Hepatotoxic Effects of Hexafluoropropylene Oxide Trimer Acid (HFPO

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Ecotoxicology and Human Environmental Health

Hepatotoxic Effects of Hexafluoropropylene Oxide Trimer Acid (HFPOTA), A Novel Perfluorooctanoic Acid (PFOA) Alternative, on Mice Nan Sheng, Yitao Pan, Yong Guo, Yan Sun, and Jiayin Dai Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01714 • Publication Date (Web): 21 Jun 2018 Downloaded from http://pubs.acs.org on June 22, 2018

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Environmental Science & Technology

Hepatotoxic Effects of Hexafluoropropylene Oxide Trimer Acid (HFPO-TA), A Novel Perfluorooctanoic Acid (PFOA) Alternative, on Mice Nan Sheng†, Yitao Pan†, 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

ACS Paragon Plus Environment


1

Abstract

2

As an alternative to perfluorooctanoic acid (PFOA), hexafluoropropylene oxide trimer

3

acid (HFPO-TA) has been increasingly used for fluoropolymer manufacture in recent

4

years. Its growing detection in environmental matrices and wildlife raises

5

considerable concern about its potential health risks. Here we investigated the effects

6

of HFPO-TA on mouse liver following 28 days of exposure to 0.02, 0.1, or 0.5

7

mg/kg/d of HFPO-TA via oral gavage. Results showed that HFPO-TA concentrations

8

increased to 1.14, 4.48, and 30.8 µg/mL in serum and 12.0, 32.2, and 100 µg/g in liver,

9

respectively. Liver injury, including hepatomegaly, necrosis, and increase in alanine

10

aminotransferase activity, was observed. Furthermore, total cholesterol and

11

triglycerides decreased in the liver in a dose-dependent manner. Liver transcriptome

12

analysis revealed that 281, 1 001, and 2 491 genes were differentially expressed (fold

13

change ≥ 2 and FDR < 0.05) in the three treated groups, respectively, compared with

14

the control group. KEGG enrichment analysis highlighted the PPAR and chemical

15

carcinogenesis pathways in all three treatment groups. Protein levels of genes

16

involved in carcinogenesis, such as AFP, p21, Sirt1 C-MYC, and PCNA, were

17

significantly increased. Compared with previously published toxicological data of

18

PFOA, HFPO-TA showed higher bioaccumulation potential and more serious

19

hepatotoxicity. Taken together, HFPO-TA does not appear to be a safer alternative to

20

PFOA.

21


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INTRODUCTION

23

Per- and polyfluoroalkyl substances (PFASs), represented by perfluorooctanoate

24

(PFOA) and perﬂuorooctane sulfonate (PFOS), have been used in industrial

25

applications for over six decades.1, 2 Based on in-depth studies, however, long-chain

26

PFASs (containing more than six perfluorinated carbons) have been found to be

27

environmentally persistent, potentially bio-accumulative, and biologically toxic,

28

resulting in their phasing out and greater regulation.3-5 In 2006, the 2010/2015 PFOA

29

Stewardship Program was signed by the U.S. Environmental Protection Agency and

30

eight related manufacturers to eliminate all usage of PFOA, a well-known C8 PFAS,

31

by 2015.6 Furthermore, in 2015, a proposal to restrict the production, usage, and

32

marketing of PFOA, its salts and related substances in the European Union was

33

accepted by the Risk Assessment Committee of the European Chemicals Agency.7

34

In light of the above regulations, the use of environmentally-friendly alternatives

35

to replace PFOA, such as shorter chain homologues and other fluorinated chemicals,

36

has become a global trend.8 PFOA alternatives used in fluoropolymer resin

37

manufacturing include perfluoroalkyl ether carboxylic acids (PFECAs), such as

38

ammonium

39

(3,5,7-trioxaoctanoic) acid (PFO3OA), perfluoro (3,5,7,9-tetraoxadecanoic) acid

40

(PFO4DA), and oligomeric hexafluoropropylene oxide (HFPO).9-12 Although many

41

alternatives have been produced and utilized, their safety to the environment, wildlife,

42

and humans remains unclear. Very little information about their environmental fate,

43

toxicokinetic behavior, or toxicity is publicly available,9 although scientific research

4,8-dioxa-3H-perfluorononanoate


(ADONA),

perfluoro


44

on these alternatives has seen some progress, especially regarding their environmental

45

occurrence. For example, seven different PFECAs have been detected in drinking

46

water treatment plants in the Cape Fear River watershed11 and ADONA has been

47

detected in the Alz River at a concentration range of 0.32–6.2 µg/L in 2008 and 2009,

48

much higher than that of PFOA in the same samples.13 A recent study also identified

49

ADONA in plasma samples collected from people living in South Germany.14 HFPO

50

dimer acid (HFPO-DA, commercial name GenX) has also been detected in recent

51

water samples from the North Sea, Rhine River, and Xiaoqing River.15 In our previous

52

study, we detected its homologue, HFPO trimer acid (HFPO-TA), at considerable

53

concentrations in water and common carp samples from Xiaoqing River and in sera

54

samples from local residents.16 It is worth noting that the concentration of HFPO-TA

55

in Xiaoqing River samples reached 68.5 µg/L, ranking second after PFOA for all

56

PFASs in both water and biological samples. HFPO-TA has also been found in frogs

57

living near fluoropolymer manufacturing plants.17

58

Due to the inserted ether oxygens in their perfluorinated carbon backbones,

59

PFECA alternatives are more hydrophilic during elimination via the kidney, and thus

60

more labile to be metabolized.10 These ether oxygens also generate structural torsion

61

and change the binding mode of PFECAs to human liver fatty binding acid

62

(hL-FABP), an essential protein in PFOA-induced hepatotoxicity.18 Whether the toxic

