Subscriber access provided by Drexel University Libraries
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 32
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:
Environmental Science & Technology
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
ACS Paragon Plus Environment
Page 2 of 32
Page 3 of 32
Environmental Science & Technology
22
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
ACS Paragon Plus Environment
alternative
to
PFOA,
Environmental Science & Technology
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
ACS Paragon Plus Environment
Page 4 of 32
Page 5 of 32
Environmental Science & Technology
66
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
ACS Paragon Plus Environment
Environmental Science & Technology
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
ACS Paragon Plus Environment
Page 6 of 32
Page 7 of 32
Environmental Science & Technology
110
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
ACS Paragon Plus Environment
Environmental Science & Technology
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
ACS Paragon Plus Environment
Page 8 of 32
Page 9 of 32
Environmental Science & Technology
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
ACS Paragon Plus Environment
Environmental Science & Technology
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
ACS Paragon Plus Environment
Page 10 of 32
Page 11 of 32
Environmental Science & Technology
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
ACS Paragon Plus Environment
Environmental Science & Technology
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
ACS Paragon Plus Environment
Page 12 of 32
Page 13 of 32
242
Environmental Science & Technology
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
ACS Paragon Plus Environment
Environmental Science & Technology
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
ACS Paragon Plus Environment
Page 14 of 32
Page 15 of 32
Environmental Science & Technology
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
ACS Paragon Plus Environment
Environmental Science & Technology
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
ACS Paragon Plus Environment
Page 16 of 32
Page 17 of 32
Environmental Science & Technology
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
ACS Paragon Plus Environment
Environmental Science & Technology
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
ACS Paragon Plus Environment
Page 18 of 32
Page 19 of 32
Environmental Science & Technology
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
ACS Paragon Plus Environment
Environmental Science & Technology
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).
REFERENCES 1. Buck, R. C.; Franklin, J.; Berger, U.; Conder, J. M.; Cousins, I. T.; de Voogt, P.; Jensen, A. A.; Kannan, K.; Mabury, S. A.; van Leeuwen, S. P. Perfluoroalkyl and polyfluoroalkyl substances in the environment: terminology, classification, and origins. Integr Environ Assess Manag 2011, 7 (4), 513-41. 2. Kissa, E. Fluorinated surfactants: synthesis properties applications (Surfactant science series 50). New York (NY): Marcel Dekker. 469 p. 1994. 3. Kissa, E. Fluorinated Surfactants and Repellents (2nd edition revised and 20
ACS Paragon Plus Environment
Page 20 of 32
Page 21 of 32
Environmental Science & Technology
