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. Author manuscript; available in PMC: 2025 Feb 1.
Published in final edited form as: Toxicology. 2024 Jan 3;502:153719. doi: 10.1016/j.tox.2023.153719

Distinct bile acid alterations in response to a single administration of PFOA and PFDA in mice

Xiaoxiao Yu a, Youcai Zhang b, Bruno Cogliati c, Curtis D Klaassen d, Sanaya Kumar a, Xingguo Cheng a, Pengli Bu a,*
PMCID: PMC10922993  NIHMSID: NIHMS1960149  PMID: 38181850

Abstract

Per- and polyfluoroalkyl substances (PFASs), a group of synthetic chemicals that were once widely used for industrial purposes and in consumer products, are widely found in the environment and in human blood due to their extraordinary resistance to degradation. Once inside the body, PFASs can activate nuclear receptors such as PPARα and CAR. The present study aimed to investigate the impact of perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA) on liver structure and functions, as well as bile acid homeostasis in mice. A single administration of 0.1 mmole/kg of PFDA, not PFOA, elevated serum ALT and bilirubin levels and caused cholestasis in WT mice. PFDA increased total and various bile acid species in serum but decreased them in the liver. Furthermore, in mouse livers, PFDA, not PFOA, down-regulated mRNA expression of uptake transporters (Ntcp, Oatp1a1, 1a4, 1b2, and 2b1) but induced efflux transporters (Bcrp, Mdr2, and Mrp2-4). In addition, PFDA, not PFOA, decreased Cyp7a1, 7b1, 8b1, and 27a1 mRNA expression in mouse livers with concomitant hepatic accumulation of cholesterol. In contrast, in PPARα-null mice, PFDA did not increase serum ALT, bilirubin, or total bile acids, but produced prominent hepatosteatosis; and the observed PFDA-induced expression changes of transporters and Cyps in WT mice were largely attenuated or abolished. In CAR-null mice, the observed PFDA-induced bile acid alterations in WT mice were mostly sustained. These results indicate that, at the dose employed, PFDA has more negative effects than PFOA on liver function. PPARα appears to play a major role in mediating most of PFDA-induced effects, which were absent or attenuated in PPARα-null mice. Lack of PPARα, however, exacerbated hepatic steatosis. Our findings indicate separated roles of PPARα in mediating the adaptive responses to PFDA: protective against hepatosteatosis but exacerbating cholestasis.

Keywords: PFASs, PFOA, PFDA, PPARα, bile acid homeostasis, hepatosteatosis

1.1. Introduction

Per- and polyfluoroalkyl substances (PFASs), such as perfluorooctanoic acid (PFOA, C8), perfluorononanoic acid (PFNA, C9) and perfluorodecanoic acid (PFDA, C10), and their respective salts, have both water and oil propellant properties, and been wildly used in various consumer products and industrial processes. Because of their extraordinary resistance to degradation, PFASs are universally detected in environmental media, as well as in humans and wildlife. As a result, exposure to PFASs poses a concern to the public health (Fenton et al. 2021). In recent years, PFASs, the so-called “forever chemicals” in the media, have attracted more and more public attention across the world. Multiple major events, including the release of the 2019 movie “Dark Waters”, the $10.3 Billion settlement in June 2023 over PFAS-contaminated water systems in the US, and the ongoing Dutch criminal investigation into pollution caused by PFAS, all pointed to a plausible link between PFAS exposure and public health. As a result, scientific evidence on PFAS toxicity and mechanistic studies are more than ever needed.

After absorption, PFASs accumulate primarily in the liver. Once inside the liver, PFASs can activate peroxisome proliferator-activated receptors (PPARs) and constitutive androstane receptor (CAR) (Cheng and Klaassen 2008a; Cheng and Klaassen 2008b; Maher et al. 2008). We previously reported that activation of PPARα or CAR alters the expression of transporters such as bile salt export pump (Bsep), multidrug-resistance-associated proteins (Mrps), sodium taurocholate cotransporting polypeptide (Ntcp), and organic anion-transporting polypeptides (Oatps) in mouse livers (Cheng et al. 2005; Maher et al. 2005). These transporters are involved in transporting xenobiotics as well as endogenous metabolites, including bile acids and bilirubin. In addition, PFASs are substrates of the Oatp and Ntcp transporters and can also modulate their activities (Ruggiero et al. 2021; Weaver et al. 2010; Zhao et al. 2015; Zhao et al. 2017).