63

effects of PFECAs decrease compared with those of PFOA and whether the toxic

64

mechanism differs require further investigation. Due to the difficulties in purchasing

65

chemicals of sufficient quantity and quality, performing appropriate toxicity tests for


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these novel alternatives can be challenging. Thus, to date, only a few studies have

67

reported on the toxicities of PFECA alternatives. As summarized by Gordon, ADONA

68

is somewhat orally toxic but not developmentally toxic in rats, and can induce mild

69

skin irritation in mice and rabbits, but is not genotoxic.19 Gannon et al. claimed that

70

HFPO-DA can be rapidly and completely absorbed in rodents without metabolism,

71

with elimination half-lives of 5 h and 20 h for rats and mice, respectively.20 Although

72

HFPO-DA appears to be of low risk to aquatic organisms, it has been reported to

73

induce liver injury in rodents after both sub-acute and chronic exposure and produce

74

benign tumors in the liver, pancreas, and testes of rats.21,22,23 Furthermore, like PFOA,

75

ADONA and HFPO-DA can induce hepatotoxicity in rodents via activation of the

76

peroxisome proliferator-activated receptor α (PPARα) pathway.19, 22, 23 Our previous

77

study showed that HFPO-TA induced more serious cytotoxicity and stronger

78

hL-FABP binding capacity than PFOA.18 Thus, research on the hepatotoxicity of

79

HFPO-TA in vivo is critical.

80

In the present study, we investigated the effects and hazards of low dose

81

HFPO-TA on the mouse liver and compared its effects with previous studies on PFOA.

82

High-throughput RNA sequencing (RNA-seq) was conducted to explore the effects of

83

HFPO-TA exposure on hepatic transcripts. The aims of this study were to (1)

84

investigate whether HFPO-TA exposure induces toxic effects on mouse liver under

85

low dose concentrations; (2) explore the possible mechanism of its hepatotoxicity;

86

and (3) determine whether HFPO-TA is a safer alternative by comparing its

87

toxicological data with that of PFOA.



88 89

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MATERIALS AND METHODS Chemicals

and

experimental

animals.

HFPO-TA

(CAS

No.

90

CAS:13252-14-7 >98.0% purity) was synthesized as described in our previous

91

study.16 Pure HFPO-TA was then dissolved with Milli-Q water to 1 g/L as stock

92

solution for the mouse exposure experiments or with methanol for the analysis of

93

HFPO-TA in mice.

94

Previous studies have reported sex differences in the bioaccumulation and

95

elimination of PFASs, which might relate to the ovulation cycle in females.24,

96

Considering our aim to measure the bioaccumulation potential of HFPO-TA, only

97

male mice were used in the present study. Male BALB/c mice (aged 6–8 weeks) were

98

purchased from the Beijing Vital River Experimental Animals Centre (Beijing, China)

99

and housed under standard conditions (temperature, 23 ± 1 °C; humidity, 60 ± 5%;

100

light/dark cycle, 12-/12-h). After one week of adaptation, 60 mice were randomly

101

divided into four groups of equal size and treated with different concentrations of

102

HFPO-TA via oral gavage. Based on previous studies24, which showed that sub-acute

103

exposure to 1 mg/kg/d of PFOA (21 days to 30 days) induced significant liver injury,

104

we choose 0, 0.02, 0.1, and 0.5 mg/kg/d as the HFPO-TA exposure dosages and 28

105

days as the exposure time. The volume of HFPO-TA given to each mouse was

106

determined by its body weight (20 µL/g body weight). Food intake was weighed

107

when the body weight of the mice in each group showed significant differences (Day

108

22 to Day 28), with details shown in the Supporting Information (SI). After 28 days of

109

continuous exposure, all mice were sacrificed and sampled for analysis. All


25

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110

procedures were approved by and performed in accordance with the Ethics

111

Committee of the Institute of Zoology, Chinese Academy of Sciences.

112

HFPO-TA content in serum and liver samples. Mouse livers were first

113

homogenized with ultrapure water (1:10 w/v) by a sonicator. The HFPO-TA in serum

114

and liver was then extracted and detected, as described in our previous study.16 Briefly,

115

serum and liver homogenate samples (20 µL) were spiked with mass-labeled standard

116

(0.5 ng), tetra-n-butylammonium hydrogen sulfate solution (0.5 M, 1 mL),

117

NaHCO3/Na2CO3 buffer (pH = 10, 2 mL), and methyl tert-butyl ether (MTBE) (4 mL).

118

Vigorous shaking and centrifugation were performed to separate the organic and

119

aqueous phases, with the resulting supernatant organic phase collected by glass

120

pipette and the remaining residue extracted with the addition of 4 mL of MTBE twice.

121

After evaporation with nitrogen, 200 µL of methanol was added for reconstitution.

122

The HFPO-TA content was analyzed by an API 5500 triple-120 quadrupole mass

123

spectrometer

(AB

SCIEX,

Framingham,

MA,

124

reaction-monitoring (MRM) in negative electrospray ionization (ESI-) mode.

125

Chromatographic separation was accomplished using an Acquity BEH C18 column

126

(100 mm × 2.1 mm, 1.7 µm, Waters, MA, USA) with mobile phases of 2 mM

127

ammonium acetate in water (A) and methanol (B) at a flow rate of 0.3 mL/min. The

128

MRM transitions were 377→293 for 6:2 FTCA (cone voltage, 8 V; collision energy,

129

22 V) and 379→294 (cone voltage, 10 V; collision energy, 22 V). Calibration curves

130

ranging from 0.05 to 20 ng/mL exhibited excellent linearity (R2 128 > 0.999). Matrix

131

recovery was 81% for serum and 79% for liver (n = 6). The limit of quantitation


USA)

under

multiple


132

(LOQ) was defined as the lowest standard having a signal-to-noise ratio greater than

133

10. In each batch, two method blanks and two matrix spiked samples were conducted

134

for quality assurance. No detectable contamination was found in any batch.

