Subscriber access provided by - Access paid by the | UCSB Libraries
Food Safety and Toxicology
Effect of propiconazole on the lipid metabolism of zebrafish embryos (Danio rerio) Miaomiao Teng, Feng Zhao, Yimeng Zhou, Sen Yan, Sinuo Tian, Jin Yan, Zhiyuan Meng, Sheng Bi, and Chengju Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00449 • Publication Date (Web): 05 Apr 2019 Downloaded from http://pubs.acs.org on April 7, 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 40
Journal of Agricultural and Food Chemistry
Effect of propiconazole on the lipid metabolism of zebrafish embryos (Danio rerio)
Miaomiao Teng1, Feng Zhao1, Yimeng Zhou1, Sen Yan2, Sinuo Tian2, Jin Yan2, Zhiyuan Meng2, Sheng Bi3, Chengju Wang1*
1. Department of Applied Chemistry, College of Science, China Agricultural University, Beijng, China 2. Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Science, China Agricultural University, Beijng, China 3. Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Correspondence author Chengju Wang Tel: +86(0)10-62733924 Fax: 010-62734294 E-mail:
[email protected] Address: Yuanmingyuan West Road 2, China Agricultural University, Beijing, 100193, People’s Republic of China
1 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
1
Abstract
2
Propiconazole is a triazole fungicide that has been widely used in agriculture and has
3
been detected in the aquatic environment. This study aimed to investigate the effects of
4
propiconazole exposure on lipid metabolism in the early life stages of zebrafish for 120
5
hours post-fertilization (hpf). Using the early life stages of zebrafish to address
6
scientific questions is lower cost, more efficient and suitable for meeting current
7
legislation than other traditional fish species. Exposure to propiconazole significantly
8
inhibited the development of zebrafish embryos and larvae. This exposure also caused
9
reduced locomotor activities in zebrafish. Furthermore, total cholesterol levels,
10
lipoprotein lipase and fatty acid synthase activities were significantly decreased. The
11
expression levels of genes involved in lipid metabolism were significantly up-regulated
12
in response to propiconazole exposure. GC-MS/MS analysis revealed that fatty acids
13
were significantly decreased. Together, the findings indicate the potential
14
environmental risk of propiconazole exposure in the aquatic ecosystem.
15
Keywords: propiconazole, zebrafish embryo, lipid metabolism, fatty acids
2 ACS Paragon Plus Environment
Page 2 of 40
Page 3 of 40
Journal of Agricultural and Food Chemistry
16
Introduction
17
Propiconazole (PCZ) (1-((2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl)methyl-
18
1H-1,2,4-trizole) is a broad-spectrum and high efficiency triazole fungicide that has
19
been widely used in agriculture and horticulture to inhibit fungal growth
20
propiconazole is a common fungicide used for crop farming and is easily transported to
21
the ecosystem through spray drift, surface run-off, and rainfall, its residues have been
22
detected in the aquatic environment (Table 1), as well as in vegetables, fruits, and
23
human sera
24
environment has a potentially adverse effect on some aquatic organisms such as
25
Daphnia manga and Channa punctata Bloch 7, 8.
26
Previous reports have also shown that propiconazole can cause toxic effects on
27
vertebrates. Levels of the reactive oxygen species (ROS) were increased in cultured
28
hepatic cells and in mouse liver after exposure to propiconazole, and the changes
29
resulting from propiconazole were also revealed in mice via genomics and proteomics
30
9, 10.
31
induces hepatocarcinogenesis of mice 11. Juvenile rainbow trout (Oncorhynchus mykiss)
32
exposed to propiconazole exhibit various physiological responses, including hepatic
33
ethoxyresorufin-O-deethylase (EROD) activity, antioxidant indices, morphological
34
indices, and hematological parameters
35
activity of lanosterol-14α-demethylase enzyme, the one that is essential for ergo sterol
36
biosynthesis, and suppresses cytochrome P450 enzyme activity (CYP51) to block
37
fungal cell wall chitin
3-6.
1, 2.
Since
Recent studies have reported that propiconazole in the water
In addition, data have shown that propiconazole affects hepatic metabolism and
13.
12.
Additionally, propiconazole inhibits the
Triazole fungicides have been considered mainly to affect 3 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 40
38
lipid biosynthesis and metabolism pathways 14-17.
39
Lipids play an important role in energy supply and maintaining normal metabolic
40
activities 18. Lipid homeostasis requires precise control of lipid process, including lipid
41
accumulation, lipogenesis, fatty acid β-oxidation, cholesterol synthesis and metabolism.
42
Disordered homeostasis leads to obesity, malnutrition, endocrine disruption, or other
43
metabolic-associated diseases
44
syndrome in 21 century. The WHO has reported that there are at least 41 million
45
children under 5 years old with obesity or overweight 23. One possibility is that a rise
46
in environmental pollutants may contribute to obesity or other health problems
47
Thus, the investigation of the effects of environmental chemical exposure on the lipid
48
metabolism of organisms in the early stages is urgent. Data have shown seasonal
49
variation in lipid metabolism of yellow perch (Perca flavescens) chronically exposed
50
to sub-lethal levels of heavy metals (Cd, Zn, Cu) (Rouyn-Noranda, Quebec)26. Studies
51
have also shown that nano-sized particles transported through the food chain affect
52
behavior and lipid metabolism in Crucian carp (genus Carassius), Bleak (Alburnus
53
alburnus), Rudd (Scardinius erythrophthalmus), Tench (Tinca tinca), Pike (Esox esox),
54
and Atlantic salmon (Salmo salar)27. Carnevali., et al have reported that the mixtures
55
of endocrine disruption chemicals (EDCs) affect lipogenesis and fat deposition in the
56
Juvenile seabream 28. Different types of toxicant substances alter lipid metabolism in
57
marine and fresh water fish. Additionally, previous studies demonstrate that
58
propiconazole increases cell proliferation and Ras farnesylation in AML12 mouse
59
hepatocytes via the dysregulation of the cholesterol biosynthesis pathway 29. Therefore,
19-22.