Enterohepatic circulation of bile acids is not only instrumental for solubilization and absorption of dietary lipids, fat-soluble vitamins, and hydrophobic drugs in the gastrointestinal tract, but also provides an elimination route, i.e., biliary excretion, for endo- and xenobiotics (Li and Chiang 2014). For example, conjugated bilirubin and porphyrins from heme catabolism, excessive cholesterol, and steroid hormones and their metabolites, are all eliminated via the biliary excretion (Boyer 2013). During cholestasis, which is when bile flow slows or stalls, metabolites that are normally excreted via the bile are returned to the blood leading to elevated serum levels of conjugated bilirubin and bile acids (Pieters et al. 2021). Bile acid homeostasis includes the active generation of bile flow by hepatocytes and cholangiocytes, and the active reabsorption of bile acids in the ileum, both of which rely on coordinated transporter activities (Boyer 2013). Consequently, alteration of these transporters interrupts bile acid homeostasis and biliary excretion of endo- and xenobiotics.

Certain PFASs may interfere with bile acid homeostasis and its own flux into and out of the liver via altering a panel of transporters through two possible mechanisms: (1) by directly modulating the transporter activities, and (2) via activating the transcription factors PPARα and/or CAR to alter transporter expression (Choudhuri and Klaassen 2021). With a specific focus on parameters of bile acid homeostasis, the present study compares the effect of a single administration of 0.1 mmole/kg of PFOA and PFDA on liver histology and physiology in mice.

2.1. Materials and Methods

Materials.

Sodium bicarbonate, sodium dodecyl sulfate, sodium chloride, 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES), HEPES sodium salt, lithium lauryl sulfate, ethylenediaminetetraacetic acid, and phenylmethylsulphonyl fluoride (PMSF) were purchased from Sigma-Aldrich (St. Louis, MO). Formaldehyde, 4-morpholinepropanesulfonic acid, and sodium citrate were purchased from Fischer Scientific (Fairlawn, NJ). Chloroform, agarose, and ethidium bromide were purchased from AMRESCO Inc. (Solon, OH). PFOA (free acid form; 96% purity; M.W. 413 g/mol) and PFDA (free acid form; 98% purity; M.W. 513 g/mol) were both purchased from Sigma-Aldrich Co. (St Louis, MO). All other chemicals, unless otherwise indicated, were purchased from Sigma-Aldrich Co. (St. Louis, MO).

Animals and Treatment.

Eight-week-old adult male C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME), and housed according to the American Animal Association Laboratory Animal Care guidance. The PPARα-null mice originally engineered in the laboratory of Dr. Frank J. Gonzalez (Lee et al. 1995), were back-crossed to a C57BL/6 background (Akiyama et al. 2001), and kindly provided by Dr. Jeffrey M. Peters (Pennsylvania State University, University Park, PA). Breeding pairs of CAR-null mice in the C57BL/6 background were obtained from Dr. Ivan Rusyn (University of North Carolina, Chapel Hill, NC), which were engineered by Tularik Inc. (South San Francisco, CA) as described previously (Ueda et al. 2002).

Adult male C57BL/6 mice, PPARα-null mice, and CAR-null mice (n = 5/genotype/treatment), at approximately 8 weeks of age, were administered a single i.p. dose of either PFOA or PFDA at 0.1 mmole/kg of body weight, whereas control mice were treated i.p. with propylene glycol: water (1:1, v/v). Two weeks later, livers were collected and snap-frozen in liquid nitrogen and stored at −80°C, and one piece of liver from each mouse was fixed in 10% Zinc formalin for histological examination. Blood was collected by cardiac puncture, allowed to coagulate, and centrifuged at 10,000 x g for 15 min. The resulting supernatant (serum) was collected for analysis.

For the longer-term studies (2, 3, and 4 months), adult male C57BL/6 mice (n = 5/treatment), at 8 weeks of age, were administered a single i.p. dose of PFOA or PFDA at 0.1 mmole/kg of body weight, and the control mice were treated with the propylene glycol: water (1:1, vol/vol). Two, three, and four months later, liver samples were collected and processed.

The animal studies were conducted at the University of Kansas Medical Center (Kansas City, KS). Collected tissue and serum samples were transferred to St. John’s University for further processing and analysis. The animal study protocols were approved by the IACUC of the University of Kansas Medical Center. All animal experiments were carried out in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. Reporting of animal experiments is in accordance with the ARRIVE guidelines.

Histopathology.

Freshly collected liver tissues, which were fixed in 10% zinc formalin, underwent routine processing and paraffin embedding. Liver sections (5 μm in thickness) were stained with hematoxylin and eosin and evaluated for hepatocellular alteration.

Quantification of conjugated bilirubin concentration and ALT activity in mouse serum.

Mouse serum was collected and analyzed for alanine aminotransferase (ALT) activity using Liquid ALT Reagent Set (Pointe Scientific Inc, Canton, MI) according to the manufacturer’s instructions. Serum conjugated bilirubin concentrations were determined using the Direct Bilirubin Reagent Set Kit (Pointe Scientific, Inc.) according to the manufacturer's protocol.