135

Serum biochemical assay and liver lipid levels. Serum enzyme levels and lipid

136

concentrations in the serum and liver samples were quantified according to our

137

previous study.26

138

RNA-seq assay and real-time PCR verification. Total liver RNA was isolated,

139

qualified, and quantified according to previous study26. Three RNA samples from

140

each group (100 µg, RIN > 8.0) were sent to Annoroad Gene Technology Co. Ltd

141

(Beijing, China) for RNA-sequencing, as described in previous studies.23, 26-27 Briefly,

142

Bowtie2 v2.2.3. was used to construct a reference genome library, with the reference

143

gene and genome annotation files downloaded from the University of California Santa

144

Cruz (UCSC). Data quality and quantity were guaranteed by performing Perl script,

145

data quantity statistics, Q30 statistics, and base content statistics. Clean data with high

146

quality and quantity were mapped into the reference genome using TopHat v2.0.12

147

software, with the mapping result viewable using the Integrative Genomics Viewer.

148

Reads per kilobase of exon model per million mapped reads (RPKM) and HTSeq

149

v0.6.0 were run to qualify and quantify the expression of genes. Differentially

150

expressed genes (DEGs) (threshold of FDR < 0.05 and absolute value of log2 (RPKM

151

ratio) ≥ 2) were then analyzed with DESeq software and used for annotation,

152

functional, and pathway enrichment analyses based on the Database for Annotation,

153

Visualization, and Integrated Discovery (DAVID), Gene Ontology (GO), and Kyoto


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154

Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, respectively,

155

using Blast2go and KAAS software.

156

Based on the RNA-seq results, real-time PCR (RT-PCR) was conducted to verify

157

the RNA sequencing assay results. RT-PCR analysis was performed as described

158

previously.24 Primer information is listed in Supplementary Table S1.

159

Histological examination of the liver. Hematoxylin-eosin staining (H&E

160

staining) was performed to measure the morphological structure of the liver after

161

HFPO-TA exposure. Oil Red O staining was used to observe lipid accumulation in the

162

mouse liver. Details on H&E and Oil Red O staining are given in the SI.

163 164

Western blot analysis. Protein isolation and Western blotting were performed according to our previous study.26 Details on antibodies are shown in Table S2.

165

Statistical analyses. All results were statistically analyzed using SPSS for

166

Windows 17.0 (SPSS Inc., Chicago, IL, USA). Differences between groups were

167

determined using one-way analysis of variance (ANOVA) followed by Duncan’s

168

multiple range test. All experimental data were represented as means with standard

169

errors (means ± SE). A p-value of < 0.05 between groups was considered statistically

170

significant and presented by different letters. All represented data from in vitro

171

experiments were assessed from at least three independent experiments.

172

RESULTS AND DISCUSSION

173

HFPO-TA content in mouse serum and liver. The HFPO-TA concentrations in

174

the serum and liver samples of mice after 28 days of exposure are shown in Figure 1.

175

In sera, the HFPO-TA concentrations in the four groups were 0.001, 1.14, 4.48, and



176

30.8 µg/ml, respectively; in the livers, the HFPO-TA concentrations were 0.004, 12.0,

177

32.2, and 100 µg/g, respectively (Figure 1A and 1B). Interestingly, for the 0.02

178

mg/kg/d- and 0.1 mg/kg/d-treated groups, the concentration of HFPO-TA in the serum

179

samples was equal to that from 186 blood samples collected from wild common carp

180

in Xiaoqing River (~1.50 µg/ml), whereas the concentration of HFPO-TA in the

181

mouse liver was many times higher than that detected in the carp liver samples.16 Due

182

to the similar structure between HFPO-TA and fatty acid, the increased total protein

183

and albumin (ALB) in the serum samples (Table 1), which participate in fatty acid

184

transportation,31, 32 were likely responsible for the accumulation of serum HFPO-TA.

185

For PFOA and PFOS, binding with ALB not only contributes to their transportation to

186

organs, but also to their accumulation in blood; thus, the increased ALB level in

187

serum after PFOA or PFOS exposure might be responsible for the increased

188

concentrations of PFOA and PFOS in serum.28-30 In our previous study on PFOA, the

189

ALB level increased in the 1.25 mg/kg/d and higher dosage groups in a dose-response

190

manner, whereas no significant changes were observed in the 0.08 or 0.31 mg/kg/d

191

exposure groups.28 As shown in Table 1, even the 0.02 mg/kg/d HFPO-TA exposure

192

group showed significantly increased TP and ALB levels, and the increasing trend in

193

all three treated groups was dose-dependent, suggesting that HFPO-TA possibly

194

exhibits higher bioaccumulation potential than PFOA in serum.

195

When we compared the concentration of HFPO-TA in mouse serum and liver

196

with that of PFOA reported by Yan et al.,28 we considered the 0.08, 0.31, and 1.25

197

mg/kg/d PFOA groups to correspond with the 0.02, 0.1, and 0.5 mg/kg/d


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HFPO-TA-treated groups, respectively. The HFPO-TA content in the serum of the

199

0.02 and 0.1 mg/kg/d groups was lower than that of PFOA in the serum of the 0.08

200

and 0.31 mg/kg/d groups, whereas the HFPO-TA content in the 0.5 mg/kg/d exposure

201

group was higher than that of PFOA in the 1.25 mg/kg/d-treated mice. The HFPO-TA

202

concentrations in the mouse livers of the three treated groups were higher than the

203

PFOA concentrations in the corresponding PFOA groups, indicating that HFPO-TA

204

might accumulate more easily than PFOA in the liver.

