Comparative Hepatotoxicity of Novel PFOA Alternatives

Mar 13, 2019 - To compare the hepatotoxicity of multiether PFECAs (e.g., PFO2HxA, PFO3OA, and PFO4DA) with PFOA, male mice were exposed to 0.4, 2, ...
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Ecotoxicology and Human Environmental Health

Comparative Hepatotoxicity of Novel PFOA Alternatives (Perfluoropolyether Carboxylic Acids) on Male Mice Hua Guo, Jinghua Wang, Jingzhi Yao, Sujie Sun, Nan Sheng, Xiaowen Zhang, Xuejiang Guo, Yong Guo, Yan Sun, and Jiayin Dai Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b00148 • Publication Date (Web): 13 Mar 2019 Downloaded from http://pubs.acs.org on March 17, 2019

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Comparative Hepatotoxicity of Novel PFOA Alternatives (Perfluoropolyether Carboxylic Acids) on Male Mice Hua Guo†, Jinghua Wang†, Jingzhi Yao†, Sujie Sun†, Nan Sheng†, Xiaowen Zhang† Xuejiang Guo,‡ Yong Guo,₤ Yan Sun,₤ and Jiayin Dai†a

†Key

Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology,

Chinese Academy of Sciences, Beijing 100101, China ₤Key

Laboratory of Organofluorine Chemistry, Shanghai Institute of Organic

Chemistry, Chinese Academy of Sciences, Shanghai 200032, China ‡State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing,

210029, China

*Correspondence author: Jiayin Dai, Institute of Zoology, Chinese Academy of Sciences,

Beijing

100101,

China.

Telephone:

+86-10-64807185.

[email protected] Competing financial interests: The authors declare no conflicts of interest.

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ABSTRACT

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As novel alternatives to perfluorooctanoic acid (PFOA), perfluoropolyether

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carboxylic acids (multi-ether PFECAs, CF3(OCF2)nCOO−, n = 2–4) have been detected

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in various environmental matrices; however, public information regarding their

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toxicities remains unavailable. To compare the hepatotoxicity of multi-ether PFECAs

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(e.g., PFO2HxA, PFO3OA, PFO4DA) with PFOA, male mice were exposed to 0.4, 2,

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or 10 mg/kg/d of each chemical for 28 d, respectively. Results demonstrated that

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PFO2HxA and PFO3OA exposure did not induce marked increases in relative liver

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weight, whereas 2 and 10 mg/kg/d of PFO4DA significantly increased relative liver

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weight. Furthermore, PFO2HxA and PFO3OA demonstrated almost no accumulation

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in the liver or serum, whereas PFO4DA was accumulated but with weaker potential

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than PFOA. Exposure to 10 mg/kg/d of PFO4DA led to 198 differentially expressed

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liver genes (56 down-regulated, 142 up-regulated), with bioinformatics analysis

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highlighting urea cycle disorder. Like PFOA, 10 mg/kg/d of PFO4DA decreased urea

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cycle-related enzyme protein levels (e.g., carbamoyl phosphate synthetase 1) and serum

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ammonia content in a dose-dependent manner. Both PFOA and PFO4DA treatment

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(highest concentration) caused a decrease in glutamate content and increase in both

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glutamine synthetase activity and aquaporin protein levels in the brain. Thus, we

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concluded that PFO4DA caused hepatotoxicity, as indicated by hepatomegaly and

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karyolysis, though to a lesser degree than PFOA, and induced urea cycle disorder,

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which may contribute to the observed toxic effects.

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INTRODUCTION

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Per- and polyfluoroalkyl substances (PFASs), a class of highly fluorinated

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aliphatic compounds, have been widely used in industrial products since the 1950s due

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to their specific physicochemical properties.1-4 PFASs (especially perfluorooctanoic

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acid (PFOA) and perfluorooctane sulfonate (PFOS)) have been detected in various

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environmental media as well as in wildlife and humans.1 Based on previous studies,

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PFOA shows environmental persistence, widespread presence, and considerable

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toxicity.5-7 In 2006, the 2010/2015 Perfluorooctanoate (PFOA) Stewardship Program

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was launched by the US Environmental Protection Agency (EPA) to eliminate the

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production and usage of PFOA.8 In 2015, a proposal was accepted by the Risk

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Assessment Committee of the European Chemicals Agency to restrict the production,

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usage, and transaction of PFOA.9

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In accordance with the above regulations, short-chain homologues and other

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PFOA alternatives with different functional groups have gained popularity due to their