For instance, obesity is an epidemic metabolic
4 ACS Paragon Plus Environment
24, 25.
Page 5 of 40
Journal of Agricultural and Food Chemistry
60
it is important to investigate the potential mechanism of the effect of propiconazole
61
exposure on lipid metabolism in the early stages of freshwater organisms.
62
The zebrafish embryo, as an ideal aquatic vertebrate model, has a rapid life cycle, and
63
is easy to observe its morphological process during the development because the
64
embryo is transparent
65
questions is more suitable for meeting current legislation
66
genes in mammals have been identified in zebrafish
67
used for investigating the toxic effects of environmental pollutants 17, 34, 35.
68
In the present study, we investigated the developmental effects, locomotor activity,
69
lipid synthesis and metabolism change, and free fatty acid alterations in the early stages
70
of zebrafish with exposure to different concentrations of propiconazole (0, 0.5 mg/L,
71
2.5 mg/, 4.5 mg/L). These results provide the insight into the underlying mechanism of
72
the effects of propiconazole exposure on lipid metabolism, predicting the potential
73
environmental risk of propiconazole to aquatic organisms.
74
Material and methods
75
Reagents. Propiconazole (CAS#: 60207-90-1; 95% purity) was obtained from China
76
Ministry of Agriculture. The stock solutions were prepared in acetone (purity > 99%).
77
Standard water was used in the lab, containing 0.5 mM Mg2+, 2 mM Ca2+, 0.074 mM
78
K+ and 0.75 mM Na+. 36. All other chemicals were at the analytical grade.
79
Experimental design and sample collection. Five-month old wild-type zebrafish (AB
80
strain, Danio rerio) were obtained from Hongdagaofeng fish shop. The parental
81
zebrafish were cultured in flow-through equipment (Esen Corp, Beijing, China) under
30.
In addition, using zebrafish embryos to address scientific
32, 33.
5 ACS Paragon Plus Environment
31.
Furthermore, most of
Therefore, the embryo was
Journal of Agricultural and Food Chemistry
82
14h:10h light/dark at 28 oC for 14 days. Zebrafish were fed with Artemia nauplii twice
83
daily. Zebrafish embryos were maintained according to our previous study 17. A range
84
of concentrations (0, 10, 11, 13.2 14.52, 15.97, 17.57 mg/L and 0, 0.42, 0.67, 1.07, 1.72,
85
2.75 mg/L) of propiconazole exposed to zebrafish embryos on the basis of pre-
86
experiment data to test acute toxicity of embryo. Six replicates were used for each test
87
solution of propiconazole (0, 0.5, 2.5, and 4.5 mg/L) based on the results from pilot
88
studies. Control and propiconazole treatments received 0.01% (v/v) acetone. Two hours
89
post fertilization (hpf), normal embryos were randomly assigned into the test groups.
90
Each replicate contained 200 embryos in 500 mL solution. During the exposure period,
91
the test solution was kept at 28 oC and the day/night cycle was 14h/10h. The exposure
92
media was changed daily. The embryos from each group were observed and recorded
93
using light microscopy at 24, 48, 72, 96, and 120 hpf. After 120 h exposure, zebrafish
94
larvae were collected and frozen in liquid nitrogen, and then stored at -80 oC for further
95
analysis.
96
Embryonic developmental test. During the propiconazole exposure, 10 larvae were
97
selected as one replicate (n = 6 replicates) and put into 24-well plates to observe the
98
embryonic development. We observed the spontaneous movement of embryos in 20s
99
at 24 hpf and counted the heartbeat number in 20s at 48 hpf. The hatching rate was
100
recorded at 72, 96, and 120 hpf. After 120 hpf, we recorded teratogenic effects and
101
measured the body length of hatched individual larvae using digital microscope (Aigo
102
GE-5, Beijing, China).
103
Determination of larval locomotor activity. Based on the previous study, the 6 ACS Paragon Plus Environment
Page 6 of 40
Page 7 of 40
Journal of Agricultural and Food Chemistry
104
locomotor activity of 120 hpf larvae was measured using the Video-Track system (UI-
105
3240CP-C-GL, IDS Imaging Development System GmbH, Obersulm, Germany)
106
following the manufacture’s protocol37. Average velocity, distance movement, active
107
and inactive time, average acceleration and deceleration were recorded from 10 larvae
108
per concentration every 10 mins (n = 6 replicates) and further analyzed using uEye
109
Cockpit software Loligo System, United Ststes).