205

The ratios of total HFPO-TA content in the liver versus serum (liver/serum ratio)

206

were calculated and compared with those of PFOA (based on our previous PFOA

207

exposure study using the same species (mice), samples (serum and liver), and

208

exposure time (28 days)) (Figure 1C)28. The liver/serum ratios of HFPO-TA in the

209

three treatment groups (0.02, 0.1, and 0.5 mg/kg/d) were 4.56, 6.15, and 3.20,

210

respectively, whereas the ratios of PFOA in the 0.08, 0.32, and 1.25 mg/kg/d-treated

211

groups increased from 0.598 to 2.17 and then decreased to ~1.00 in the 5 and 20

212

mg/kg/d-treated groups.28, 33 Although comparing these two chemicals under different

213

dosages is somewhat imprecise, the higher liver/serum ratio of HFPO-TA implies that

214

it may be more easily accumulated than PFOA in the liver. Moreover, higher

215

HFPO-TA liver/serum ratios compared with PFOA were been found in our common

216

carp study.16 In that research, we hypothesized that protein binding capacity and

217

hydrophobic properties may lead to the distribution of PFASs in sera and organs.16

218

HFPO-TA reportedly has a stronger binding capacity to human liver fatty acid binding

219

protein (lower dissociation constant) and is more hydrophobic (higher estimated log



220

KOW) than PFOA.16, 18 It is worth noting that the liver/serum ratio decreased in the

221

higher dose groups for both HFPO-TA and PFOA. Considering that PFAS chemicals

222

can bind to proteins due to their similar structure to fatty acids, we proposed a binding

223

saturation hypothesis for PFAS: that is, with higher exposure dosage, the liver/serum

224

ratio will increase due to increased binding to proteins at the beginning, until a peak

225

value is reached, after which the ratio will decrease once the liver proteins are fully

226

bound, with a final balance occurring after the binding saturation of PFAS to serum

227

proteins. As shown in Figure 1C, the ratio increased from the 0.08 to 1.25

228

mg/kg/d-treated groups and then declined to ~1.00 (5 and 20 mg/kg/d-group ratios

229

were 1.25 and 1.16, respectively), supporting the above hypothesis. The peak value

230

for PFOA and HFPO-TA exposure occurred in the 1.25 mg/kg/d and 0.1

231

mg/kg/d-treated groups, respectively, suggesting that HFPO-TA more easily saturated

232

liver protein compared with PFOA. Along with the higher HFPO-TA absolute level in

233

the liver and the liver/serum ratio, it is reasonable to assume that HFPO-TA exhibits

234

higher accumulation potential than PFOA in mouse liver.

235

HFPO-TA exposure induced liver injury. As shown in Table 1, after exposure

236

to 0.02 mg/kg/d of HFPO-TA for 28 days, although no significant changes in body

237

weight and remaining body weight after liver removal were observed, liver weight

238

and relative liver weight were significantly increased by 50.9% and 48.8%,

239

respectively, compared with the control group. The significantly increased body

240

weight in the 0.1 mg/kg/d HFPO-TA group was responsible for the strikingly high

241

liver weight (~149.7%) (Table 1). For the 0.5 mg/kg/d HFPO-TA-treated group, body


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weight and remaining weight both decreased significantly. Compared with mice in the

243

other groups, the decreased food intake of mice in the 0.5 mg/kg/d HFPO-TA group

244

(Figure S1) provides a possible explanation for their decreased body weight. In

245

addition to their enlarged livers (almost four times larger), the significantly decreased

246

body weights of mice in this group suggests a profound impact on mouse health

247

(Table 1). Hepatomegaly, a dominant effect of PFAS exposure, has been observed in

248

mice, rats, and monkeys.34, 35 In our previous study, after PFOA exposure for 28 days,

249

no significant toxic effects were observed in the 0.08 mg/kg/d group; however, PFOA

250

exposure at 0.31 and 1.25 mg/kg/d induced a 27.1% and 105% increase in liver

251

weight, though no significant changes in body weight.28 In comparison, more

252

extensive hepatomegaly was induced in the 0.1 and 0.5 mg/kg/d HFPO-TA-treated

253

groups following exposure than was induced by similar doses of PFOA.

254

In addition to hepatomegaly, pathological changes such as hepatocellular

255

hypertrophy, necrosis, and apoptosis can be induced by exposure to long-chain

256

PFASs.5, 36, 37 As shown in Figure 2A, karyolysis occurred in the three HFPO-TA

257

treatment groups, necrosis appeared in the 0.1 and 0.5 mg/kg/d exposure groups, and

258

obvious cytoplasmic vacuolation and focal necrosis were observed in the 0.5 mg/kg/d

259

exposure group. In addition, the decreased number of cell nuclei within defined liver

260

slice areas from the three treatment groups suggested the induction of enlarged

261

hepatocytes by HFPO-TA exposure (Figure 2B and 2C). Corresponding to the

262

pathological changes observed in the liver sections, serum alkaline phosphatase (ALP)

263

and alanine aminotransferase (ALT) increased significantly and dose-dependently in



264

all three treatment groups and aspartate aminotransferase (AST) increased

265

significantly in the 0.5 mg/kg/d HFPO-TA exposure group compared with the control

266

(Table 1). ALT and AST are two transaminases that can be used as biochemical

267

markers for early liver injury. They are stored in the cytoplasm and are released into

268

the bloodstream after liver damage has occurred.38-40 PFOA can induce elevated levels

269

of ALT and AST and other serum parameters associated with liver function such as

270

ALP and total bile acid (TBA).28, 41, 42 Epidemiological studies have also reported

271

positive correlation between PFOA and levels of serum ALT and AST.43-45 Here, the

272

obvious pathological changes and elevated ALT and ALP levels in serum observed in

273

the 0.02 and 0.1 mg/kg/d-treated mice implied that HFPO-TA could induce early liver

274

injury, even under environmentally-relevant exposure doses. Compared with

275

toxicological study of low-dose PFOA, in which no changes of the above parameters

276

were observed following 1.25 mg/kg/d exposure,28 HFPO-TA induced considerably

277

more severe effects on the mouse liver.