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perceived

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perfluoropolyether carboxylic acids (PFECAs), such as ammonium 4,8-dioxa-3H-

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perfluorononanoate (ADONA), hexafluoropropylene oxide dimer acid (HFPO-DA,

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also known as GenX), and hexafluoropropylene oxide trimer acid (HFPO-TA),11-15

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have been subsequently detected in the environment. For example, following its

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introduction as an emulsifier in manufacturing, ADONA has been detected in the Alz

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River at a concentration range of 0.8–7.5 μg/L.16 HFPO-DA has also been detected in

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samples from the North Sea, Rhine River, Scheur River, and Xiaoqing River at much

environmental

friendliness.10

As

a

new

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to

PFOA,

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higher concentrations than PFOA.17 We also previously detected HFPO-TA in water

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and biological samples from Xiaoqing River at a concentration of 68.5 μg/L, much

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higher than that of other PFASs, except for PFOA.14 Furthermore, perfluoro-(3,5,7,9-

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tetraoxadecanoic) acid (PFO4DA), perfluoro-(3,5-dioxahexanoic) acid (PFO2HxA),

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and perfluoro-(3,5,7-trioxaoctanoic) acid (PFO3OA), which are new types of PFECAs,

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have been detected in the Cape Fear River.18 However, although these alternatives have

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been utilized in industry and many studies have reported their occurrence in the

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environment, their environmental fate and risk to health remains unclear.

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Compared to PFOA, PFECAs, which have an ether oxygen inserted between the

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perfluorinated carbon backbone, may be more hydrophilic during metabolism and thus

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more easily eliminated in organisms. The additional ether oxygen in the backbone of

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PFECAs may also result in differences in biological properties compared with PFOA,

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such as alterations in protein binding affinity and toxic effects and mechanisms. For

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example, ADONA is somewhat orally toxic in rats and induces moderate to severe eye

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irritation in rabbits.11 HFPO-DA is absorbed rapidly and eliminated exclusively in

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mouse and rat urine19 and the bioaccumulation potential and hepatotoxicity of HFPO-

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TA is much higher than that of PFOA.15 Furthermore, like PFOA, hepatotoxicity and

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lipid dysregulation appear to be the most important impairments induced by PFECAs.

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Specifically, due to the similar structure of PFECAs with fatty acids, lipid dysregulation

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can be induced by activation of the peroxisome proliferator-activated receptor α

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(PPARα) pathway.15, 20 Urea cycle disorders in general are another form of liver injury,

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which result in the accumulation of ammonia and other nitrogenous compounds.21 Of 4

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concern, the major sequelae of hyperammonemia are cerebral injuries, such as cerebral

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edema and metabolic disorders.21 However, despite the possible environmental and

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health issues related to PFOA alternatives, much remains unclear. In this study, we

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aimed to (1) compare the hepatotoxicity of PFOA with multi-ether PFECAs (e.g.,

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PFO2HxA, PFO3OA, PFO4DA) in mice; (2) investigate the effect of PFO4DA

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exposure on the liver protein profile using quantitative proteomics; and, (3) explore

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liver urea cycle dysfunction induced by PFECA exposure.

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MATERIALS AND METHODS

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Chemicals and animal treatment. The potassium salt of PFO2HxA, PFO3OA, and

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PFO4DA (> 97.0%) were obtained from Dr. Guo Yong (Shanghai Institute of Organic

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Chemistry, Chinese Academy of Sciences, China). The PFOA (ammonium salt, CAS:

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3825-26-1, > 98.0% purity) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

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The PFOA and PFECA stock solutions (1 g/L) were dissolved in Milli-Q water for all

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exposure experiments.

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We obtained 6−8-week-old male BALB/c mice from the Weitong Lihua

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Experimental Animal Center (Beijing, China). The mice were housed under stable

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conditions (12:12 h light:dark cycle, 23 ± 1 ℃, and 40%−60% relative humidity). After

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one week of adaptation, the mice were randomly divided into thirteen groups (n = 12

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per group) and dosed by oral gavage with PFOA, PFO2HxA, PFO3OA, or PFO4DA at

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different concentrations (0, 0.04, 2, and 10 mg/kg body weight, respectively) once a

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day for 28 d. The dosing volume of all compounds was 10 mL/kg body weight. The 5

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doses were chosen according to our previous study. After treatment, all animals were

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weighed, bled by orbital puncture, and sacrificed for sample collection (blood, liver,

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and brain) after a night of fasting. Blood was centrifuged for 15 min at 3 000 rpm after

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clotting at room temperature for 3 h. The samples were stored at −80 °C until further

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use.22 This experiment and all procedures were approved by the Ethics Committee of

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the Institute of Zoology, Chinese Academy of Sciences, China (Approval number:

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IOZ14048).