Quantification of Individual Bile Acids in Mouse Serum and Livers.

Total bile acids were extracted from mouse serum and liver, as previously described (Alnouti et al. 2008). Individual bile acids, including cholic acid (CA), alpha-muricholic acid (α-MCA), β-MCA, chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), and deoxycholic acid (DCA) and their glycine- and taurine-conjugates were quantified by a UPLC-MS/MS method (Alnouti et al. 2008).

Total RNA Isolation.

Total RNAs were extracted using the TRIzol reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. RNA pellets were resuspended in diethyl pyrocarbonate (DEPC)-treated deionized water. Total RNA concentrations were quantified spectrophotometrically at 260 nm. Only RNA samples with an A260/A280 ratio between 1.8 and 2.0 were used for further analysis.

Real-time quantitative PCR (RT-qPCR) assay.

Total RNA was reverse transcribed into cDNA using Superscript II or IV reverse transcriptase (Life Technologies, Carlsbad, CA) following the manufacturer’s instructions. Quantitative PCR (qPCR) was performed using SYBR Select Master Mix (Life Technologies, Carlsbad, CA) in an AriaMx Real-Time qPCR system (Agilent Technologies, Santa Clara, CA). Data were calculated according to the comparative delta-delta CT method and presented as a relative fold of the control. Primers used in quantitative real-time qPCR were designed with Primer3 software (version 4), and synthesized by Integrated DNA Technologies (Coralville, Iowa) as listed in our previous paper (Zhang et al. 2018b). A total of six housekeeping genes were included (Gapdh, 18s, β-actin, Hprt1, Ubc, Tbp) with RT-qPCR assay (raw data are included in supplementary data). Gapdh was selected as the most representative housekeeping gene and used in the calculation of relative fold change.

Quantification of free and total cholesterol in mouse serum and livers.

Liver lipid content, including cholesterol, was extracted as previously described (Tanaka et al. 2008). Serum and liver lipid samples were analyzed spectrophotometrically for free and total cholesterol (505 nm) in accordance with the manufacturer's protocols (Wako Chemicals USA, Inc, Richmond, VA).

Statistical Analysis.

Data are expressed as Mean ± S.E.M. Data were analyzed by one-way analysis of variance, followed by Tukey's post hoc test using GraphPad Prism statistics software. Statistical significance was considered when p < 0.05.

3.1. Results

PFDA caused more severe liver injury than PFOA in mice.

Two weeks after a single i.p. administration, both PFOA and PFDA caused hepatocytes around the central vein to become enlarged, with concomitant denser eosin staining of the cytoplasm (Fig. 1A, left column). PFDA caused a more prominent change than PFOA in WT and PPARα-null mice. The hepatocytes of PFDA-treated PPARα-null mice were markedly swollen with apparent and prevalent microvesicular steatosis. Such hepatic changes were particularly more noticeable around the central vein (Fig. 1A, middle column). PFDA did not cause apparent hepatic steatosis in WT mice (Fig. 1A, left column) or in CAR-null mice (Fig. 1A, right column), but caused marked and prevalent hepatic steatosis in PPARα-null mice (Fig. 1A, middle column). Overall, PFDA caused more conspicuous histological changes than PFOA in the livers of WT, PPARα-null, and CAR-null mice. The absence of PPARα, as reflected in the PPARα-null mice, exacerbated PFDA-induced hepatic steatosis two weeks after exposure.

Fig. 1. Histological alterations of mouse livers following a single administration of PFOA and PFDA in WT, PPARα-null, and CAR-null mice.

Fig. 1.

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A, Adult male C57BL/6J WT, PPARα-null (PPARα−/−), and CAR-null (CAR−/−) mice were given a single i.p. administration of 0.1 mmole/kg PFOA, PFDA, or vehicle (n=5/treatment/genotype). Representative images of liver histology two weeks after treatment with hematoxylin and eosin staining are shown in 200 X magnification. B, Adult male C57BL/6J WT mice were given a single i.p. administration of 0.1 mmole/kg PFOA, PFDA, or vehicle. Representative liver histological images with hematoxylin and eosin staining at two, three, and four months post-treatment. Images are shown in 200X magnification. Yellow arrows indicate cytoplasmic vacuoles; blue arrows indicate bile plugs.

A single dose of PFOA and PFDA caused a long-term impact on the WT mouse liver.