278

HFPO-TA exposure affected lipid metabolism in mouse liver. Lipid

279

concentrations in the mouse liver are shown in Table 1. With the increase in

280

HFPO-TA dose, both total cholesterol (TCHO) and triglycerides (TG) in the liver

281

decreased. In serum, except for the significantly decreased TG level in the 0.5

282

mg/kg/d exposure group, no obvious changes in TG and TCHO levels were observed

283

(Table 1). As shown in Table 1, low-density lipoprotein (LDL) in serum was

284

significantly increased in the 0.1 and 0.5 mg/kg/d exposure groups, whereas

285

high-density lipoprotein (HDL) changed irregularly. Studies focusing on the effects of


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286

PFOA on human health have confirmed the positive association between PFOA and

287

high cholesterol and TG levels in serum,45-48 in line with the increased TG levels

288

observed in mouse serum after low-dose PFOA exposure (0.31 and 1.25 mg/kg/d for

289

28 days).28 In laboratory animals, exposure to high doses of PFOA has been shown to

290

decrease the levels of serum TCHO, TG, HDL, and LDL.49, 50 In the present study, the

291

changes in lipid levels in serum after HFPO-TA exposure were similar to those after

292

high-dose PFOA exposure28. PFOA treatment can also increase liver lipid levels,

293

which are stored as lipid drops (LDs) in liver cells and transported into the nucleus.49

294

Generally, TG and TCHO in hepatocytes can be secreted into blood circulation by

295

binding with very low-density lipoproteins (VLDLs).49,

296

converted to LDL and HDL and return cholesterol to the liver.51 Although the

297

decreased LDL and HDL levels in the serum after HFPO-TA exposure were in

298

keeping with levels after PFOA exposure, the significantly decreased liver lipid levels

299

detected by absolute concentration and Oil Red O staining (Table 1 and Figure S3)

300

suggest promotion of lipid metabolism rather than blocking secretion of liver lipids

301

that have been observed in PFOA-treated mice28. The contrasting effects on liver

302

lipids between HFPO-TA and PFOA suggest a possible different mechanism for

303

interrupted lipid metabolism. The decreased liver lipid content after both HFPO-TA

304

and PFOA treatment was in keeping with that observed in mice after WY14643

305

treatment,52,

306

PPARα pathway to enhance lipid metabolism in the liver.

307

53

51

VLDLs can then be

a PPARα activator, suggesting that HFPO-TA might activate the

HFPO-TA exposure influenced transcriptome in the liver. RNA transcripts for



308

each exposure group were deeply sequenced. Compared with the control group, we

309

identified 281 (157 upregulated, 124 downregulated), 1 001 (620 upregulated, 381

310

downregulated), and 2 491 (1 479 upregulated, 1 012 downregulated) DEGs in the

311

0.02, 0.1, and 0.5 mg/kg/d HFPO-TA exposure groups, respectively (Figure 3A). In

312

total, 184 (123 upregulated, 61 downregulated) DEGs were identified in all three

313

treatment groups (Figure 3A). Enrichment analyses in DAVID and GO terms were

314

carried out for these DEGs, with the changed biological processes shown in Figure 3B,

315

Figure S1, and Table S3. For each exposure group, 64, 206, and 428 biological

316

processes were recognized. A high percentage of DEGs were assigned to: “metabolic

317

processes” such as single-organism metabolic processes, lipid metabolic processes,

318

and long-chain fatty acid metabolic processes; “cellular processes” such as cell cycle,

319

mitotic nuclear division, and cell division; and “regulation of immune system

320

processes”. Based on KEGG pathway analysis of the DEGs, 15, 26, and 52 pathways

321

were significantly enriched in the three HFPO-TA treatment groups, respectively

322

(Figure 3C and Table S4). Ten enriched pathways, mostly involved in metabolism

323

processes, were shared by all three exposure groups. The enriched pathways related to

324

fatty acid metabolism, including retinol metabolism, arachidonic acid metabolism,

325

linoleic acid metabolism, PPAR signaling pathways, and fatty acid degradation,

326

suggest that HFPO-TA exposure could alter lipid metabolism processes in the liver

327

(Table S3). These findings are consistent with the results obtained in laboratory

328

animals following PFOA treatment, which showed that exposure strongly influences

329

liver lipid metabolism.54, 55


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330

Previous studies have revealed that activation of the PPARα pathway in rodents is

331

an important way in which PFASs elicit toxic effects.45, 50, 54, 56 As an activator of

332

PPARα, PFASs can bind to the ligand binding domain and then activate target gene

333

expression in the PPARα pathway by binding to peroxisome proliferator response

334

elements.56 These target genes are involved in many processes, such as fatty acid

335

uptake, TG catabolism, mitochondrial β-oxidation, and lipoprotein assembly and

336

transport.57,

337

genes activated by PPARγ and other transcriptional regulation factors.59 In the present

338

study, we first measured the expression levels of Ppar genes and PPARα in the mouse

339

liver. Results showed that the mRNA levels of Pparα and Pparγ remained stable after

340

HFPO-TA exposure (Table 2), whereas the protein level of PPARα was increased

341

significantly in the 0.1 and 0.5 mg/kg/d exposure groups, suggesting activation of the