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PFAS content in liver and serum sample. The PFOA concentrations in liver and

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serum were analyzed using high-performance liquid chromatograph-tandem mass

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spectrometry (UPLC-MS/MS, Xevo TQ-S Waters, Milford, MA, USA). The multi-

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ether PFECAs were analyzed using UPLC-tandem mass spectrometry (UPLC-MS/MS,

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Exion LC-Triple Quad 5500, AB SCIEX, Framingham, MA, USA). Detailed

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information on this experiment can be found in our previous study.23 Details on the

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multi-ether PFECA measurements in liver and serum are provided in the Supporting

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Information.

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Serum and brain biochemical assay. The serum parameters were assayed using a

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HITAC7170A automatic analyzer (Hitachi, Japan). The levels of ammonia and urea

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nitrogen in serum were detected using a blood ammonia assay kit (Njjcbio, A086, China)

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and urea assay kit (Njjcbio, China), respectively. Glutamic acid levels in the brain were

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determined using a glutamic acid determination reagent kit (Cominbio, China) and

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glutamine synthetase (GS) activity was examined using a GS kit (Cominbio, China)

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according to the manufacturer’s recommendations. Protein quantification was 6

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performed using a bicinchoninic acid (BCA) assay, as described previously.24

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Liver protein preparation and isobaric tags for relative and absolute quantitation

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(iTRAQ) analysis of the PFO4DA group. Based on relative liver weight and serum

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parameters, we selected the 10 mg/kg/d PFO4DA group for iTRAQ analysis to explore

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the effect of PFO4DA on the global profile of the differentially expressed proteins

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(DEPs) in the liver. Total protein was extracted from six individual liver samples in the

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control and 10 mg PFO4DA/kg/d groups. The same amount of protein from two

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individual liver samples in each group was pooled randomly, resulting in three pooled

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samples in each group for iTRAQ analysis. The pooled protein samples were alkylated,

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quantified, enzymolyzed, labeled, and analyzed. The iTRAQ analysis details are

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described in our previous study.25

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The DEPs obtained from iTRAQ were defined as: unique peptide count of ≥ 1, p

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< 0.05, and fold-change ratio ≥ 1.2 or ≤ 0.83. To better understand the relationship

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between DEPs and epidemic disease, disease ontology analysis was performed using

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DisGeNET (http://www.disgenet.org)26 and verified using Disease Ontology

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(http://www.disease-ontology.org/).27-29 In addition, interactions between key urea

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cycle proteins and DEPs were obtained using String (https://string-db.org/).30 We used

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Cytoscape software to visualize the complex networks between key urea cycle proteins

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and other DEPs.31 The degrees of centrality of the key urea cycle proteins were

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calculated using CentiScaPe.32 The degree of centrality was defined as the number of

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edges connected to a node, and included Betweenness Centrality (BC), Closeness

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Centrality (CC), and Degree Centrality (DC). The higher the centrality degree of a node, 7

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the greater the impact of the node on the entire network.30

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Western blotting. Proteins isolated from the samples were prepared as per previous

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research.33 The urea cycle proteins and aquaporin were selected to compare the toxicity

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of PFOA and PFO4DA. GAPDH was used as an internal reference. Information on

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antibodies used for Western blotting are given in Table S1.

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Statistical analyses. All data analyses were performed using SPSS 23.0 software for

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Windows (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) and

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Duncan’s multiple range test were used to test significant differences between groups.

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Values (means ± SE) of p < 0.05 were considered statistically significant.

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RESULTS

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Effects of PFOA and multi-ether PFECAs on the liver

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Body weight, liver weight, and relative liver weight were unchanged after 28 d of

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exposure to PFO2HxA and PFO3OA (Table S2). Furthermore, no liver injury was

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observed according to histopathological section examination (Figure S2). Compared to

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the control group, the liver weights and relative liver weights increased significantly in

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both the 2 and 10 mg/kg/d PFO4DA groups (Table S3 and Figure 1B), whereas the

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body weights were unchanged. For PFOA, the liver and relative liver weights increased

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in all groups, whereas body weights decreased but only in the highest group (10

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mg/kg/d) (Figure S1D). The increase in relative liver weight in the 10 mg/kg/d

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PFO4DA group was almost the same as that of the 0.4 mg/kg/d PFOA group.