Two months after a single i.p. administration of 0.1 mmole/kg of PFOA, mild hepatocyte enlargement was observed in WT mice (Fig. 1B, left column, middle image). In contrast, a single i.p. administration of 0.1 mmole/kg of PFDA caused much more prominent changes in hepatocytes with some cytoplasmic vacuoles (Fig. 1B, left column, bottom image, yellow arrows). Three months after a single treatment, bile plugs (a characteristic of cholestasis) were observed in the livers of WT mice treated with PFDA but not PFOA (Fig. 1B, middle column, bottom image, blue arrows). No bile plugs were observed in PFOA- or PFDA-treated PPARα-null or CAR-null mice (data not shown). Four months post-treatment, liver histology became progressively worse in both PFOA- and PFDA-treated WT mice, with hepatic nodularity becoming vague, and hepatocytes around the central vein becoming enlarged with abundant eosinophilic granules (Fig. 1B, right column, middle and bottom images). The hepatic vacuoles remained in PFDA-treated WT livers (Fig. 1B, right column, bottom images, yellow arrows). Overall, PFDA appeared to negatively affect the liver to a greater degree than PFOA, both short-term and long-term, in the three genotypes of mice examined.

PFOA and PFDA distinctly affected serum alanine aminotransaminase (ALT) and bilirubin concentrations.

Increased serum levels of ALT and bilirubin usually indicate liver injury. PFDA produced a marked increase in serum ALT levels, with the greatest increase in WT mice, less increase in CAR-null mice, and the least increase in PPARα-null mice (Fig. 2A). In contrast, PFOA did not increase serum ALT in either WT or PPARα-null mice and moderately increased ALT in CAR-null mice (Fig. 2A, right group). Therefore, although very similar in structure, PFDA, but not PFOA, caused these changes in WT mice. The PFDA-induced ALT elevation was dependent on both PPARα and CAR, as in either null mouse model, PFDA increased ALT to a lesser degree compared to WT mice.

Fig. 2. PFOA and PFDA distinctly affected serum ALT and bilirubin levels in mice.

Fig. 2.

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Serum concentrations of ALT (A) and conjugated bilirubin (B) in adult male C57BL/6J WT, PPARα-null (PPARα−/−), and CAR-null (CAR−/−) mice two weeks after a single administration of 0.1 mmole/kg PFOA, PFDA, or vehicle. Data are presented as mean ± SEM. Asterisk (*) represents a statistically significant difference (p<0.05) between the specified treatment and control of the same genotype. Dagger (†) represents a statistically significant difference (p<0.05) of the same treatment between the indicated null and WT mice.

A similar pattern was seen with serum levels of conjugated bilirubin (Fig. 2B). PFDA increased serum bilirubin in WT mice, but PFOA did not. PFDA-induced increase in serum bilirubin level was partially reduced in the CAR-null mice but completely abolished in the PPARα-null mice. These results suggest that both PPARα and CAR contribute to PFDA-induced increase in serum bilirubin, and that PPARα plays a predominant role in this effect (Fig. 2B). Taken together, the attenuated induction of ALT (Fig. 2A, middle group) and the lack of increase of bilirubin (Fig. 2B, middle group) in PPARα-null mice suggest that PPARα is mediating PFDA-induced hepatic injuries.

PFDA, not PFOA, increased serum bile acid levels.

Histological analysis showed that a single injection of PFDA, not PFOA, resulted in cholestasis, as indicated by bile plugs three months later (Fig. 1B, middle column). In addition to bile plugs, cholestasis is typically characterized by elevated serum ALT, bilirubin, and bile acids. As expected, PFDA markedly increased serum total bile acids (25-fold), unconjugated bile acid, and glycine- and taurine-conjugated bile acids (2-fold and 53-fold, respectively) in WT mice two weeks after exposure (Fig. 3A). In contrast, PFOA did not increase serum bile acids in WT mice (Fig. 3A). The PFDA-induced increase in serum bile acids was attenuated or abolished in PPARα-null mice but remained in CAR-null mice (Fig. 3A). This indicates that PPARα, not CAR, was primarily responsible for PFDA-induced bile acid elevation in serum. For individual bile acids, including both conjugated and unconjugated forms, PFOA and PFDA had opposite effects. PFOA decreased serum levels of CDCA, CA, MCA, and UDCA in WT mice (Fig. 3B). In contrast, PFDA increased serum levels of CDCA, CA, MCA, and UDCA in WT mice (Fig. 3B). In PPARα-null mice, the alterations of serum concentrations of individual BAs produced by either PFOA or PFDA in the WT mice were either abolished or reversed (Fig. 3B). In CAR-null mice, the effects of both PFOA and PFDA on serum levels of individual bile acids was similar to those observed in WT mice (Fig. 3B).

Fig. 3. PFDA and PFOA differentially altered serum bile acid levels in mice.

Fig. 3.