110
Measurement of triglyceride (TG) and total cholesterol (TCHO) content. 30 larvae
111
(n = 6 replicates) were homogenized in 270 μL cell lysates (Applygen, Beijing, China)
112
and centrifuged at 3000 g for 10 min at 4 oC. Thus the supernatant was used for
113
determinations of total protein using a bicinchoninic acid (BCA) protein assay kit
114
(Cwbiotech, Beijing, China)16. And then the remained samples were incubated for 10
115
min in 70 oC water to detect TG and TCHO contents according to the enzymatic kits
116
(Applygen, Beijing, China) according to previous studies16. TG and TCHO were
117
normalized to the sample protein concentration. The content of was measured
118
Absorbance of TCHO, TG and total protein were measured using the microplate reader
119
(Multiscan MK3, Thermo Scientific) at 540, 540, 595 nm, respectively. The calibration
120
of protein was shown in Figure S1.
121
Lipoprotein lipase (LPL) and fatty acid synthase (FASN) activity assay.
122
Lipoprotein lipase and fatty acid synthase were extracted from 120 hpf larvae samples
123
(30 larvae, n = 6 replicates). Samples were homogenized in 270 μL saline and
124
centrifuged at 3000 g for 10 min at 4 oC. The supernatant was used to enzymatic activity
125
analysis. The LPL and FASN activity assays were determined using enzyme-linked 7 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 8 of 40
126
immunosorbent assay (ELISA) kits (Fu life Industry Co., Ltd. Shanghai, China)38. The
127
range suitability of LPL and FASN were 1.6-65 U/mL, 10-360 U/L, respectively. The
128
standard calibration of LPL and FASN were shown in Figure S2.
129
Quantitative real-time polymerase chain reaction (qRT-PCR) assay. After 120 h
130
exposure, 30 embryos were collected and dissolved in Trizol reagent for total RNA
131
extraction according to previous method
132
using FastQuant RTase kit (Tiangen Biotech, Beijing, China). Quantitative real-time
133
polymerase chain reaction (qRT-PCR) was conducted using SYBR Green PCR Master
134
Mix reagent kit (Tiangen Biotech) and performed using an ABI 7500 PCR system
135
(Advanced Biosystems, Foster City, CA, USA). Thermal cycling was set at 95 °C for
136
15 min, followed by 40 cycles at 95 °C for 10 s, 60°C for 20s, and 72°C for 32s.
137
Transcription of target genes was calculated using the 2−ΔΔCt method. β-actin was
138
chosen as the house-keeping gene. All primers of target genes were designed using
139
Primer 6.0 software and synthesized by Sangon Biotechology (Shanghai, China) (Table
140
S1).
141
Metabolomics analysis based Gas chromatography-mass spectrometry (GC-MS).
142
Embryos (80 embryo, n = 6 replicates) were extracted as previously described
143
Briefly, 80 embryos were collected and homogenized in 20 μL internal standard (50
144
μg/L C15:0 fatty acid and C17:0 methyl ester) and 600 μL extracting solution
145
(chloroform : methanol = 1:2, v/v). Then, the homogenization was added into 200 μL
146
chloroform and 200 μL water, vortexed for 2 min, and centrifuged at 18001 g for 10
147
min. Extraction was repeated again and evaporated to dryness under a stream of
22 39
The first-strand cDNA was synthesized
8 ACS Paragon Plus Environment
40, 41.
Page 9 of 40
Journal of Agricultural and Food Chemistry
148
nitrogen, following derivatization with 1 m L methanol/hydrochloric acid (41.5/9.7 mL)
149
for 12 h at 60 oC. The solvent was cleaned up by hexane-saline (1:1). After extracts
150
were evaporated by nitrogen, each solvent was dissolved with 200 μL hexane and then
151
transferred to an injection vial for analysis. Free fatty acids were measured using a
152
Thermo Fisher Scientific Trace GC gas chromatograph coupled to a Quantum XLS
153
mass spectrometer (Thermo Scientific, Waltham, MA, USA) using a DB-5 column (30
154
m×0.31 mm×0.25 mm), running in full scan mode. Data analysis was conducted with
155
Thermo Xcalibur software. Fatty acids were quantified by normalizing the integrated
156
peak areas to the internal standards.
157
Propiconazole in water analysis
158
Water samples were collected at the beginning of exposure (0 h) and at 24 h. All
159
exposure solutions were analyzed for all treatments. The experimental solutions were
160
filtered with a 0.22 μm filtration membrane and then determined using ultra-high
161
performance liqud chromatography – tandem mass spectrometry (UHPLC-MS/MS)
162
(ultiMate 3000 system,
163
described 42, 43.
164
Statistical analysis. All statistical analyses were performed with SPSS 19.0 (IBM,
165
USA). Significant differences were determined by one-way ANOVA analysis on the
166
basis of Dunnett post hoc comparison (P < 0.05). All values were presented as the mean
167
± standard deviation (SD).
168
Results
169
Assay validation
Thermo Scientific, Waltham, MA, USA) as previously
9 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
170
Spiked samples with 1, 50, and 2500 μ,/L of propiconazole were analyzed. The
171
recovery of propiconazole in solutions ranged from 93.8% to 106.9%. (Table S2). An
172
external standard calibration curve was used to calculate the amount of propiconazole
173
(Figure S3). The range of propiconazole concentrations and linear regression equations
174
was 1-1000 μg/L, R2=0.99, respectively. Concentrations of propiconazole in water (0,
175
0.5, 2.5, 4.5mg/L) were detected. The measured propiconazole concentrations in water
176
samples were 80-120% of nominal values (Table S3).