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In addition to the increase in liver and relative liver weights, pathological changes, 8

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including hepatocellular hypertrophy, were found in liver sections from all PFO4DA

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and PFOA exposure groups (Figure S2). Furthermore, total nucleus number per unit

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area was significantly decreased in all PFO4DA and PFOA exposure groups. Compared

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to PFO4DA at the same dose (2 mg/kg/d), the hepatocellular hypertrophy induced by

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PFOA was more severe at the 2 mg/kg/d dose. Karyolysis was also observed in the 10

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mg/kg/d PFO4DA group and all PFOA-treated groups, with steatosis and acidophilic

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bodies further detected in the 10 mg/kg/d PFOA group (Figure S2).

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Serum biochemical parameters, such as levels of alanine aminotransferase (ALT)

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and aspartate aminotransferase (AST), are important markers of liver injury. In the

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PFO2HxA and PFO3OA exposure groups, the activities of ALT and AST were

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unchanged (Table S2). Following PFO4DA exposure, ALT activity only increased in

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the 10 mg/kg/d group (Figure 1C), whereas AST activity increased in all groups (Table

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S3). For the PFOA-treated groups, ALT and AST activity both increased dose

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dependently. The activities of ALT and AST in the 10 mg/kg/d PFOA group were

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significantly higher than their activities in the 10 mg/kg/d PFO4DA group. In addition

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to the increase in liver enzymes, the levels of albumin (ALB) in serum were also

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increased in the 2 and 10 mg/kg/d PFO4DA groups and all PFOA-treated groups.

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Contents of PFOA and multi-ether PFECAs in liver and serum

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The liver and serum contents of PFOA and multi-ether PFECAs are shown in

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Figure 2. In both serum and liver, the levels of PFOA and multi-ether PFECAs

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increased dose dependently (Figure 2A and 2B). Under the same exposure dose, serum

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content of the tested compounds was ranked PFOA > PFO4DA > PFO3OA > 9

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PFO2HxA. Furthermore, the PFECA liver/serum ratio (< 1.0) was much lower than

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that of PFOA. This is consistent with PFECAs exhibiting higher water solubility (>

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1000 g/L, supporting information) than PFOA (> 500 g/L),34 which resulted in the

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stronger accumulation of PFOA compared to PFECAs (Figure 2C). In addition, the

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relationship between total PFAS mass in the liver and total exposure mass was

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determined to assess the accumulation of PFASs in the liver. Figure 2D shows that

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PFOA had the highest accumulation level (~10%), followed by PFO4DA (~1%),

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PFO3OA (> 0.001%), and PFO2HxA (~ 0.001%).

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Bioinformatics analysis of DEPs in the liver after PFO4DA exposure

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iTRAQ analysis was performed to analyze the global change in mouse liver

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proteins after exposure to 10 mg/kg/d PFO4DA, from which we identified 13 649

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peptides from 2 226 proteins. After differential analysis, 198 proteins were identified

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as DEPs, including 142 up-regulated and 56 down-regulated proteins (Table S4). To

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validate the iTRAQ results, three proteins were randomly chosen for Western blot

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analysis (Figure S3). Solute carrier family 2 (facilitated glucose transporter member 2,

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SLC2A2) levels decreased, peroxisomal acyl-coenzyme A oxidase 1 (ACOX1) levels

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increased, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels remained

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unchanged. These protein levels were consistent with the iTRAQ results, showing that

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the iTRAQ data could represent the global changes in proteins in PFO4DA-exposed

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mouse liver.

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GO and KEGG pathway analyses both revealed enrichment in fatty acid

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metabolism, amino acid metabolism, and the PPAR signaling pathway (Figure S4-S5). 10

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We investigated the association between DEPs and human diseases using DisGeNET.

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The results indicated that most of the up-regulated proteins were associated with

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diseases related to lipid metabolism, whereas most of the down-regulated proteins were

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associated with diseases related to nitrogen metabolism, especially the urea cycle. Here,

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we focused on the DEPs related to the urea cycle. As shown in Figure 3A, gene-disease

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association analysis demonstrated that lipid metabolism disorders, acetyl-CoA

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acetyltransferase deficiency, and bifunctional peroxisomal enzyme deficiency were the

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most overrepresented diseases related to the up-regulated proteins. In the down-

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regulated proteins, nitrogen metabolism was one of the most overrepresented pathways

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(another was fatty liver), and related diseases included urea cycle disorder, carbamoyl-

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phosphate synthase I (CPS1) deficiency, and hyperammonemia. The Disease Ontology