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Adult male C57BL/6J WT, PPARα-null (PPARα−/−), and CAR-null (CAR−/−) mice received a single i.p. administration of 0.1 mmole/kg PFOA, PFDA, or vehicle (n=5/treatment/genotype). Two weeks later sera were collected and processed for quantification of total (A) and individual (B) bile acids. Concentrations of various bile acids in mouse serum are presented as mean ± SEM. Asterisk (*) represents a statistically significant difference (p<0.05) between the specified treatment and control of the same genotype.

These findings suggest that (1) PFOA and PFDA, although very similar in structure, have distinct effects on serum bile acid homeostasis, and (2) PPARα, not CAR, mediated PFAS-induced increase in serum bile acid concentrations.

Both PFOA and PFDA caused an overall decrease in bile acids in mouse livers.

Both PFOA and PFDA decreased total bile acids, unconjugated and glycine-conjugated bile acids in the livers of WT mice (Fig. 4A). PFOA, not PFDA, decreased taurine-conjugated bile acids in mouse livers (Fig. 4A). Similar to what was observed with serum bile acids, the decreases in hepatic bile acids produced by the PFASs in WT mice was almost completely abolished in the PPARα-null mice, but remained, for the most part, in the CAR-null mice (Fig. 4A). For individual bile acids, both PFOA and PFDA decreased CA, DCA, MCA, and UDCA in the livers of WT mice and CAR-null mice, but not in PPARα-null mice (Fig. 4B). Therefore, PPARα, but not CAR, played a pivotal role in mediating PFAS-induced bile acid alterations in both serum and liver in mice.

Fig. 4. PFDA and PFOA differentially alter bile acid levels in mouse liver.

Fig. 4.

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Adult male C57BL/6J WT, PPARα-null (PPARα−/−), and CAR-null (CAR−/−) mice received a single i.p. administration of 0.1 mmole/kg PFOA, PFDA, or vehicle (n=5/treatment/genotype). Two weeks later, mouse livers were collected and processed for quantification of total (A) and individual (B) bile acids. Bile acids in mouse livers are presented as mean ± SEM. Asterisk (*) represents a statistically significant difference (p<0.05) between the specified treatment and control of the same genotype. Dagger (†) represents a statistically significant difference (p<0.05) of the same treatment between the indicated null and WT mice.

In addition, lack of PPARα, as reflected in PPARα-null mice, decreased concentrations of liver total bile acids, unconjugated bile acids, taurine-conjugated bile acids, CDCA, CA, MCA, and UDCA (Fig. 4). In contrast, lack of CAR, as reflected in the CAR-null mice, decreased liver total bile acids, taurine-conjugated bile acids, CDCA, CA, MCA, and UDCA.

PFOA and PFDA altered transporter expression in mouse livers primarily in a PPARα-dependent manner.

Hepatic transporters mediate the uptake and efflux of endobiotics (such as bile acids and bilirubin) (Klaassen and Aleksunes 2010) and xenobiotics (such as PFASs) (Ruggiero et al. 2021; Weaver et al. 2010; Zhao et al. 2015; Zhao et al. 2017) into and out of the liver. Because PFDA elevated bile acids (Fig. 3) and bilirubin in serum (Fig. 2B) of WT mice, the impact of PFAS on the expression of hepatic transporters was also examined. Uptake transporters (including Ntcp, Oatp1a1, Oatp1a4, Oatp1b2, and Oatp2b1), which are localized on the sinusoidal side of hepatocytes, are responsible for the hepatic uptake of various substrates (bile acids, bilirubin, and certain PFASs) from the portal blood (Klaassen and Aleksunes 2010). PFOA did not affect Ntcp or Oatp1a1, but reduced Oatp1a4, and increased Oatp1b2 and Oatp2b1 mRNA expression (Fig. 5A, PFOA treatment). In contrast, PFDA decreased the mRNA expression of all uptake transporters examined (Ntcp, Oatp1a1, Oatp1a4, Oatp1b2, and Oatp2b1) in WT mouse liver (Fig. 5A, PFDA treatment). It has been previously reported that PFOA, PFNA, and PFDA are substrates for Ntcp (Ruggiero et al. 2021) as well as Oatp1a1, 1b2, and 2b1 (Weaver et al. 2010; Zhao et al. 2017). Therefore, these data suggest that PFDA downregulates the expression of uptake transporters in mouse livers, which might in turn reduce its own hepatic uptake and accumulation.

Fig. 5. PFAS altered transporter expression in mouse liver.

Fig. 5.