177
Lethal effect of propiconazole
178
According to the results, the 96 h half dose/lethal concentration (LD50/LC50) value of
179
embryo (95% conference limit) was 12.90 (12.40-13.40) mg/L with linear equation Y=
180
14.33X-15.92 (R2 = 0.97). The low observed effect concentration (LOEC) was 1.72
181
mg/L and the no observed effect concentration (NOEC) was 1.07 mg/L at 96 hpf
182
(Figure S4). There is no significance in other parameters.
183
Toxicological endpoints in embryos.
184
The effects of propiconazole exposure on the spontaneous movement, heartbeat,
185
hatching rate, and body length of embryos and larvae were shown in Figure 1.
186
Propiconazole exposure resulted in significant increases in the spontaneous movement
187
at 24 hpf and significant decreases in the number of heartbeat at 48 hpf in the 2.5 mg/L
188
and 4.5 mg/L propiconazole groups. Compared with the control group, the hatching
189
rate was inhibited at 72, 96, and 120 hpf by 4.5 mg/L propiconazole exposure, but was
190
not affected by other doses. We also observed reductions of body length in larvae
191
exposed to 2.5 and 4.5 mg/L propiconazole for 120 h. Larvae with malformation 10 ACS Paragon Plus Environment
Page 10 of 40
Page 11 of 40
Journal of Agricultural and Food Chemistry
192
following exposure to propiconazole at 120 hpf were shown in Figure 2. In the
193
propiconazole-exposed groups, the spine deformation (Sd) and tail malformation (Tm)
194
were observed, indicating that propiconazole induced the developmental toxicity of
195
zebrafish embryos. Although the malformation rate was observed in the groups treated
196
with high doses, there were no statistical significances (Figure 2H).
197
Locomotor activity analysis.
198
Locomotor activity was conducted in 120 hpf larvae (Figure 3). Under the light, average
199
velocity and moving distance were significantly decreased in 2.5 and 4.5 mg/L
200
propiconazole groups. As the exposure concentrations increased, active times of larvae
201
were significantly increased and inactive times were significantly decreased. Therefore,
202
compared with the control group, the average acceleration was significantly decreased
203
and the average deceleration was significantly increased in a dose-dependent manner.
204
TG and TCHO contents.
205
The effects of propiconazole exposure on TG and TCHO contents in larvae were shown
206
in Figure 4. 120 h exposure to 2.5 mg/L propiconazole significantly reduced the levels
207
of TCHO, while no significant changes of TG contents were observed in larvae exposed
208
to propiconazole.
209
LPL and FASN activity.
210
After 120 h exposure, LPL enzyme activity of zebrafish larvae was significantly
211
decreased in the 2.5 and 4.5 mg/L propiconazole groups (Figure 5A). The FASN
212
activity was also significantly decreased in the larvae following propiconazole exposure
213
(Figure 5B). 11 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 12 of 40
214
Gene expression analysis.
215
To further identify the effects of propiconazole on lipid metabolism in larvae, the
216
transcription levels of genes associated with lipid metabolism were determined in the
217
larvae with propiconazole exposure (Figure 6). As described in previous literatures,
218
these genes are involved in lipid accumulation, lipogenesis, fatty acid (FA) β-oxidation,
219
and cholesterol metabolism 16, 33, 44. At 120 hpf, compared with the control group, the
220
transcription
221
palmitoyltransferase 1 (cpt1), and hydroxymethyl glutaryl coenzyme A reductase b
222
(HMGCRb) were significantly increased in larvae exposed to 0.5 and 4.5 mg/L
223
propiconazole.
224
acyltransferase (apgat4), carbohydrate response element binding protein (chrebp),
225
acetyl-CoA carboxylase 1(acc1), peroxisome proliferator-activated receptor-α (pparα),
226
sterol 14α-demethylase – cytochrome P51(CYP51), and cytochrome P7A1 (CYP7A1)
227
were significantly upregulated in all three groups of larvae after propiconazole exposure.
228
The expression levels of sterol regulatory element-binding protein 1(screbf1), acyl-
229
CoA oxidase 1 (acox1), and hydroxymethyl glutaryl coenzyme A reductase a
230
(HMGCRa) were significantly upregulated in the 4.5 mg/L propiconazole group.
231
Upregulation of 7-dehydrocholesterol reductase (DHCR7) was also observed in the 2.5
232
mg/L propiconazole group.
233
Fatty acids analysis
234
We further semi-quantitatively analyzed the composition of free fatty acids in larvae
235
(Figure 7 and Table S4). The typical curve of a control (A) and 4.5 mg/L propiconazole
levels
The
of
diglyceride
transcription
levels
acyltransferase
of
(dgat2),
carnitine
1-acylglycerol-3-phosphate
12 ACS Paragon Plus Environment
o-
Page 13 of 40
Journal of Agricultural and Food Chemistry
236
treatment (B) were showed in Figure S4. Compared with the control group, most of
237
saturated fatty acids (C16:0, C18:0, C20:0, Figure 7A) and monounsaturated fatty acids
238
(C20:1N9, C20:1n11, Figure 7B) were significantly decreased in zebrafish following
239
propiconazole exposure for 120 hpf. In contrast, the content of C18:1N9 was increased
240
in 2.5, and 4.5 mg/l propiconazole-treated groups. We did not detect polyunsaturated
241
fatty acids.