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27-29

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regulated proteins. The results were consistent with those of DisGeNET (Figure S5).

database was used to verify the gene-disease association analysis using the down-

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Protein-protein interaction (PPI) network analysis was performed to assess the

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association of key enzymes related to the urea cycle with other DEPs. As shown in

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Figure 3B, 32 DEGs (20 up-regulated and 12 down-regulated) were related to the five

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key urea cycle proteins. Usually, the higher the centrality values, the more important

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the protein is in the pathway. According to the centrality values of the key urea cycle

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proteins, their importance was ranked: CPS1 > ornithine transcarbamylase (OTC) >

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argininosuccinate lyase (ASL) > argininosuccinate synthetase 1 (ASS1) > arginase 1

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(ARG1) (Table S5).

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Comparative urea cycle disorder induced by PFOA and PFO4DA in mouse liver 11

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Further detection of the expression levels of urea cycle proteins confirmed the

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iTRAQ results. As shown in Figure 4, the level of CPS1 significantly decreased in all

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PFOA exposure groups. For PFO4DA, the level of CPS1 only decreased in the 10

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mg/kg/d group. To further illustrate the regulation of CPS1 in the urea cycle, the levels

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of N-acetyl-L-glutamate synthase (NAGS) and sirtuin (silent mating type information

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regulation 2 homolog) 5 (SIRT5) were analyzed. Results showed that NAGS was

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unchanged in the PFOA and PFO4DA-treated groups, whereas the level of SIRT5

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decreased in all PFOA exposure groups and in the 10 mg/kg/d PFO4DA group. These

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findings are consistent with the expression pattern of CPS1. Other proteins located

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downstream of CPS1 also decreased. For PFO4DA, the expression levels of OTC,

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ASS1, and ASL decreased in the 2 and 10 mg/kg/d groups, whereas the expression level

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of ARG1 only decreased in the 10 mg/kg/d group. For PFOA, the expression levels of

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OTC, ASL, and ARG1 decreased in all groups, whereas the expression level of ASS1

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only decreased in the 2 and 10 mg/kg/d groups.

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Effects of PFOA and multi-PFECAs on ammonia and blood urea nitrogen content

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The levels of ammonia and blood urea nitrogen in serum can reflect the effects on

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urea cycle dysfunction.35 As shown in Figure 5, the serum ammonia content increased

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in a dose-dependent manner in all PFO4DA and PFOA groups, though the content was

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lower in the PFO4DA than in the PFOA groups at the same dose. In contrast, the serum

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urea level decreased in all PFO4DA and PFOA exposure groups. For the PFO2HxA

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and PFO3OA groups, the serum levels of ammonia and urea were unchanged (Figure

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S6). 12

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Effects of PFO4DA on glutamate and aquaporin content in the brain

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Glutamate concentration and glutamine synthetase activity in the brain were

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analyzed to assess the impact of urea cycle disorder on mice (Figure 6). Glutamate

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content in the brain decreased significantly in the 10 mg/kg/d PFOA and PFO4DA

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groups, although no difference was observed between PFO4DA and PFOA. While body

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weight was reduced by more than 10% in the 10 mg/kg/d PFOA group. The changes in

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glutamine synthetase activity were contrary to that of glutamate content.

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As ammonia content can affect the rapid transportation of water,36-38 we

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determined changes in the expressions of aquaporins (AQP1 and AQP4) in the brain

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(Figure S8). Results demonstrated that AQP1 expression increased in the 10 mg/kg/d

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PFO4DA and PFOA groups and APQ4 expression increased in the 2 and 10 mg/kg/d

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PFOA and PFO4DA groups, although no significant differences were observed

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between PFO4DA and PFOA. However, body weight was reduced by more than 10%

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in the 10 mg/kg/d PFOA group only.

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DISCUSSION

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Multi-ether PFECAs have been introduced as PFOA replacements in the

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manufacture of fluoropolymer and can be generated as byproducts during this process.

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While concentrations of multi-ether PFECAs are reported to be 2–113 times greater

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(PFO2HxA, PFO3OA) or similar (PFO4DA) to that of GenX in certain areas (e.g., Cape

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Fear River Watershed),18 no data regarding their toxicities are currently available. To

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the best of our knowledge, this is the first study to report on the toxic effects of multi13

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ether PFECAs on mice, which is expected to help clarify their potential health risks.