expanded) (Surfactant science series 97). New York (NY): Marcel Dekker. 640 p. 2001. 4. Smart, B. E., Characteristics of C-F Systems. In Organofluorine Chemistry: Principles and Commercial Applications, Banks, R. E.; Smart, B. E.; Tatlow, J. C., Eds. Springer US: Boston, MA, 1994; pp 57-88. 5. Steenland, K.; Fletcher, T.; Savitz, D. A. Epidemiologic evidence on the health effects of perfluorooctanoic acid (PFOA). Environ Health Perspect 2010, 118 (8), 1100-8. 6. Guruge, K. S.; Yeung, L. W. Y.; Yamanaka, N.; Miyazaki, S.; Lam, P. K. S.; Giesy, J. P.; Jones, P. D.; Yamashita, N. Gene Expression Profiles in Rat Liver Treated With Perfluorooctanoic Acid (PFOA). Toxicological Sciences 2006, 89 (1), 93-107. 7. Hines, E. P.; White, S. S.; Stanko, J. P.; Gibbs-Flournoy, E. A.; Lau, C.; Fenton, S. E. Phenotypic dichotomy following developmental exposure to perfluorooctanoic acid (PFOA) in female CD-1 mice: Low doses induce elevated serum leptin and insulin, and overweight in mid-life. Molecular & Cellular Endocrinology 2009, 304 (1), 97-105. 8. U.S., E. P. A. 2010/2015 PFOA stewardship program. 2006. 9. Assessment., C. f. R. Background document to the opinion on the Annex XV dossier proposing restrictions on perfluorooctanoic acid (PFOA), PFOA salts and PFOA-related substances. 2015. 10. Ritter, S. K. Fluorochemicals go short. Chemical & Engineering News 2010, 88 (5), 12-17. 11. Gordon, S. C. Toxicological evaluation of ammonium 4,8-dioxa-3Hperfluorononanoate, a new emulsifier to replace ammonium perfluorooctanoate in fluoropolymer manufacturing. Regul Toxicol Pharmacol 2011, 59 (1), 64-80. 12. Strynar, M.; Dagnino, S.; Lindstrom, A.; Andersen, E.; McMillan, L.; Thurman, M.; Ferrer, I.; Ball, C. In Identification of novel polyfluorinated compounds in natural waters using accurate mass TOFMS, 33rd SETAC North America Annual Meeting, 2012; 2012; pp 11-15. 13. Strynar, M.; Dagnino, S.; McMahen, R.; Liang, S.; Lindstrom, A.; Andersen, E.; McMillan, L.; Thurman, M.; Ferrer, I.; Ball, C. Identification of Novel Perfluoroalkyl Ether Carboxylic Acids (PFECAs) and Sulfonic Acids (PFESAs) in Natural Waters Using Accurate Mass Time-of-Flight Mass Spectrometry (TOFMS). Environ Sci Technol 2015, 49 (19), 11622-30. 14. Pan, Y. T.; Zhang, H. X.; Cui, Q. Q.; Sheng, N.; Yeung, L. W. Y.; Guo, Y.; Sun, Y.; Dai, J. Y. First Report on the Occurrence and Bioaccumulation of Hexafluoropropylene Oxide Trimer Acid: An Emerging Concern. Environ Sci Technol 2017, 51 (17), 9553-9560. 15. Sheng, N.; Pan, Y. T.; Guo, Y.; Sun, Y.; Dai, J. Y. Hepatotoxic Effects of Hexafluoropropylene Oxide Trimer Acid (HFPO-TA), A Novel Perfluorooctanoic Acid (PFOA) Alternative, on Mice. Environ Sci Technol 2018, 52 (14), 8005-8015. 16. LfU (Bavarian Enviroment Agency) PFOA Und ADONA Messungen an Der Probenahmestelle Alz. Online: http://www.lfu.bayern.de/analytik stoffe/analytik org stoffe perfluorierte chemikalien/doc/pfoa adona alz.pdf. 2016. 17. Heydebreck, F.; Tang, J.; Xie, Z.; Ebinghaus, R. Alternative and Legacy Perfluoroalkyl Substances: Differences between European and Chinese River/Estuary 21
ACS Paragon Plus Environment
Environmental Science & Technology
Systems. Environ Sci Technol 2015, 49 (14), 8386-95. 18. Sun, M.; Arevalo, E.; Strynar, M.; Lindstrom, A.; Richardson, M.; Kearns, B.; Pickett, A.; Smith, C.; Knappe, D. R. U. Legacy and Emerging Perfluoroalkyl Substances Are Important Drinking Water Contaminants in the Cape Fear River Watershed of North Carolina. Environmental Science & Technology Letters 2016, 3 (12), 415-419. 19. Gannon, S. A.; Fasano, W. J.; Mawn, M. P.; Nabb, D. L.; Buck, R. C.; Buxton, L. W.; Jepson, G. W.; Frame, S. R. Absorption, distribution, metabolism, excretion, and kinetics of 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoic acid ammonium salt following a single dose in rat, mouse, and cynomolgus monkey. Toxicology 2016, 340, 1-9. 20. Wang, J. S.; Wang, X. Y.; Sheng, N.; Zhou, X. J.; Cui, R. N.; Zhang, H. X.; Dai, J. Y. RNA-sequencing analysis reveals the hepatotoxic mechanism of perfluoroalkyl alternatives, HFPO2 and HFPO4, following exposure in mice. J Appl Toxicol 2017, 37 (4), 436-444. 21. Mew, N. A.; Pappa, M. B.; Gropman, A. L., Chapter 57 - Urea Cycle Disorders. In Rosenberg's Molecular and Genetic Basis of Neurological and Psychiatric Disease (Fifth Edition), Rosenberg, R. N.; Pascual, J. M., Eds. Academic Press: Boston, 2015; pp 633-647. 