242
Discussion
243
In the present study, we investigated the developmental toxicity of the fungicide
244
propiconazole and its particular effects on lipid metabolism in the early life stages of
245
zebrafish. Propiconazole exposure significantly inhibited the development of zebrafish
246
embryos and larvae, showing lowered heartbeat, hatching rate, and body length and
247
decreased locomotor activities. Analysis of lipid metabolism revealed that
248
propiconazole exposure resulted in decreased activity of LPL and FASN.
249
Propiconazole exposure also altered the expression of genes related to lipid
250
accumulation, lipogenesis, fatty acids β-oxidation, as well as cholesterol synthesis and
251
metabolism. Together, these results indicate that propiconazole has developmental
252
toxic effects likely via affecting lipid metabolism.
253
Total cholesterol, as a multifunctional molecule, is an important constituent of lipid
254
related to lipid raft45. In addition, TCHO was the tetracyclic hydrocarbon chemicals to
255
be essential for the component of cell members and signal transduction between cells46.
256
HMGCR plays a crucial role in cholesterol synthesis and is a limiting step of cholesterol
257
biosynthesis
47.
CYP51, as the objective of triazole fungicides, catalyzes lanosterol 13 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
258
demethylation in the process of cholesterol synthesis 16. The DHCR7 gene is the last
259
step in cholesterol synthesis, which catalyzes the conversion of 7-dehydrocholesterol
260
into cholesterol 48. In our study, the mRNA expression levels of four critical cholesterol
261
synthesis transcripts (HMGCRa, HMGCRb, CYP51, and DHCR7) were significantly
262
up-regulated, leading to increased content of TCHO. In contrast, CYP7A1, as a key
263
rate-limiting enzyme gene, plays an important role in maintaining the homeostasis of
264
lipids and participates in the cholesterol metabolism, through which process bile acids
265
are generated
266
resulted in significantly decreased levels of TCHO following exposure to propiconazole,
267
suggesting that propiconazole affects lipid metabolism in the zebrafish larvae. Similarly,
268
the expression levels of HMGCRa, HMGCRb, CYP51, CYP7A1, and DHCR7 were
269
significantly increased in female zebrafish exposed to triazole fungicide difenoconazole
270
at environmentally relative concentrations for 15 days influenced the content of
271
TCHO16. Skolness et al. also demonstrated that the reduction of TCHO content was
272
found in the fathead minnow (Pimephales promelas) after three weeks exposure of
273
propiconazole
274
TCHO and affected lipid metabolism in adult zebrafish liver, due to the lack of energy
275
supply 51. It would be of interest to examine any alterations in glucose metabolism in
276
zebrafish following propiconazole exposure.
277
FASN, as a multifunctional single-chain protein, catalyzes saturated fatty acids
278
synthesis in cells 52, 53. Palmitate, a 16-carbon long-chain fatty acid (C16:0), is the major
279
product of fasn, which can undergo elongation by fasn Ⅲ-forming stearate (C18:0) 54.
49.
50.
Comprehensively, the alterations of genes related to cholesterol
Young et al. reported that thifluzamide caused decreased levels of
14 ACS Paragon Plus Environment
Page 14 of 40
Page 15 of 40
Journal of Agricultural and Food Chemistry
280
Acox1 encodes a key lipogenic enzyme that catalyzes the conversion of acetyl-CoA to
281
malnoyl-CoA and further converted by FASN to fatty acids 55, 56. The reduced activity
282
of FASN led to decreased levels of fatty acids in zebrafish following exposure to
283
propiconazole. Furthermore, the expression of genes associated with fatty acids β-
284
oxidation process were altered by propiconazole. The first step of acyl-CoA was
285
conversed to acyl-carnitine by cpt1 catalysis that transports fatty acids from the external
286
membrane into the mitochondrial, and the pparα induces these oxidations
287
pparα could be activated by free fatty acids or lipids, which reduces the rate of lipid
288
degeneration by increasing the rate of lipid metabolism in organisms 59, 60. In our study,
289
the reductions of fatty acids were in response to the down-regulation of cpt1 and pparα.
290
The transcription factors screbf1 and chrebp are necessary regulators involved in fatty
291
acids synthesis. Glucose regulates the expression of chrebp gene, which stimulates the
292
process of lipogenesis 61. The apgat4 gene, codes the protein for catalyzing the glycerol
293
phosphate 62. DGAT, as the last step of TG biosynthesis, which catalyzes the conversion
294
of fatty acids and glycerol into triglyceride
295
catalyzes the decomposition of TG into fatty acids and monoglycerides in very low-
296
density lipoprotein (VLDL) to be used for tissue oxidation energy supply and storage
297
64.
298
the up-regulation of screbf1, chrebp, apgat4, dgat2 expression and decreased LPL
299
activity cause increased TG content in zebrafish larvae. Overall, we found that the
300
expression levels of genes related to fatty acid β-oxidation, lipogenesis, and cholesterol
301
metabolism were significantly increased and the enzyme activities of LPL and FASN
63.
57, 58.
The
In addition, LPL, as a glycoprotein,
The activity of LPL was decreased in zebrafish exposed to propiconazole. In general,
15 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 16 of 40
302
was markedly decreased, suggesting that such changes resulted in reduced contents of
303
fatty acids and disrupted lipid metabolism.
304
Lipids are associated with biomembrane composition, energy metabolism, and other
305
physiological processes, which plays a crucial role in zebrafish development
306
According to the previous study, triazole fungicide difenoconazole could induce the
307
disruption of lipid metabolism and impair embryonic development on the basis of
308
transcriptomics and metabolomics by showing changes in expression levels of genes
309
involved in lipogenesis and lipolysis 17. Similarly, in this work, we observed that the
310
inhibition of developmental parameters were related to the disturbance of lipids
311
metabolism.
