Comparative Hepatotoxicity of Novel PFOA Alternatives

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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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Environmental Science & Technology

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.

1

ACS Paragon Plus Environment

E-mail:


1

ABSTRACT

2

As novel alternatives to perfluorooctanoic acid (PFOA), perfluoropolyether

3

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

4

in various environmental matrices; however, public information regarding their

5

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

6

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

7

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

8

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

9

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

10

weight. Furthermore, PFO2HxA and PFO3OA demonstrated almost no accumulation

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

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

19

concluded that PFO4DA caused hepatotoxicity, as indicated by hepatomegaly and

20

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

21

which may contribute to the observed toxic effects.

2


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INTRODUCTION

23

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

24

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

25

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

26

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

27

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

28

PFOA shows environmental persistence, widespread presence, and considerable

29

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

30

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

31

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

32

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

33

usage, and transaction of PFOA.9

34

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

35

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

36

perceived

37

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

38

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

39

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

40

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

41

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

42

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

43

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

environmental

friendliness.10

As

a

new

3


alternative

to

PFOA,


44

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

45

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

46

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

47

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

48

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

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59

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

60

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

61

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

62

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

63

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

64

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

65

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

67

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

68

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

69

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

70

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

71

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

72

liver urea cycle dysfunction induced by PFECA exposure.

73 74

MATERIALS AND METHODS

75

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

76

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

77

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

78

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

79

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

80

exposure experiments.

81

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

82

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

83

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

84

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

85

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

86

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

87

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



88

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

89

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

90

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

91

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

92

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

93

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

94

IOZ14048).

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

Information.

103

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

104

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

105

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

106

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

107

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

108

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

109

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

111

Liver protein preparation and isobaric tags for relative and absolute quantitation

112

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

113

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

114

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

115

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

116

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

117

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

118

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

119

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

120

described in our previous study.25

121

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

122

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

123

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

124

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

125

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

126

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

127

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

128

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

129

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

130

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

131

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



132

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

133

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

134

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

135

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

136

antibodies used for Western blotting are given in Table S1.

137

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

138

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

139

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

140

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

141 142

RESULTS

143

Effects of PFOA and multi-ether PFECAs on the liver

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

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


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154

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

155

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

156

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

157

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

158

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

159

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

160

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

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

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

170

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

171

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

172

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

173

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

174

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

175

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



176

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

177

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

178

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

179

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

180

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

181

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

182

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

183

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

184

Bioinformatics analysis of DEPs in the liver after PFO4DA exposure

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

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

195

mouse liver.

196

GO and KEGG pathway analyses both revealed enrichment in fatty acid

197

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


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198

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

199

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

200

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

201

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

202

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

203

association analysis demonstrated that lipid metabolism disorders, acetyl-CoA

204

acetyltransferase deficiency, and bifunctional peroxisomal enzyme deficiency were the

205

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

206

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

207

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

208

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

209

27-29

210

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-

211

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

212

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

213

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

214

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

215

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

216

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

217

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

218

(ARG1) (Table S5).

219

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



220

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

221

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

222

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

223

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

224

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

225

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

226

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

227

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

228

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

229

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

230

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

231

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

232

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

233

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

234

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

235

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

236

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

237

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

238

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

239

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

240

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

241

S6). 12


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Effects of PFO4DA on glutamate and aquaporin content in the brain

243

Glutamate concentration and glutamine synthetase activity in the brain were

244

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

245

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

246

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

247

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

248

glutamine synthetase activity were contrary to that of glutamate content.

249

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

250

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

251

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

252

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

253

PFOA and PFO4DA groups, although no significant differences were observed

254

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

255

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

256 257

DISCUSSION

258

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

259

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

260

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

261

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

262

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

263

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



264

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

265

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

266

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

267

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

268

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

269

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

270

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

271

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

272

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

273

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

274

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

275

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

276

hydrophilicity and easier elimination from biotic systems compared with perfluorinated

277

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

278

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

279

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

280

As structurally similar chemicals often cause similarly adverse effects on

281

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

282

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

283

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

284

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

285

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


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286

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

287

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

288

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

289

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

290

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

291

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

292

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

293

and PFO3OA groups (Figure S2).

294

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

295

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

296

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

297

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

298

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

299

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

300

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

301

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

15



308

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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330

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

17



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



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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25



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.



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.



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.



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


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Comparative Hepatotoxicity of Novel PFOA Alternatives

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