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Analysis of the content and ratios of multi-ether PFECAs in organs and serum

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demonstrated that multi-ether PFECAs had a stronger preference for serum and the

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cumulative mass ratio in the liver increased with OCF2 moiety. The cumulative mass

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ratio of PFOA in the liver was 10-fold that of PFO4DA and 10 000-fold that of

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PFO2HxA and PFO3OA, indicating that PFO2HxA and PFO3OA did not accumulate

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in the liver to any significant degree. We further calculated the octanol-water partition

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coefficient (Kow) values for PFOA, PFO2HxA, PFO3OA, and PFO4DA using EPI

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Suite 4.1 from the US EPA. The obtained values were 4.81, 2.63, 3.94, and 5.25,

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respectively, which did not explain why PFO4DA was less accumulative than PFOA in

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the liver. Therefore, it may be that, due to the inserted oxygen, PFECAs can more easily

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form hydrogen bonds with water in biotic environments. This could result in increased

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hydrophilicity and easier elimination from biotic systems compared with perfluorinated

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carboxylic acids. Our results showed that PFOA binding affinity to albumin was

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stronger than that of PFO4DA (data not shown), indirectly supporting our current

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finding that PFO4DA is less accumulative than PFOA in the liver.

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As structurally similar chemicals often cause similarly adverse effects on

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organisms,39 it is reasonable to hypothesize that multi-ether PFECAs and PFCAs with

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the same number of perfluorinated carbons in the backbone may have similar toxicities.

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After exposure to legacy PFASs, the mouse liver shows varying degrees of

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enlargement.15, 23, 25, 40 In the present study, however, enlargement induced by multi-

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ether PFECAs was lower than that of PFOA; in particular, the relative liver weights 14

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were unchanged for all PFO2HxA and PFO3OA-treated groups. Comparing the

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changes in serum ALT and AST activities induced by PFOA exposure with those

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induced by multi-ether PFECAs, the hepatotoxicity of PFOA was found to be much

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higher than that of the multi-ether PFECAs, with very weak hepatotoxicity detected in

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the PFO2HxA- and PFO3OA-treated groups. In addition to relative liver weight and

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biochemical parameters, liver sections stained by hematoxylin and eosin (H&E) also

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confirmed that hepatomegaly and other pathologies were not found in the PFO2HxA

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and PFO3OA groups (Figure S2).

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To further explore how the presence of ether groups in multi-ether PFECAs

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affected toxicity, we conducted proteomic analysis of mouse livers treated with

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PFO4DA. Both GO and KEGG pathway analyses demonstrated that most DEPs

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induced by PFO4DA were related to fatty acid metabolism and the PPAR signaling

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pathway, consistent with previous research on legacy PFASs.15, 20, 25, 33, 40 In the current

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study, the most obvious effect of PFO4DA was disruption of fatty acid metabolism;

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however, we highlighted the effect of PFO4DA on the urea cycle and subsequent

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nitrogen metabolism disruption. For the PFO4DA-treated groups, the expression levels

302

of OTC, ASS1, and ASL decreased significantly in the higher dose groups (2 and 10

303

mg/kg/d), with decreased ARG1 expression only observed at the highest dose (10

304

mg/kg/d). Following PFOA exposure, the expression levels of OTC, ASL, and ARG1

305

decreased in all treated groups, whereas ASS1 expression only decreased in the higher

306

dose groups (2 and 10 mg/kg/d). In previous studies, CPS1 transcript levels were shown

307

to decrease with increasing PFAS dosage.15,

25, 40, 41

CPS1, OTC, ASS1, ASL, and

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ARG1 are the five key enzymes of the urea cycle, which is an important detoxification

309

pathway in the liver and converts toxic ammonium derived from the dysregulation of

310

metabolites into less toxic urea for excretion.42-45 Ammonia exists in the form of NH4+

311

in mammals and is generated during nitrogen metabolism.46 Previous studies have

312

shown that abnormal expression and dysfunction of any of the five key urea cycle

313

enzymes may lead to many kinds of disease. In addition, a decrease in ureagenesis will

314

increase blood ammonium levels, causing lethargy, central nervous system dysfunction,

315

and brain damage.47 CPS1 is the most important and rate-limiting enzyme in the urea

316

cycle and serves to catalyze the synthesis of carbamoyl phosphate (CP) from ammonia

317

and carbon dioxide.48, 49 CPS1 activity is associated with NAG and SIRT5 content.50-53

318

As a substrate, NAG is vital for carbamoyl phosphate synthesis.54 As a sirtuin, SIRT5

319

regulates CPS1 by deacetylation,53 which is an essential modification for CPS1 activity.