342

PPARα pathway (Figure S5). The expression levels of genes involved in the PPARα

343

pathway were investigated by RNA-seq, followed by RT-PCR verification. No

344

significant changes in Pparα or Pparγ gene expression were observed, whereas

345

downstream genes such as Cyp4a10, Acox1, Scd1, Pltp, Cd36, Slc27a1, and Fabp

346

were significantly increased (q-value or p-value < 0.05) (Table 2). As Cd36, Slc27a1,

347

FABP, and Pltp participate in lipid transportation,60-62 their strong upregulation might

348

be responsible for the uptake of HFPO-TA into hepatocytes and even into nuclei. A

349

similar increase in these genes has also been observed in PFOA-exposed mice.63 In

350

addition to lipid uptake, Slc27a1 also plays a central role in the metabolism of

351

long-chain fatty acids by increasing activity of lipolytic catalyzation.64 The significant

58

Further studies using PPARα knockout mice reported alteration in



352

increase in expression levels of Slc27a1 after HFPO-TA exposure was responsible for

353

the significantly decreased liver lipid levels. In addition, Cyp4a10, Acox1, and Scd1

354

are involved in fatty acid oxidation and lipogenesis;65 thus, the induction of these

355

genes suggests enhanced oxidation of fatty acids after HFPO-TA exposure, which

356

might be a possible reason for the reduced lipid content in the mouse liver.

357

HFPO-TA disrupted the expression of genes and proteins involved in

358

hepatocellular carcinoma. Increased liver weights and hepatocellular hypertrophy

359

are associated with increased incidences of hepatocellular tumors in rodents.

360

Epidemiological studies have reported positive relationships between PFAS exposure

361

and cancers, especially in occupational workers, including kidney, testicular, and liver

362

cancers.45, 66, 67 In rodents, PFOA exposure is reported to induce tumors in the liver,

363

testicle, pancreas, and breast. In addition, chronic and shorter-term toxicity studies on

364

HFPO-DA have also observed benign tumors in the liver, testes, and pancreas of mice.

365

A possible mechanism for these effects is activation of the PPAR pathways.45, 50, 54 In

366

this study, using KEGG enrichment analysis, we identified chemical carcinogenesis

367

processes at a ratio equal to that of the PPAR signaling pathway, even in the 0.02

368

mg/kg/d HFPO-TA exposure group (Figure 3C and 3D, Table 2). Except for the

369

significantly decreased expression of Cyp2e1, other genes involved in chemical

370

carcinogenesis processes, including Gstt2, Gstt3, Nqo1, Cyp2b C-myc, and Acaa1,

371

were significantly increased, though Afp gene expression was not markedly changed.

372

Among the genes involved in chemical carcinogenesis processes, the GST family is

373

involved in the metabolism and detoxification of numerous endogenous toxins and


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374

xenobiotics, whereas C-myc affects cell proliferation by influencing p21 expression.68

375

C-myc was one of the first oncogenes identified for its high expression levels in

376

hepatocellular carcinoma (HCC), and its interaction with p53 is extremely important

377

for HCC by driving DNA-damaged cells into the cell cycle.69 In the present study,

378

although mRNA levels of p53 were not significantly changed, the markedly increased

379

expression level of C-myc suggested high possibility for HCC. With their

380

upregulation, Cyp2e1 and Cyp2b are reported to play important roles in the

381

carcinogenesis process.70-72 As summarized in a review on chemical carcinogenesis

382

induced by different exposures (particulate matter, benzene, and polycyclic aromatic

383

hydrocarbons), the GST family, Cyp2e1, and Nqo1 are related to these effects.71 In the

384

present study, our data indicated the potential for HFPO-TA to induce hepatocellular

385

tumors by affecting the chemical carcinogenesis pathways.

386

Further detection of the expression levels of related proteins involved in chemical

387

carcinogenesis processes was performed to confirm the effects on mouse liver. As

388

shown in Figure 4, significantly increased AFP levels were observed in the livers of

389

the 0.1 and 0.5 mg/kg/d HFPO-TA exposure groups compared with the control group,

390

indicating the possibility of liver cancer as AFP is a hepatocellular carcinoma

391

bio-marker.73 As a marker of cell proliferation and DNA replication, PCNA was

392

upregulated following PFOA exposure in mice.52 Its upregulation in the present study

393

suggests enhanced effects on mitosis after 0.1 and 0.5 mg/kg/d HFPO-TA exposure.

394

Significantly increased Sirt1 and decreased p21 and MTA2 levels were also observed

395

in the three treatment groups in a dose-dependent manner. Furthermore, C-MYC and



396

MDM4 levels were increased significantly in the livers of the 0.1 and 0.5 mg/kg/d

397

groups. As reported previously, deficient expression of tumor suppressors such as p53,

398

RB, p21, and p27 has been detected in human liver cancers.74, 75 The expression of

399

p21 is regulated by p53 and C-MYC, whereas p53 is negatively regulated by

400

MDM4.76-78 The increased MDM4 and C-MYC protein levels observed in this study

401

were co-responsible for the decrease of p21 in the liver, suggesting that HFPO-TA

402

exposure may induce abnormal cell proliferation, which may lead to the possible

403

generation of tumors in the liver. In addition, other studies have reported the opposite

404

effects of Sirt1 and MTA2: overexpression of Sirt1 and/or inhibition of MTA2 can

405

inhibit cell growth in cancer.68,

406

decreased MTA2 levels observed in the current study may protect the liver from the

407

generation of tumors.

