treatment*Genotype -0.31 (0.27) 0.26 0.02 (0.40) 0.96 -0.76 (0.29) 0.02 Male Sex -0.30 (0.13) 0.03 - - - -

Figure Legends

Fig. 1. Weight gain in PFOA-exposed mice.

Three-week-old male and female hPPARα and PPARα null mice were treated with either vehicle

(Vh, NERL water with 5% sucrose) or PFOA (8 µM in NERL water with 5% sucrose) as

drinking water for 6 weeks. During treatment, the mice were fed an American Diet (see Table

S1). a, c Body weight (reported as percent increase from initial weight measured at weaning)

was measured weekly. Data are presented as mean ± SE. N = 5-9. b, d Body composition was

measured by EchoMRI. Data are from individual mice, with the mean indicated by a line. N = 5-

Fig. 2. Liver/body weight in PFOA-exposed mice.

hPPARα and PPARα null mice were exposed to Vh or PFOA in their drinking water for 6

weeks, as described in Fig. 1. a, c Liver to body weight ratios were determined at euthanasia. b,

d H&E staining of representative liver sections. Data are from individual mice, with the mean

indicated by a line. N = 5-9. Significantly different from Vh (**** p<0.0001, t-test).

Fig. 3. PPARα-target gene expression in liver of PFOA-exposed mice.

hPPARα and PPARα null mice were exposed to Vh or PFOA in their drinking water for 6

weeks, as described in Fig. 1. Following isolation of RNA from liver, gene expression was

determined by RT-qPCR. Data are from individual mice, with the mean indicated by a line. N =

5-9. Significantly different from Vh (* p<0.05, ** p<0.01, **** p<0.0001, t-test).

Fig. 4. Alternative nuclear receptor-target gene expression in liver of PFOA-exposed mice.

hPPARα and PPARα null mice were exposed to Vh or PFOA in their drinking water for 6

weeks, as described in Fig. 1. Following isolation of RNA from liver, gene expression was

determined by RT-qPCR. a PPARγ (Nr1c3) and its target genes. b CAR (Nr1i3) and its target

genes. Data are from individual mice, with the mean indicated by a line. N = 5-9. Significantly

different from Vh (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, t-test).

Fig. 5. Cholesterol homeostasis-related gene expression in liver of PFOA-exposed mice.

hPPARα and PPARα null mice were exposed to Vh or PFOA in their drinking water for 6

weeks, as described in Fig. 1. Following isolation of RNA from liver, gene expression was

determined by RT-qPCR. The hypothetical model indicates biomarker genes for each of the

pathways. a Cholesterol synthesis. b Cholesterol export. c Cholesterol import. d Cholesterol

efflux. Data are from individual mice, with the mean indicated by a line. N = 5-9. Significantly

different from Vh (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, t-test).

Fig. 6. Expression of transcription factors that regulate cholesterol homeostasis in liver of

PFOA-exposed mice.

hPPARα and PPARα null mice were exposed to Vh or PFOA in their drinking water for 6

weeks, as described in Fig. 1. Following isolation of RNA from liver, gene expression was

determined by RT-qPCR. Data are from individual mice, with the mean indicated by a line. N =

5-9. No significant differences were detected (t-test).

Figure 1 a Female b

Vh Female PFOA hPPARa PPARa null % Body Fat 200 200 40

150 150 30

100 100 20

50 50 10 % Body Weight % Increase in Body Weight % Increase in Body Weight 0 0 0 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 hPPARa Null Weeks of Treatment Weeks of Treatment c d Male Male hPPARa PPARa null % Body Fat 200 200 40