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Messenger RNA expression of uptake (A) and efflux (B) transporters in adult male C57BL/6J WT, PPARα-null (PPARα−/−), and CAR-null (CAR−/−) mice two weeks after a single i.p. administration of 0.1 mmole/kg PFOA, PFDA, or vehicle (n=5/treatment/genotype). Data are presented as relative folds of the same transporter expression level in control WT mice. Asterisk (*) indicates a statistically significant difference (p<0.05) between the specified treatment and control of the same genotype.

PFOA decreased Ntcp, Oatp1a1, 1a4, and 1b2 mRNA expression in both PPARα-null and CAR-null mouse livers (Fig. 5A). PFOA decreased Oatp1b2 mRNA in PPARα-null but not in CAR-null mouse livers. In contrast, PFOA decreased Oatp2b1 mRNA in CAR-null but not in PPARα-null mouse livers. PFDA decreased Ntcp, Oatp1a1, 1a4, 1b2, and 2b1 mRNA expression in WT and CAR-null mouse livers but affected PPARα-null mouse livers to a much lesser degree (Fig. 5A).

Once inside hepatocytes, chemicals, including bile acids, are exported via efflux transporters on the canalicular membrane (such as Bsep, Mrp2, Bcrp, and Mate1) into the bile, or via efflux transporters on the sinusoidal side (such as Ostα/β, Mrp3, and Mrp4) back into blood. In addition, sterols and phospholipids are exported from the canalicular side by Abcg5/8 and Mdr2, respectively, into bile (Arab et al. 2017; Zollner and Trauner 2008). PFOA and PFDA induced Abcg8, Bcrp, Mdr2, and Mrp2-4 mRNA expression, with PFDA inducing Mrp4 over 75-fold (Fig. 5B), providing another mechanism (i.e., active efflux) for the elevated serum levels of bile acids in addition to reduced uptake by Ntcp and Oatps (Fig. 5A). Furthermore, PFOA and PFDA decreased Bsep mRNA expression, by 25% and 90%, respectively. Most of the PFAS-induced alterations in WT liver were either abolished or reduced in PPRAα-null mouse livers but remained in CAR-null mouse livers (Fig. 5).

PFDA markedly decreased the expression of Cyp enzymes that are required for BA synthesis in mouse liver.

Bile acid homeostasis is achieved not only via transporter-driven enterohepatic circulation but also through de novo bile acid synthesis from cholesterol in the liver (Li and Dawson 2019). Cyp7a1, 7b1, 8b1, and 27a1 play important roles in bile acid biosynthesis, with Cyp7a1 and Cyp8b1 being required for the classic bile acid biosynthesis pathway. Amongst these Cyps, PFOA only mildly inhibited Cyp7b1 mRNA expression (Fig. 6A). In contrast, PFDA down-regulated the mRNA expression of Cyp7a1, 7b1, 8b1, and 27a1 (Fig. 6A), and similarly decreased the mRNA expression of Cyp7a1, 7b1, 8b1 and 27a1 in PPARα-null and CAR-null mouse liver, indicating a mechanism independent of PPARα or CAR.

Fig. 6. PFDA, not PFOA, markedly decreased BA synthesis in mouse liver.

Fig. 6.

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Adult male C57BL/6J WT, PPARα-null (PPARα−/−), and CAR-null (CAR−/−) mice received a single i.p. administration of PFOA, PFDA, or vehicle (n=5/treatment/genotype). Two weeks later, mouse livers and sera were collected and processed to determine the mRNA expression of liver Cyp enzymes that are required for bile acid biosynthesis (A), and free and total cholesterol concentrations in mouse liver (B) and serum (C). Gene expression data are presented as relative folds of the same Cyp gene expression level in the control WT mice. Asterisk (*) indicates a statistically significant difference (p<0.05) between the specified treatment and control of the same genotype.

Cholesterol is primarily detoxified in the liver via conversion to bile acids by serving as the substrate for bile acid biosynthesis (Li and Chiang 2014). Inhibition of bile acid synthesis in the liver could result in the accumulation of cholesterol. As expected, PFDA, not PFOA, caused higher free and total cholesterol levels in the liver (Fig. 6B), without affecting free or total cholesterol in the serum (Fig. 6C).

4.1. Discussion

In the present study, a single high dose of PFOA and PFDA was administered i.p. to laboratory mice to investigate their impact on bile acid homeostasis. Although regimens of high doses in a short period of exposure are commonly utilized in toxicological studies, it is worth noting that high-dose regimens may mimic earlier occupational exposure, residence exposure near PFAS production sites, or living in areas heavily contaminated by PFAS waste depot (as depicted in the movie Dark Waters), but is quite different from what the general population is exposed to, which is much lower concentrations but can last decades or lifetime (Fromme et al. 2009).