76, 79

Thus, the significantly increased Sirt1 and

408

To the best of our knowledge, this is the first report on the toxic effects of

409

HFPO-TA on mice, especially at low exposure doses. Collectively, after 28 days of

410

exposure, HFPO-TA exhibited higher bioaccumulation potential than PFOA in mice

411

and was more easily accumulated in the liver. Liver injury, along with decreased lipid

412

content in both serum and liver, was observed even in the 0.02 mg/kg/d HFPO-TA

413

group, indicating that HFPO-TA exposure may result in more serious hepatotoxicity

414

in mice than that of PFOA. Further investigation on the liver transcriptome showed

415

that HFPO-TA exposure enhanced lipid metabolism via activation of the PPAR

416

pathways. In addition, although no tumors were detected in the mouse livers, the

417

changed expression of genes and proteins involved in chemical carcinogenesis


Page 20 of 40

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418

pathways and/or tumor generation suggest that HFPO-TA has strong carcinogenic

419

potential. Thus, we concluded that HFPO-TA might not be a safe alternative to PFASs.

420

In view of the considerable concentrations of HFPO-TA detected in the environment

421

and its significant hepatotoxicity in mice, further investigations are urgently required,

422

including the toxic effects of HFPO-TA under long-term exposure, its metabolism and

423

half-life in animals, as well as sex and species differences.

ACKNOWLEDGMENTS

This work was supported by National Natural Science Foundation of China (grants 21737004 and 31320103915) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB14040202).

SUPPORTING INFORMATION AVAILABLE Table S1. Sequences of primers used for real-time PCR amplification. Table S2. Information on antibodies used for Western blotting. Table S3. Altered biological processes highlighted by DAVID. Table S4. Enriched KEGG pathways of differentially expressed hepatic sequences after HFPO-TA exposure. Fig. S1. Changes in body weight (A) and food intake (B) of mice during exposure. Fig. S2. Linear fitting for HFPO-TA exposure dosage and HFPO-TA concentration in serum (A) and liver (B), and HFPO-TA mass in the liver (C).



Fig. S3. Analysis of liver sections stained with Oil Red O. Fig. S4. Highest ranked biological processes enriched by GO analysis for each exposure group. Fig. S5. Protein expression levels for PPARα, representative blots from three experiments, and mean levels of protein bands compared with the control. Values indicate means ± SE (n = 3); error bars indicate standard errors; different letters represent significance between groups at p < 0.05 by ANOVA and Duncan’s multiple range tests.


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Page 23 of 40


Figure Legend Figure 1. HFPO-TA concentrations in the serum (A) and liver (B) of mice and liver/serum ratios of HFPO-TA and PFOA# (C). Error bars indicate standard errors. Different letters represent statistical significance between groups. # indicates that data were obtained from Yan et al. (2014). Figure 2. Histopathological analysis of liver sections stained with hematoxylin and eosin (H&E; 200×) (A). Red arrow indicates karyolysis; black arrow indicates necrosis; blue arrow indicates cytoplasmic vacuolation. Comparison of the average number of cell nuclei within a defined area (1392 × 1040) acquired by inForm v2.0 (B) and manual counting (C). Different letters represent statistical significance between groups.

Figure 3. Effects of HFPO-TA on liver gene expressions obtained by RNA-seq. Venn diagram for differentially expressed genes determined by RNA sequencing (q value < 0.05), red represents upregulated genes, blue represents downregulated genes (A); Number of changed biological processes highlighted by DAVID in the three HFPO-TA-treated groups (B); Heat map of KEGG enrichment pathways in the three treatment groups (C); Enrichment ratio of pathways by KEGG analysis in the 0.02 mg/kg/d group (D).

Figure 4. Protein expression levels in each treated group: representative blots from three experiments and mean levels of protein bands compared with the control. Values indicate means ± SE (n = 3); error bars indicate standard errors; different letters



represent significance between groups at p < 0.05 by ANOVA and Duncan’s multiple range tests.


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Table 1. Body weights (BW), liver weights (LW), remaining body weight after removing liver (BW-LW), serum biochemical levels (n = 10), and liver lipid concentrations after HFPO-TA exposure. Data are means ± SE (n = 10), significantly different from control group, different letter represent significance between groups at p < 0.05 by ANOVA and Duncan's multiple range tests.

Ctrl

0.02 a

0.1 b

19.6 ± 0.432d

Body weight (g)

22.8 ± 0.319

23.1 ± 0.319

Liver weight (g)

0.917 ± 0.0133a

1.38 ± 0.0342b

2.29 ± 0.0297c

3.19 ± 0.0997d

BW - LW (g)

21.9 ± 0.306a

21. 7 ± 0.284a

21.9 ± 0.258a

16.4 ± 0.332b

Relative liver weight (%)

4.03 ± 0.0560a

6.00 ± 0.102b

9.47 ± 0.0730c

16.3 ± 0.405d

ALT (IU/L)

25.4 ± 1.42a

36.9 ± 2.71b

86.6 ± 23.3c

448 ± 79.5d

AST (IU/L)

113 ± 16.1a

101 ± 8.96a

114 ± 6.67a

181 ± 17.2b

ALP (IU/L)

147 ± 2.38a

179 ± 5.64b

430 ± 15.1c

3250 ± 118d

0.940 ± 0.0826a

1.23 ± 0.570a

2.27 ± 0.83a

16.5 ± 3.46b

TP (g/L)

52.7 ± 0.377a

54.7 ± 0.681b

54.8 ± 0.37b

57.8 ± 0.727c

ALB (g/L)

24.7 ± 0.194a

26.1 ± 0.390b

26.2 ± 0.180b

27.8 ± 0.323c

HDL (mmol/L)

3.78 ± 0.0502a

4.09 ± 0.110b

3.39 ± 0.09c

3.74 ± 0.135a

LDL (mmol/L)

0.250 ± 0.0134a

0.310 ± 0.0214a

0.650 ± 0.0350c

0.42 ± 0.0527b

TCHO (µmol/L)