312
Fatty acids are aliphatic acids and key components of cells membranes that play a key
313
role in energy transport and storage, cell structure, and intermediates that provide
314
hormone synthesis 66-69. Fatty acid metabolism usually takes place in mitochondria and
315
peroxisome. Saturated fatty acids may ameliorate environmental heat stress by
316
affecting mitochondrial energetics. Moreover, there is a close relationship between fatty
317
acids and cardiovascular function 70, 71. Locomotor activity, as a quantitative endpoint,
318
is used for measuring testing behavioral toxicity in aquatic organism72. The reduction
319
of fatty acids affected the energy metabolism and then further influenced locomotor
320
activity of embryo and heartbeat number, which may cause decreased heartbeat,
321
lowered average velocity, short in moving distance, more inactive time and less active
322
time on larvae after propiconazole exposure for 120 hpf, compared to the control group.
323
Previous studies have shown that fatty acids are involved in growth, cognition, and 16 ACS Paragon Plus Environment
65.
Page 17 of 40
Journal of Agricultural and Food Chemistry
324
stunting in the early development of organisms 73-75. In general, embryos have abundant
325
TG, TCHO, and phosphatidylcholine during embryogenesis and larval development
326
period from 0 hpf to 120 hpf
327
acids may inhibit the growth performance of zebrafish embryos following
328
propiconazole exposure, showing decreases in hatching rate, and body length.
329
Furthermore, the levels of lipid species are associated with fish quality and nutrient
330
retention 76. Therefore, these results indicate that we should pay great attention to the
331
impact of environmental pollutants on aquatic organisms.
332
In the present study, we demonstrate that exposure to propiconazole alters the
333
expression of genes associated with the lipogenesis and lipolysis pathway, such changes
334
leading to decreasing fatty acid synthesis, and enhancing cholesterol metabolism and
335
fatty acid β-oxidation, overall causing the disruption of lipid metabolism in zebrafish.
336
Both decreased activity of LPL and FASN and increased expression of lipolysis may
337
contribute to the reduction of TCHO and fatty acids in larvae. Taken together, our
338
results indicate that propiconazole is a contributing factor to the abnormal development
339
at the early life stages of organisms.
340
environmental and health risks of propiconazole should be considered, especially at the
341
early life stages of aquatic organisms.
342
Acknowledgements
343
Conflicts of interest
344
The authors declare that there are no conflicts of interest.
65.
However, in this report, decreased contents of fatty
Therefore, we suggest that the potential
17 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
345
Supporting Information
346
More experimental details, nucleotide sequences of primers, percentage recovery of
347
propiconazole, GC-MS instrumental parameters.
348
References
349
1.
350
S. A., Side effects of the sterol biosynthesis inhibitor fungicide, propiconazole, on a
351
beneficial arbuscular mycorrhizal fungus. Communications in Agricultural & Applied
352
Biological Sciences 2011, 76, 891-902.
353
2.
354
J. M.; Durmaçay, S.; Gerardin, P., Inhibition of fungi with wood extractives and natural
355
durability of five Cameroonian wood species. Industrial Crops & Products 2018, 123,
356
183-191.
357
3.
358
biotransformation of chiral triazole fungicides in rainbow trout ( Oncorhynchus mykiss
359
). Aquatic Toxicology 2006, 80, 372-381.
360
4.
361
Sufyani, O.; Hakami, A.; Hadadi, A., Pesticide Residues Determination in Vegetables
362
from South Western Region of Saudi Arabia. Advances in Environmental Biology 2016.
363
5.
364
detectability of pesticides in fruits and vegetables analysed by high-performance liquid
365
chromatography – Time-of-flight. Journal of Chromatography A 2018, 1542.
366
6.
Calonne, M.; Fontaine, J.; Debiane, D.; Laruelle, F.; Grandmougin, A.; Louneshadj,
Tchinda, J. B. S.; Ndikontar, M. K.; Belinga, A. D. F.; Mounguengui, S.; Njankouo,
Konwick, B. J.; Garrison, A. W.; Avants, J. K.; Fisk, A. T., Bioaccumulation and
Hassan, A. A.; Marwan, A. M.; Khardali, I. A.; Oraiby, M. E.; Al-Khairat, M.;
Muehlwald, S.; Buchner, N.; Kroh, L. W., Investigating the causes of low
Shin, Y.; Lee, J.; Lee, J. H.; Lee, J.; Kim, E.; Liu, K. H.; Lee, H. S.; Kim, J. H., 18 ACS Paragon Plus Environment
Page 18 of 40
Page 19 of 40
Journal of Agricultural and Food Chemistry
367
Validation of a Multiresidue Analysis Method for 379 Pesticides in Human Serum
368
Using Liquid Chromatography-Tandem Mass Spectrometry. J Agric Food Chem 2018,
369
66.
370
7.
371
difference in synergistic potentials of propiconazole and prochloraz toward pyrethroids
372
in Daphnia magna? Aquatic toxicology (Amsterdam, Netherlands) 2016, 172, 95-102.
373
8.
374
induced toxicological alterations in brain of freshwater fish Channa punctata Bloch.
375
Ecological Indicators 2016, 62, 242-248.
376
9.