We show that a single dose of PFDA, not PFOA, caused a long-term impact on mouse liver, including cholestasis in WT mice and hepatic steatosis in PPARα-null mice. Upon being exposed to PFDA, WT mice manifested cholestasis, as evidenced by elevations in serum ALT, conjugated bilirubin, and bile acids, as well as associated transporter changes. However, the lack of PPARα, as reflected in the PPARα-null mice, protected the mice against cholestasis by preventing almost all of the above changes.

The elimination of PPARα in the PPARα-null mice reduced or decreased a number of the adverse effects of PFDA. In response to PFDA, PPARα mediated transporter changes led to reduced uptake and enhanced efflux of hepatic contents (i.e., conjugated bilirubin (Fig. 2B), serum bile acids (Fig. 3), and presumably PFDA) into either bile (via Bcrp, Mdr2, and Mrp2) or blood (via Mrp 3 and 4). However, lack of PPARα exacerbated hepatic steatosis, consistent with PPARα playing a critical role in lipid metabolism in the liver (Pawlak et al. 2015). A schematic diagram (Fig. 7) summarizes the different outcomes between WT and PPARα-null mice in response to PFDA.

Fig. 7.

Fig. 7.

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A schematic diagram summarizes the effects of PFDA on hepatic transporter expression and its correlation with the development of cholestasis in WT and PPARα null (PPARα−/−) mice. The increased susceptibility of PPARα null mice to PFDA-induced hepatosteatosis is also depicted.

In this study, while PFDA increased serum concentration of bile acids (Fig. 3), it decreased liver concentration of bile acids (Fig. 4). This is in line with clinical observations that elevated serum bile-acid concentrations are standard indicators of cholestasis, while liver bile-acid concentrations may vary depending on the specific etiology (Pieters et al. 2021).

Once PFASs are absorbed, they are resistant to metabolism and likely undergo continual enterohepatic circulation and remain in the body for a long time (Ruggiero et al. 2021; Weaver et al. 2010; Zhao et al. 2015; Zhao et al. 2017). The long elimination half-lives of PFASs are partially responsible for these long-term effects, such as cholestasis in WT mice and hepatosteatosis in PPARα-null mice.

We previously reported that PFNA alters the synthesis and transport of bile acids and cholesterol in a PPARα-dependent manner (Zhang et al. 2018b). In the present study, PFOA (C8) and PFDA (C10) were compared on how they affect bile acid homeostasis after a single i.p. administration. Although very similar in structure with only a two-carbon difference in their carbon chain, PFOA and PFDA exhibited different hepatotoxicities and distinct impacts on bile acid homeostasis. For example, PFDA caused cholestasis and mild hepatosteatosis, similar to PFNA (Zhang et al. 2018b), whereas PFOA did not. With regard to transporter expression, PFDA markedly downregulated the expression of most of the uptake transporters, including Oatp1a1, 1a4, 1b2, and 2b1 (Fig. 5A), whereas PFOA only mildly decreased Oatp1a4 and increased Oatp1b2 and 2b1 mRNA expression (Fig. 5A). Among Oatps, Oatp1b2 is important for uptake of unconjugated bile acids into liver (Csanaky et al. 2011). As a result, serum unconjugated bile acids were not changed in PFOA-treated mice but significantly elevated in PFDA-treated mice, because PFDA, not PFOA, decreases Oatp1b2 (Fig. 3, WT). Because PFOA is a substrate of the OATP/Oatp1b family of transporters (Zhao et al. 2017), one might speculate that the liver is attempting to detoxify PFOA by increasing its hepatic extraction and biliary excretion. Quantification of PFOA in liver and bile would provide direct evidence to support or disprove this hypothesis. These adaptive responses to PFOA may help to mitigate liver injury as reflected by only mild changes in liver histology (Fig. 1) and no change in serum ALT or bilirubin levels (Fig. 2) in WT mice.

PFDA affected the liver more severely than PFOA. According to biomonitoring studies, PFOA, PFNA, PFOS, and PFHxS are the four most abundant PFASs responsible for most human exposure (Costello et al. 2022). On the sinusoidal side, PFDA down-regulated uptake transporters while it induced efflux transporters (Fig. 5), resulting in less liver burden of conjugated bilirubin and bile acids (Figs. 2 and 3) and presumably PFDA. Another unique response to PFDA was the marked suppression of the expression of hepatic Cyp enzymes for bile acid de novo synthesis, which was not seen in PFOA treatment group (Fig. 6A). The reduced concentrations of bile acids in the livers of PFDA-treated mice (Fig. 4) was likely due to decreased uptake and production, while the lower concentrations of bile acids in the liver of PFOA-treated mice (Fig. 4) was more likely a result of enhanced biliary excretion, because most uptake and efflux transporters expressions were not decreased nor was bile acid synthesis inhibited (Fig. 6A).