320

In the present study, SIRT5 protein levels were decreased, which may have contributed

321

to the decreased expression of CPS1. As another key enzyme of the urea cycle, OTC

322

serves to catalyze the transition of ornithine and carbamyl phosphate to citrulline.55 56

323

Previous research has shown that abnormal expression of CPS1 and OTC can impair

324

liver function.56 Regarding other key enzymes of the urea cycle, impediment of ASS1

325

and ASL function will disturb the synthesis of urea, polyamines, glutamate, creatine,

326

and many other metabolic pathways.43, 57-59

327

Similar to the liver, the concentration of ammonia in serum is normally very low

328

due to intricately coordinated processes such as enzyme activity and transport systems.

329

60

In the current study, serum ammonia levels were increased in both PFO4DA- and 16

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PFOA-treated groups in a dose-dependent manner, although the levels in the PFO4DA-

331

treated groups were lower than those in the PFOA-treated groups at the same dose. In

332

addition, the level of serum urea decreased after PFO4DA and PFOA exposure,

333

contrary to the level of serum ammonia. In the PFO2HxA and PFO3OA groups, the

334

levels of serum ammonia and urea were unchanged. These results indicated that, similar

335

to PFOA, PFO4DA can disrupt the urea cycle and increase serum ammonia levels.

336

In the brain, ammonia detoxication is mainly carried out by astrocytes, a group of

337

supportive glial cells.61 Ammonia is removed by converting glutamate to glutamine, a

338

process that is catalyzed by glutamine synthetase. In the current study, like PFOA,

339

PFO4DA exposure significantly decreased brain glutamate content in the 10 mg/kg/d

340

group, which was accompanied by an increase in glutamine synthetase activity.

341

Previous studies on cultured astrocytic cells have reported cellular swelling under high

342

ammonium concentrations.36,

343

AQP4, mainly facilitate water influx into cells. In the brain, AQP1 primarily

344

participates in cerebrospinal fluid regulation.63 In the rat, AQP1 is also detected in

345

astrocytic cells after brain injury, with a relationship found between high AQP1 levels

346

and brain edema.36-38, 64, 65 AQP4 is also primarily expressed in astrocytic cells,38, 66 and

347

overexpression of the Aqp4 gene in brain astrocytic cells can also result in cellular

348

swelling.67 In our study, the APQ1 protein level only increased in the 10 mg/kg/d

349

PFOA- and PFO4DA-treated groups, whereas the APQ4 protein level increased in both

350

the 2 and 10 mg/kg/d PFOA- and PFO4DA-treated groups. Although body weight

351

decreased by more than 10% in the 10 mg/kg/d PFOA group, there were no significant

37, 62

The two primary aquaporin proteins, AQP1 and

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352

differences between the PFO4DA and PFOA groups regarding the expression of AQPs.

353

Previous research has shown that high ammonia concentrations induce metabolic

354

alterations in the brain, which are associated with aquaporin dysfunction.68 Our results

355

indicated that urea cycle dysfunction is a key event that can be used to assess the effect

356

of PFO4DA on the liver, and that increased serum ammonia can affect the brain.

357

However, the detailed mechanisms need further exploration.

358

In summary, after 28 d of exposure, PFO4DA and PFOA exhibited similar toxic

359

mechanisms leading to liver dysfunction. PFO4DA displayed a weaker accumulation

360

potential than PFOA, whereas PFO2HxA and PFO3OA demonstrated no accumulation

361

and thus did not cause liver injury. Relative liver weights increased significantly in the

362

2 and 10 mg/kg/d PFO4DA groups and all of PFOA treated groups. Like PFOA,

363

PFO4DA exposure decreased the protein levels of the five main urea cycle-related

364

enzymes. Furthermore, the serum ammonia level increased, whereas the serum urea

365

level decreased following PFO4DA exposure. Accompanied with serum ammonia

366

increase, glutamate content decreased and glutamine synthetase activity and aquaporin

367

protein level increased in the brain. Taken together, PFO4DA still caused

368

hepatotoxicity, although with less severity than the same dosage of PFOA. PFO4DA

369

exposure also induced urea cycle dysregulation, which may have contributed to the

370

observed hepatotoxicity. Our data implied that PFO4DA may not be a suitable

371

alternative to PFOA. Considering that PFO4DA has been detected at much higher

372

concentrations than PFOA in raw water samples from a drinking water treatment plant

373

(downstream of a PFAS manufacturer),18 efforts to remove or at least decrease its 18

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374

occurrence in drinking water should be made urgently. For PFO2HxA and PFO3OA,

375

further investigations on their effects on other organs and aquatic toxicity are required

376

to assess their safety as PFOA alternatives.