22. Yan, S.; Wang, J.; Zhang, W.; Dai, J. Circulating microRNA profiles altered in mice after 28 d exposure to perfluorooctanoic acid. Toxicology Letters 2014, 224 (1), 24-31. 23. Wang, J.; Zhang, Y.; Zhang, W.; Jin, Y.; Dai, J. Association of perfluorooctanoic acid with HDL cholesterol and circulating miR-26b and miR-199-3p in workers of a fluorochemical plant and nearby residents. Environ Sci Technol 2012, 46 (17), 927481. 24. Walker, J. M. The bicinchoninic acid (BCA) assay for protein quantitation. Methods Mol Biol 1994, 32, 5-8. 25. Zhang, H.; Zhou, X.; Sheng, N.; Cui, R.; Cui, Q.; Guo, H.; Guo, Y.; Sun, Y.; Dai, J. Subchronic Hepatotoxicity Effects of 6:2 Chlorinated Polyfluorinated Ether Sulfonate (6:2 Cl-PFESA), a Novel Perfluorooctanesulfonate (PFOS) Alternative, on Adult Male Mice. Environmental Science & Technology 2018, 52 (21), 12809-12818. 26. Pinero, J.; Queralt-Rosinach, N.; Bravo, A.; Deu-Pons, J.; Bauer-Mehren, A.; Baron, M.; Sanz, F.; Furlong, L. I. DisGeNET: a discovery platform for the dynamical exploration of human diseases and their genes. Database 2015, 2015 (0), bav028bav028. 27. Osborne, J. D.; Flatow, J.; Holko, M.; Lin, S. M.; Kibbe, W. A.; Zhu, L.; Danila, M. I.; Feng, G.; Chisholm, R. L. Annotating the human genome with Disease Ontology. BMC Genomics 2009, 10 (Suppl 1). 28. Kibbe, W. A.; Arze, C.; Felix, V.; Mitraka, E.; Bolton, E.; Fu, G.; Mungall, C. J.; Binder, J. X.; Malone, J.; Vasant, D.; Parkinson, H.; Schriml, L. M. Disease Ontology 2015 update: an expanded and updated database of human diseases for linking biomedical knowledge through disease data. Nucleic Acids Res 2015, 43 (Database issue), D1071-8. 22
ACS Paragon Plus Environment
Page 22 of 32
Page 23 of 32
Environmental Science & Technology
29. Schriml, L. M.; Arze, C.; Nadendla, S.; Chang, Y. W.; Mazaitis, M.; Felix, V.; Feng, G.; Kibbe, W. A. Disease Ontology: a backbone for disease semantic integration. Nucleic Acids Res 2012, 40 (Database issue), D940-6. 30. Szklarczyk, D.; Franceschini, A.; Kuhn, M.; Simonovic, M.; Roth, A.; Minguez, P.; Doerks, T.; Stark, M.; Muller, J.; Bork, P.; Jensen, L. J.; von Mering, C. The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Research 2011, 39, D561-D568. 31. Kohl, M.; Wiese, S.; Warscheid, B., Cytoscape: Software for Visualization and Analysis of Biological Networks. In Data Mining in Proteomics: From Standards to Applications, Hamacher, M.; Eisenacher, M.; Stephan, C., Eds. Humana Press: Totowa, NJ, 2011; pp 291-303. 32. Scardoni, G.; Petterlini, M.; Laudanna, C. Analyzing biological network parameters with CentiScaPe. Bioinformatics 2009, 25 (21), 2857-9. 33. Yan, S. M.; Wang, J. S.; Dai, J. Y. Activation of sterol regulatory element-binding proteins in mice exposed to perfluorooctanoic acid for 28 days. Arch Toxicol 2015, 89 (9), 1569-78. 34. ECHA Substance Name: Ammonium pentadecafluorooctanoate (APFO) EC Number: 223-320-4 CAS Number: 3825-26-1. 35. Lee, B.; Diaz, G. A.; Rhead, W.; Lichter-Konecki, U.; Feigenbaum, A.; Berry, S. A.; Le Mons, C.; Bartley, J. A.; Longo, N.; Nagamani, S. C.; Berquist, W.; Gallagher, R.; Bartholomew, D.; Harding, C. O.; Korson, M. S.; McCandless, S. E.; Smith, W.; Cederbaum, S.; Wong, D.; Merritt, J. L., II; Schulze, A.; Vockley, G.; Kronn, D.; Zori, R.; Summar, M.; Milikien, D. A.; Marino, M.; Coakley, D. F.; Mokhtarani, M.; Scharschmidt, B. F.; Consortium, U. C. D. Blood ammonia and glutamine as predictors of hyperammonemic crises in patients with urea cycle disorder. Genetics in Medicine 2015, 17 (7), 561-568. 36. Swain, M.; Butterworth, R. F.; Blei, A. T. Ammonia and related amino-acids in the pathogenesis of brain edema in acute ischemic liver-failure in rats. Hepatology 1992, 15 (3), 449-453. 