Given the crucial role bile acid homeostasis plays in physiology, it is not surprising that multiple layers of regulation are in place to ensure its functioning, especially in the face of stressors. One important layer of regulation is conferred by nuclear receptors. Nuclear receptors are powerful regulators involved in many fundamental physiological processes, and, among them, the Farnesoid X receptor (FXR) has been well established as the sensor for bile acids (Claudel et al. 2005; Rizzo et al. 2005; Wang et al. 1999). A different, perhaps broader, perspective aiming to link bile acid homeostasis to detoxification allows one to consider potential contributions from the so-called “xenobiotic receptors” such as PPARα, CAR, and Pregnane X receptor (PXR), which are best known for their pivotal roles in sensing and handling of foreign substances (Kliewer et al. 1999; Timsit and Negishi 2007). The rationale to connect detoxification with bile acid homeostasis is supported by an emerging body of evidence: (1) biliary excretion is one major route of toxicant elimination and is also a component of bile acid homeostasis (Gregus and Klaassen 1987; Thomas et al. 2008); (2) activation of nuclear receptors FXR, PXR, and CAR regulates the expression of detoxifying enzymes and transporters (Cheng et al. 2007; Cheng and Klaassen 2006; Cheng and Klaassen 2008b; Guo et al. 2003; Klaassen 2002; Schuetz et al. 2001); (3) activation of the nuclear receptor PPARα suppresses bile acid synthesis by downregulating transcription of Cyp7a1, the rate-limiting enzyme in bile acid synthesis (Marrapodi and Chiang 2000; Post et al. 2001); (4) activation of PPARα increases biliary excretion and reduces bile acid concentration in the liver (Zhang et al. 2017; Zhang et al. 2018a); (5) additional evidence on how activation of PPARα regulates bile acid synthesis, conjugation, and transport (Li and Chiang 2009; Zhang et al. 2017; Zhang et al. 2018a). Despite the differences in liver toxicity elicited by PFOA and PFDA, the nuclear receptor PPARα, but not CAR, appears to be essential in mediating most adaptive responses. Furthermore, PPARα appears to play a dual role in the case of PFDA: lack of PPARα protected mice from cholestasis that was prominent in WT mice, but exacerbated hepatic steatosis (profound and prevalent in PPARα-null mice after two weeks, Fig. 1A), which, after four months, was only mild in WT mice (Fig. 1B).

Although there is known species difference between mouse and human PPARα in promoting hepatocyte proliferation, hepatomegaly, and liver tumorigenesis, the ability of PPARα to regulate lipid metabolism appears to be very similar (Foreman et al. 2021; Morimura et al. 2006; Yang et al. 2008). The transporters assessed in the current study are mouse-specific, their human counterparts also play important roles in handling endo- and xenobiotics in the liver (Hagenbuch and Meier 2004; Klaassen and Aleksunes 2010).

Supplementary Material

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Acknowledgements

The authors thank previous members of Dr. Klaassen’s laboratory for help with sample collection and processing, as well as members of Dr. Cheng’s laboratory for assisting with the execution of experiments, data analysis, and helpful discussion on the manuscript.

Funding

This work was supported by St. John’s University New Faculty Start-up Fund, Seed Grant, and Summer Support of Research Award (to P.B.), and a grant from the National Institutes of Health (R01 ES09649 to C.D.K).

Abbreviation:

Abcg5

ATP-binding cassette transporter g5

ALT

alanine aminotransferase

BA

bile acid

Bcrp

breast cancer resistance protein

Bsep

bile salt export pump

CA

cholic acid

CAR

constitutive androstane receptor

CDCA

chenodeoxycholic acid

Cyp

cytochrome p450 enzyme

DCA

deoxycholic acid

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

LCA

lithocholic acid

MCA

muricholic acid

Mate1

multidrug and toxin extrusion transporter

Mdr2

multidrug resistance transporter 2

Ntcp

sodium/taurocholate cotransporting polypeptide

Oat

organic anion transporter

Oatp

organic anion-transporting polypeptide

Oct

organic cation transporter

Ost

organic solute transporter

PFASs

Per- and polyfluoroalkyl substances

PFDA

perfluorodecanoic acid

PFHxS

perfluorohexanesulfonic acid

PFNA

perfluorononanoic acid

PFOA

perfluorooctanoic acid

PPARα

peroxisome proliferator-activated receptor alpha

UDCA

ursodeoxycholic acid

WT

wild type

Footnotes

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Conflicts of Interests

All authors declare that there is no conflict of interest.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Supplementary Materials

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NIHMS1960149-supplement-1.docx (33.1KB, docx)
2
NIHMS1960149-supplement-2.pdf (5.6MB, pdf)

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