Supporting Information Details on PFECA analyses are provided in the Supporting Information. The variation in body weight, histopathological analysis validation of iTRAQ results, GO and KEGG pathway analyses, gene-disease association, ammonia and urea contents in PFO2HxA and PFO3OA groups, and association between expose dosage and content are shown in Figures S1–S7. Antibody information, physiological indices, serum biochemical levels, DEPs, and centrality of key urea cycle proteins are shown in Tables S1–S5 (Excel). Figure legends in Supporting Information. Pg S3. Analysis of PFECA levels in serum and liver. Pg S4. Details for hematoxylin and eosin staining. Pg S5. Table S1. Information on antibodies used for Western blotting. Pg S6. Table S2. Body weights, liver weights, and serum biochemical levels after PFO2HxA and PFO3OA exposure. Pg S7. Table S3. Body weights, liver weights, and serum biochemical levels after PFOA and PFO4DA exposure. Pg S8. Table S4. Protein names in Figure 3B. Pg S9. Table S5. Differentially expressed proteins highlighted by iTRAQ. 19

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Pg S10. Table S6. Centrality of key urea cycle proteins. Pg S11. Fig. S1. Variation in body weight (n = 12) following exposure to PFO2HxA, PFO3OA, PFO4DA, or PFOA. Pg S12-13. Fig. S2. Histopathological analysis of liver sections stained with hematoxylin and eosin. Pg S14. Fig. S3. Western blotting for validation of iTRAQ results. Pg S15. Fig. S4 GO analysis results. Pg S16. Fig. S5. KEGG pathway and gene-disease association results. Pg S17. Fig. S6. Ammonia and urea content in serum from PFO2HxA and PFO3OA groups. Pg S18. Fig. S7. Best models for PFAS expose dosage and content in liver and serum. Pg S19. Fig. S8. Western blotting for aquaporin in the brain.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21737004) and Strategic Priority Research Program of the Chinese Academy of Sciences (XDB14040202).

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Fig. 1. Compound structure, relative liver weight, and alanine transaminase (ALT) activity in serum. Structures of PFOA, PFO2HxA, PFO3OA, and PFO4DA (A); relative liver weights of mice treated with PFOA, PFO2HxA, PFO3OA, and PFO4DA (B); serum ALT activity in mice treated with PFOA, PFO2HxA, PFO3OA, and PFO4DA (C). All data are means ± SE (n = 10–12), different letters represent significant differences between groups at p < 0.05 by ANOVA and Duncan’s multiple range tests.

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Fig. 2. Content and cumulative rate of PFOA and multi-ether PFECAs in liver and serum of male mice after 28 d of exposure. PFAS content in serum (A); PFAS content in liver (B); content ratio of PFASs in liver and serum (C); cumulative rate of PFASs in liver (D). Data are means ± SE (n = 6–18), double asterisks indicate significant difference at p < 0.01, single asterisk indicates significant difference at p < 0.05.

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Fig. 3. Disease ontology terms determined by DisGeNET and protein-protein interaction between key urea cycle proteins and other differentially expressed proteins (DEPs). Enrichment of DEPs related to protein metabolism diseases in mice exposed to PFO4DA (A). Protein-protein interaction between key urea cycle proteins and other DEPs (B). ACAT, Acetyl-CoA acetyltransferase; BPED, bifunctional peroxisomal enzyme deficiency; CPS1, carbamoyl-phosphate synthase 1. Protein names in Figure 3B were provided in Table S4.

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Fig. 4. Protein profile changes in urea cycle induced by PFOA and PFO4DA exposure. Western blotting of urea cycle proteins (left panel), relative fold change of each protein compared to GAPDH in each group (right panel). All graphs depict means ± SE (n = 3), different letters represent significant differences between groups at p < 0.05 by ANOVA and Duncan’s multiple range tests.

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Fig. 5. Ammonia and urea content in mouse serum following PFOA and PFO4DA exposure. Ammonia content in serum (A) and blood urea nitrogen (BUN) content in serum (B). All graphs depict means ± SE (n = 6), different letters represent significant differences between groups at p < 0.05 by ANOVA and Duncan’s multiple range tests.

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Fig. 6. Glutamic content (A) and glutamine synthetase (GS) activity (B) in the brain. Data are means ± SE (n = 6), different letters represent significant differences between groups at p < 0.05 by ANOVA and Duncan’s multiple range tests.

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For TOC art only 84x47mm (300 x 300 DPI)

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