37. Norenberg, M. D.; Baker, L.; Norenberg, L. O. B.; Blicharska, J.; Brucegregorios, J. H.; Neary, J. T. Ammonia-induced astrocyte swelling in primary culture. Neurochemical Research 1991, 16 (7), 833-836. 38. Eefsen, M.; Jelnes, P.; Schmidt, L. E.; Vainer, B.; Bisgaard, H. C.; Larsen, F. S. Brain expression of the water channels Aquaporin-1 and-4 in mice with acute liver injury, hyperammonemia and brain edema. Metabolic Brain Disease 2010, 25 (3), 315323. 39. Lajiness, M. S. In Molecular similarity-based methods for selecting compounds for screening, Computational Chemical Graph Theory, 1990; 1990. 40. Zhang, H. X.; Zhou, X. J.; Sheng, N.; Cui, R. N.; Cui, Q. Q.; Guo, H.; Guo, Y.; Sun, Y.; Dai, J. Y. Subchronic Hepatotoxicity Effects of 6:2 Chlorinated Polyfluorinated Ether Sulfonate (6:2 Cl-PFESA), a Novel Perfluorooctane Sulfonate (PFOS) Alternative, on Adult Male Mice. Environ Sci Technol 2018. 41. Walters, M. W.; Wallace, K. B. Urea cycle gene expression is suppressed by PFOA treatment in rats. Toxicol Lett 2010, 197 (1), 46-50. 23
ACS Paragon Plus Environment
Environmental Science & Technology
42. Liu, H.; Dong, H.; Robertson, K.; Liu, C. DNA methylation suppresses expression of the urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) in human hepatocellular carcinoma. Am J Pathol 2011, 178 (2), 652-61. 43. Mew, N. A.; Pappa, M. B.; Gropman, A. L., Urea Cycle Disorders. In Rosenberg's Molecular and Genetic Basis of Neurological and Psychiatric Disease, 2015; pp 633647. 44. Pagon, R. A.; Adam, M. P.; Ardinger, H. H.; Bird, T. D.; Dolan, C. R.; Fong, C. T.; Rjh, S.; Stephens, K. Urea Cycle Disorders Overview -- GeneReviews(®). 1993. 45. Lee, J. S.; Adler, L.; Karathia, H.; Carmel, N.; Rabinovich, S.; Auslander, N.; Keshet, R.; Stettner, N.; Silberman, A.; Agemy, L.; Helbling, D.; Eilam, R.; Sun, Q.; Brandis, A.; Malitsky, S.; Itkin, M.; Weiss, H.; Pinto, S.; Kalaora, S.; Levy, R.; Barnea, E.; Admon, A.; Dimmock, D.; Stern-Ginossar, N.; Scherz, A.; Nagamani, S. C. S.; Unda, M.; Wilson, D. M., 3rd; Elhasid, R.; Carracedo, A.; Samuels, Y.; Hannenhalli, S.; Ruppin, E.; Erez, A. Urea Cycle Dysregulation Generates Clinically Relevant Genomic and Biochemical Signatures. Cell 2018, 174 (6), 1559-1570 e22. 46. Souba, W. W. Interorgan ammonia metabolism in health and disease: a surgeon's view. Journal of Parenteral & Enteral Nutrition 1987, 11 (6), 569-579. 47. Burton, B. K. Urea cycle disorders. Clinics in Liver Disease 2000, 4 (4), 815-830. 48. Zhuang, Z.; Lin, Y.; Yang, C.; Yuan, J.; Wang, S. Study on CPS1: The key gene of urea cycle under the stress of aflatoxin B1. Asian Journal of Chemistry 2014, 26 (11), 3305-3310. 49. Diez-Fernandez, C.; Hu, L.; Cervera, J.; Haberle, J.; Rubio, V. Understanding carbamoyl phosphate synthetase (CPS1) deficiency by using the recombinantly purified human enzyme: effects of CPS1 mutations that concentrate in a central domain of unknown function. Mol Genet Metab 2014, 112 (2), 123-32. 50. Ah Mew, N.; Caldovic, L. N-acetylglutamate synthase deficiency: an insight into the genetics, epidemiology, pathophysiology, and treatment. Appl Clin Genet 2011, 4, 127-35. 51. Caldovic, L.; Ah Mew, N.; Shi, D.; Morizono, H.; Yudkoff, M.; Tuchman, M. Nacetylglutamate synthase: structure, function and defects. Mol Genet Metab 2010, 100 Suppl 1, S13-9. 52. Shigesada, K. T., M. N-Acetylglutamate synthetase from rat-liver mitochondria. Partial purification and catalytic properties. European journal of biochemistry / FEBS 1978, (84. 285-91. ). 53. Nakagawa, T.; Lomb, D. J.; Haigis, M. C.; Guarente, L. SIRT5 Deacetylates carbamoyl phosphate synthetase 1 and regulates the urea cycle. Cell 2009, 137 (3), 56070. 54. Shi, D.; Allewell, N. M.; Tuchman, M. The N-Acetylglutamate Synthase Family: Structures, Function and Mechanisms. International Journal of Molecular Sciences 2015, 16 (6), 13004-13022. 55. Yamaguchi, S.; Brailey, L. L.; Morizono, H.; Bale, A. E.; Tuchman, M. Mutations and polymorphisms in the human ornithine transcarbamylase (OTC) gene. Hum Mutat 2006, 27 (7), 626-32. 56. Philip, J. S. M. D. Ornithine Transcarbamylase. Encyclopedia of Molecular 24