3.480 ± 0.0522a

3.89 ± 0.131b

3.70 ± 0.125ab

3.81 ± 0.125ab

TG (mmol/L)

2.15 ± 0.220a

2.11 ± 0.0769a

2.10 ± 0.125a

0.700 ± 0.0735b

TCHO in liver (µmol/g)

9.39 ± 0.260a

5.76 ± 0.420b

3.29 ± 0.357c

1.21 ± 0.141d

TG in liver (mmol/g)

45.5 ± 1.327a

42.6 ± 3.08a

35.2 ± 1.88b

24.1 ± 2.09c

TBA (µmol/L)

24.2 ± 0.288

0.5 c


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Table 2. Verification results by real-time PCR 0.02 mg/kg/d RNA sequencing Fold

q value

change

PPAR

0.1 mg/kg/d RT-PCR

Fold

p value

change

RNA sequencing Fold

q value

change

0.5 mg/kg/d RT-PCR

Fold

p value

change

RNA sequencing Fold

q value

change

RT-PCR Fold

p value

change

Pparα

0.942

0.372

0.84

0.490

1.20

1.00

0.892

0.580

0.971

1.00

0.640

0.0174

Pparγ

1.15

0.552

1.32

0.320

0.990

1.00

0.784

0.210

0.860

0.561

1.12

0.557

Cyp4a10

1.67

0.195

2.82

5.90E-04

4.29

0.0390

7.21

3.40E-05

4.05

0.0430

8.30

5.90E-07

Acox1

2.78

6.10E-04

2.78

0.0375

2.85

6.70E-23

12.3

1.70E-04

4.29

4. 80E-30

21.6

3.80E-05

Scd1

2.67

1.70E-06

1.46

0.231

6.24

1.80E-32

7.34

0.00300

7.68

1.10E-08

13.9

8.90E-05

Pltp

2.64

5.90E-08

2.10

0.0264

10.3

4.30E-83

11.9

2.60E-06

9.73

9.70E-68

17.0

8.20E-07

Cd36

17.6

5.20E-217

20.74

1.50E-04

32.5

4.40E-295

46.7

3.20E-05

43.4

0.00

90.2

1.70E-04

Slc27a1

6.62

1.43E-61

7.50

0.00318

34.9

5.10E-202

104

2.80E-07

30.2

2.80E-164

119

2.58E-06

Fabp

1.21

0.721

1.09

0.873

2.24

4.40E-17

3.45

0.0280

9.02

2.30E-62

4.90

0.0164

Afp

0.380

0.0372

0.79

0.560

1.15

1.00

0.684

0.380

1.15

1.00

1.48

0.174

Gstt3

4.11

1.43E-17

1.06

0.811

6.49

2.90E-36

1.01

0.980

9.54

6.70E-28

2.59

0.00300

Gstt2

2.03

7.51E-17

2.44

0.002

2.89

1.10E-40

3.82

5.50E-04

2.81

1.10E-30

8.64

1.00E-04

Nqo1

2.12

0.013

2.20

4.40E-04

2.77

3.90E-07

3.39

3.70E-04

11.3

6.90E-32

31.0

1.60E-06

Acaa1

2.95

3.30E-28

2.51

1.40E-04

4.66

8.90E-64

4.55

2.40E-05

4.75

1.50E-46

12.0

1.00E-06

Cyp2e

0.794

0.0974

0.31

1.20E-04

0.57

0.0760

0.225

4.20E-05

0.393

1.20E-07

0.190

1.70E-05

Cyp2b

20.1

8.90E-19

5.79

1.00E-04

56.0

1.90E-23

19.1

3.20E-04

157

5.14E-35

47.7

4.60E-04

p53

0.971

1.00

1.18

0.652

1.16

0.574

1.21

0.720

1.09

0.690

1.34

0.411

C-myc

3.19

0.0162

83.27

1.70E-04

15.1

4.20E-05

347

1.50E-05

34.5

2.30E-09

648

1.10E-05

pathway

Chemical carcinogenesis



Figure 1. HFPO-TA concentrations in the serum (A) and liver (B) of mice and liver/serum ratios of HFPO-TA and PFOA# (C). Error bars indicate standard errors. Different letters represent statistical significance between groups. # indicates that data were obtained from Yan et al. (2014). 83x27mm (300 x 300 DPI)


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Figure 2. Histopathological analysis of liver sections stained with hematoxylin and eosin (H&E; 200×) (A). Red arrow indicates karyolysis; black arrow indicates necrosis; blue arrow indicates cytoplasmic vacuolation. Comparison of the average number of cell nuclei within a defined area (1392 × 1040) acquired by inForm v2.0 (B) and manual counting (C). Different letters represent statistical significance between groups. 136x85mm (300 x 300 DPI)



Figure 3. Effects of HFPO-TA on liver gene expressions obtained by RNA-seq. Venn diagram for differentially expressed genes determined by RNA sequencing (q value < 0.05), red represents upregulated genes, blue represents downregulated genes (A); Number of changed biological processes highlighted by DAVID in the three HFPO-TA-treated groups (B); Heat map of KEGG enrichment pathways in the three treatment groups (C); Enrichment ratio of pathways by KEGG analysis in the 0.02 mg/kg/d group (D). 177x157mm (300 x 300 DPI)


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Figure 4. Protein expression levels in each treated group: representative blots from three experiments and mean levels of protein bands compared with the control. Values indicate means ± SE (n = 3); error bars indicate standard errors; different letters represent significance between groups at p < 0.05 by ANOVA and Duncan’s multiple range tests. 92x42mm (300 x 300 DPI)



TOC 47x26mm (300 x 300 DPI)


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Hepatotoxic Effects of Hexafluoropropylene Oxide Trimer Acid (HFPO

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