377
Chen, P. J.; Wood, C. E.; Murphy, L., Propiconazole increases reactive oxygen species
378
levels in mouse hepatic cells in culture and in mouse liver by a cytochrome P450
379
enzyme mediated process. Chemico-biological interactions 2011, 194, 79.
380
10. Ortiz, P. A.; Bruno, M. E.; Moore, T.; Nesnow, S.; Winnik, W.; Ge, Y., Proteomic
381
analysis of propiconazole responses in mouse liver: comparison of genomic and
382
proteomic profiles. Journal of proteome research 2010, 9, 1268.
383
11. Nesnow, S.; Padgett, W. T.; Moore, T., Propiconazole Induces Alterations in the
384
Hepatic
385
Hepatocarcinogenesis. Toxicological Sciences An Official Journal of the Society of
386
Toxicology 2011, 120, 297.
387
12. Li, Z. H.; Zlabek, V.; Velisek, J.; Grabic, R.; Machova, J.; Kolarova, J.; Li, P.;
388
Randak, T., Multiple biomarkers responses in juvenile rainbow trout, Oncorhynchus
Dalhoff, K.; Gottardi, M.; Kretschmann, A.; Cedergreen, N., What causes the
Tabassum, H.; Khan, J.; Salman, M.; Raisuddin, S.; Parvez, S., Propiconazole
Nesnow, S.; Grindstaff, R. D.; Lambert, G.; Padgett, W. T.; Bruno, M.; Ge, Y.;
Metabolome
of
Mice:
Relevance
19 ACS Paragon Plus Environment
to
Propiconazole-Induced
Journal of Agricultural and Food Chemistry
389
mykiss, after acute exposure to a fungicide propiconazole. Environmental Toxicology
390
2013, 28, 119.
391
13. Roberts, T. R.; Hutson, D. H., Metabolic pathways of agrochemicals. Part 2:
392
insecticides and fungicides. 1999; p 48-48.
393
14. Goetz, A. K.; Dix, D. J., Toxicogenomic effects common to triazole antifungals
394
and conserved between rats and humans. Toxicology & Applied Pharmacology 2009,
395
238, 80-89.
396
15. Hermsen, S. A. B.; Pronk, T. E.; Brandhof, E. J. V. D.; Ven, L. T. M. V. D.; Piersma,
397
A. H., Chemical class-specific gene expression changes in the zebrafish embryo after
398
exposure to glycol ether alkoxy acids and 1,2,4-triazole antifungals. Reproductive
399
Toxicology 2011, 32, 245-252.
400
16. Mu, X.; Wang, K.; Chai, T.; Zhu, L.; Yang, Y.; Zhang, J.; Pang, S.; Wang, C.; Li,
401
X., Sex specific response in cholesterol level in zebrafish (Danio rerio) after long-term
402
exposure of difenoconazole. Environmental pollution (Barking, Essex : 1987) 2015,
403
197, 278-286.
404
17. Teng, M.; Zhu, W.; Wang, D.; Qi, S.; Wang, Y.; Yan, J.; Dong, K.; Zheng, M.;
405
Wang, C., Metabolomics and transcriptomics reveal the toxicity of difenoconazole to
406
the early life stages of zebrafish (Danio rerio). Aquatic Toxicology 2018, 194, 112.
407
18. Birsoy, K.; Festuccia, W. T.; Laplante, M., A comparative perspective on lipid
408
storage in animals. Journal of Cell Science 2013, 126, 1541-1552.
409
19. Grün, F.; Blumberg, B., Environmental obesogens: organotins and endocrine
410
disruption via nuclear receptor signaling. Endocrinology 2006, 147, S50. 20 ACS Paragon Plus Environment
Page 20 of 40
Page 21 of 40
Journal of Agricultural and Food Chemistry
411
20. Ouadahboussouf, N.; Babin, P. J., Pharmacological evaluation of the mechanisms
412
involved in increased adiposity in zebrafish triggered by the environmental contaminant
413
tributyltin. Toxicology & Applied Pharmacology 2016, 294, 32-42.
414
21. De, C. M.; Van, d. B. M., Obesogenic effects of endocrine disruptors, what do we
415
know from animal and human studies? Environment International 2014, 70, 15-24.
416
22. Teng, M.; Qi, S.; Zhu, W.; Wang, Y.; Wang, D.; Dong, K.; Wang, C., Effects of
417
the bioconcentration and parental transfer of environmentally relevant concentrations
418
of difenoconazole on endocrine disruption in zebrafish (Danio rerio). Environmental
419
pollution (Barking, Essex : 1987) 2017, 233, 208-217.
420
23. World
421
http://www.who.int/mediacentre/factsheets/fs311/en/. 2015, (Accessed 3 March 2016).
422
24. Heber, D.; Apovian, C. M., An integrative view of obesity. American Journal of
423
Clinical Nutrition 2010, 91, 280S.
424
25. Capitão, A.; Lyssimachou, A.; Lfc, C.; Santos, M. M., Obesogens in the aquatic
425
environment: an evolutionary and toxicological perspective. Environment International
426
2017, 106, 153.
427
26. Levesque, H. M.; Moon, T. W.; Campbell, P. G. C.; Hontela, A., Seasonal variation
428
in carbohydrate and lipid metabolism of yellow perch (Perca flavescens) chronically
429
exposed to metals in the field. Aquatic Toxicology 2002, 60, 257-267.