150 150 30

100 100 20

50 50 10 % Body Weight % Increase in Body Weight 0 % Increase in Body Weight 0 0 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 hPPARa Null Weeks of Treatment Weeks of Treatment bioRxiv preprint doi: https://doi.org/10.1101/2020.01.30.926642; this version posted January 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure 2

b Female a hPPARa PPARa null Female Vh PFOA

Liver/Body Weight 15 **** ****

5 % Body Weight

0 hPPARa Null

d c Male Male hPPARa PPARa null Vh PFOA

Liver/Body Weight 15 **** ****

5 % Body Weight

0 hPPARa Null bioRxiv preprint doi: https://doi.org/10.1101/2020.01.30.926642; this version posted January 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure 3

Female Vh PFOA PPARA Acox Adrp Mogat1 Vnn1 Pdk4 800 8 ** 8 ** 200 **** 15 *** 35 ** *

600 6 6 150 10 25

400 4 4 100 15 5 200 2 2 50

Relative Expression 5 0 0 0 0 0 hPPARa Null hPPARa Null hPPARa Null hPPARa Null hPPARa Null hPPARa Null Male PPARA Acox Adrp Mogat1 Vnn1 Pdk4 800 8 ** 8 **** 200 *** 15 **** * 35 **

600 6 6 150 10 25

400 4 4 100 15 5 200 2 2 50

Relative Expression 5 0 0 0 0 0 hPPARa Null hPPARa Null hPPARa Null hPPARa Null hPPARa Null hPPARa Null bioRxiv preprint doi: https://doi.org/10.1101/2020.01.30.926642; this version posted January 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure 4 Vh a PFOA Female Nr1c3 Fabp4 Cd36 15 **** ** 4 * 25 **** ** 20 3 10 15 2 10 5 1 5 Relative Expression

0 0 0 hPPARa Null hPPARa Null hPPARa Null

Male Nr1c3 Fabp4 Cd36 15 **** ** 4 ** 25 **** ** 20 3 10 15 2 10 5 1 5 Relative Expression 0 0 0 hPPARa Null hPPARa Null hPPARa Null b Female Nr1i3 Cyp2b10 Gstm3 2.5 100 **** **** 100 ** *** 2.0 75 80

1.5 60 50 1.0 40 25 0.5 20 Relative Expression 0 0.0 0 hPPARa Null hPPARa Null hPPARa Null

Male Nr1i3 Cyp2b10 Gstm3 2.5 * 100 **** **** 100 **** **

2.0 75 80

1.5 60 50 1.0 40 25 0.5 20 Relative Expression 0 0.0 0 hPPARa Null hPPARa Null hPPARa Null bioRxiv preprint doi: https://doi.org/10.1101/2020.01.30.926642; this version posted January 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure 5

a Vh b c d PFOA Hmgcr Apob Ldlr Cyp7a1 Female 2.0 * 2.5 0.8 ** ** 3 **** * 2.0 1.5 0.6 2 1.5 1.0 0.4 1.0 1 0.5 0.5 0.2 Relative Expression 0.0 0.0 0.0 0 hPPARa Null hPPARa Null hPPARa Null hPPARa Null Male Hmgcr Apob Ldlr Cyp7a1 2.0 2.5 0.8 3

2.0 1.5 0.6 2 1.5 1.0 0.4 1.0 1 0.5 0.2 0.5 Relative Expression

0.0 0.0 0.0 0 hPPARa Null hPPARa Null hPPARa Null hPPARa Null bioRxiv preprint doi: https://doi.org/10.1101/2020.01.30.926642; this version posted January 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure 6

Vh PFOA Female Cebpa Srebf1 Srebf2 Hnf4a 1.0 8 1.0 2.0

0.8 0.8 6 1.5 0.6 0.6 4 1.0 0.4 0.4 2 0.5 0.2 0.2 Relative Expression

0.0 0 0.0 0.0 hPPARa Null hPPARa Null hPPARa Null hPPARa Null

Male Cebpa Srebf1 Srebf2 Hnf4a 0.8 8 1.0 2.0

0.8 0.6 6 1.5 0.6 0.4 4 1.0 0.4 0.2 2 0.5 0.2 Relative Expression 0.0 0 0.0 0.0 hPPARa Null hPPARa Null hPPARa Null hPPARa Null