ACS Paragon Plus Environment
Page 24 of 32
Page 25 of 32
Environmental Science & Technology
Mechanisms of Disease 2004, 1531-1531. 57. Mori, M.; Gotoh, T. Arginine metabolic enzymes, nitric oxide and infection. Journal of Nutrition 2004, 134 (10), 2820S-2825S. 58. Erez, A.; Nagamani, S. C. S.; Shchelochkov, O. A.; Premkumar, M. H.; Campeau, P. M.; Chen, Y.; Garg, H. K.; Li, L.; Mian, A.; Bertin, T. K.; Black, J. O.; Zeng, H.; Tang, Y.; Reddy, A. K.; Summar, M.; O'Brien, W. E.; Harrison, D. G.; Mitch, W. E.; Marini, J. C.; Aschner, J. L.; Bryan, N. S.; Lee, B. Requirement of argininosuccinate lyase for systemic nitric oxide production. Nature Medicine 2011, 17 (12), 1619-U134. 59. Mian, A.; Lee, B. Urea-cycle disorders as a paradigm for inborn errors of hepatocyte metabolism. Trends in Molecular Medicine 2002, 8 (12), 583-589. 60. Frisell, W.; Frisell, W. Metabolic fate of nitrogen. Human Biochemistry. Frisell WR, ed. New York: MacMillan 1982, 236-249. 61. Sofroniew, M. V.; Vinters, H. V. Astrocytes: biology and pathology. Acta Neuropathol 2010, 119 (1), 7-35. 62. Rao, K. V. R.; Chen, M.; Simard, J. M.; Norenberg, M. D. Increased aquaporin-4 expression in ammonia-treated cultured astrocytes. Neuroreport 2003, 14 (18), 23792382. 63. McCoy, E.; Sontheimer, H. MAPK induces AQP1 expression in astrocytes following injury. Glia 2010, 58 (2), 209-17. 64. Badaut, J.; Brunet, J. F.; Grollimund, L.; Hamou, M. F.; Magistretti, P. J.; Villemure, J. G.; Regli, L., Aquaporin 1 and aquaporin 4 expression in human brain after subarachnoid hemorrhage and in peritumoral tissue. In Brain Edema Xii, Kuroiwa, T.; Baethmann, A.; Czernicki, Z.; Hoff, J. T.; Ito, U.; Katayama, Y.; Mararou, A.; Mendelow, A. D.; Reulen, H. J., Eds. 2003; Vol. 86, pp 495-498. 65. Oshio, K.; Binder, D. K.; Liang, Y.; Bollen, A.; Feuerstein, B.; Berger, M. S.; Manley, G. T. Expression of the aquaporin-1 water channel in human glial tumors. Neurosurgery 2005, 56 (2), 375-380. 66. Badaut, T.; Lasbennes, T.; Magistretti, P. J.; Regli, L. Aquaporins in brain: Distribution, physiology, and pathophysiology. Journal of Cerebral Blood Flow and Metabolism 2002, 22 (4), 367-378. 67. Yang, B.; Zador, Z.; Verkman, A. S. Glial cell aquaporin-4 overexpression in transgenic mice accelerates cytotoxic brain swelling. J Biol Chem 2008, 283 (22), 15280-6. 68. Lemberg, A.; Fernandez, A., Hepatic encephalopathy, ammonia, glutamate, glutamine and oxidative stress. 2009; Vol. 8, p 95-102.
25
ACS Paragon Plus Environment
Environmental Science & Technology
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.
ACS Paragon Plus Environment
Page 26 of 32
Page 27 of 32
Environmental Science & Technology
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.
ACS Paragon Plus Environment
Environmental Science & Technology
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.
ACS Paragon Plus Environment
Page 28 of 32
Page 29 of 32
Environmental Science & Technology
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.
ACS Paragon Plus Environment
Environmental Science & Technology
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.
ACS Paragon Plus Environment
Page 30 of 32
Page 31 of 32
Environmental Science & Technology
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.
ACS Paragon Plus Environment
Environmental Science & Technology
For TOC art only 84x47mm (300 x 300 DPI)
ACS Paragon Plus Environment
Page 32 of 32