430
27. Cedervall, T.; Hansson, L.-A.; Lard, M.; Frohm, B.; Linse, S., Food Chain
431
Transport of Nanoparticles Affects Behaviour and Fat Metabolism in Fish. PLOS ONE
432
2012, 7, e32254.
Health
Organization
(WHO),
Obesity
21 ACS Paragon Plus Environment
and
Overweight.
Journal of Agricultural and Food Chemistry
433
28. Carnevali, O.; Notarstefano, V.; Olivotto, I.; Graziano, M.; Gallo, P.; Di Marco
434
Pisciottano, I.; Vaccari, L.; Mandich, A.; Giorgini, E.; Maradonna, F., Dietary
435
administration of EDC mixtures: A focus on fish lipid metabolism. Aquatic Toxicology
436
2017, 185, 95-104.
437
29. Murphy, L. A.; Moore, T.; Nesnow, S., Propiconazole-enhanced hepatic cell
438
proliferation is associated with dysregulation of the cholesterol biosynthesis pathway
439
leading to activation of Erk1/2 through Ras farnesylation. Toxicology & Applied
440
Pharmacology 2012, 260, 146.
441
30. Volz, D. C.; Belanger, S.; Embry, M.; Padilla, S.; Sanderson, H.; Schirmer, K.;
442
Scholz, S.; Villeneuve, D., Adverse outcome pathways during early fish development:
443
a conceptual framework for identification of chemical screening and prioritization
444
strategies. Toxicological Sciences An Official Journal of the Society of Toxicology 2011,
445
123, 349-58.
446
31. Strähle, U.; Scholz, S.; Geisler, R.; Greiner, P.; Hollert, H.; Rastegar, S.;
447
Schumacher, A.; Selderslaghs, I.; Weiss, C.; Witters, H., Zebrafish embryos as an
448
alternative to animal experiments—A commentary on the definition of the onset of
449
protected life stages in animal welfare regulations. Reproductive Toxicology 2012, 33,
450
128–132.
451
32. Seth, A.; Stemple, D. L.; Barroso, I., The emerging use of zebrafish to model
452
metabolic disease. Disease Models & Mechanisms 2013, 6, 1080-1088.
453
33. Ho, J. C. H.; Hsiao, C. D.; Kawakami, K.; Tse, W. K. F., Triclosan (TCS) exposure
454
impairs lipid metabolism in zebrafish embryos. Aquatic toxicology (Amsterdam, 22 ACS Paragon Plus Environment
Page 22 of 40
Page 23 of 40
Journal of Agricultural and Food Chemistry
455
Netherlands) 2016, 173, 29-35.
456
34. Dasgupta, S.; Cheng, V.; Vliet, S. M. F.; Mitchell, C. A.; Volz, D. C., Tris(1,3-
457
dichloro-2-propyl) phosphate Exposure During Early-Blastula Alters the Normal
458
Trajectory of Zebrafish Embryogenesis. Environ Sci Technol 2018.
459
35. Xing, X.; Kang, J.; Qiu, J.; Zhong, X.; Shi, X.; Zhou, B.; Wei, Y., Waterborne
460
exposure to low concentrations of BDE-47 impedes early vascular development in
461
zebrafish embryos/larvae. Aquatic Toxicology 2018, 203, 19-27.
462
36. ISO., [Water quality. Determination of the acute lethal toxicity of substances to a
463
freshwater fish (Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)). Pt. 1:
464
Static method.-pt. 2: Semi-static method.- pt. 3: Flow-through method]. Norme
465
Internationale Iso 1996.
466
37. Qian, L.; Qi, S.; Cao, F.; Zhang, J.; Li, C.; Song, M.; Wang, C., Effects of
467
penthiopyrad on the development and behaviour of zebrafish in early-life stages.
468
Chemosphere 2019, 214, 184-194.
469
38. Zhang, J.; Qian, L.; Teng, M.; Mu, X.; Qi, S.; Chen, X.; Zhou, Y.; Cheng, Y.; Pang,
470
S.; Li, X.; Wang, C., The lipid metabolism alteration of three spirocyclic tetramic acids
471
on zebrafish (Danio rerio) embryos. Environmental Pollution 2019, 248, 715-725.
472
39. Teng, M.; Qi, S.; Zhu, W.; Wang, Y.; Wang, D.; Yang, Y.; Li, H.; Li, C.; Dong,
473
K.; Wang, C., Sex-specific effects of difenoconazole on the growth hormone endocrine
474
axis in adult zebrafish (Danio rerio). Ecotoxicology & Environmental Safety 2017, 144,
475
402.
476
40. Zhang, L.; Hatzakis, E.; Nichols, R. G.; Hao, R.; Correll, J.; Smith, P. B.; Chiaro, 23 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
477
C. R.; Perdew, G. H.; Patterson, A. D., Metabolomics Reveals that Aryl Hydrocarbon
478
Receptor Activation by Environmental Chemicals Induces Systemic Metabolic
479
Dysfunction in Mice. Environmental Science & Technology 2015, 49, 8067.
480
41. Wang, D.; Yan, J.; Teng, M.; Yan, S.; Zhou, Z.; Zhu, W., In utero and lactational
481
exposure to BDE-47 promotes obesity development in mouse offspring fed a high-fat
482
diet: impaired lipid metabolism and intestinal dysbiosis. Archives of Toxicology 2018.
483
42. Jadhav, M. R.; Pudale, A.; Raut, P.; Utture, S.; Ahammed Shabeer, T. P.; Banerjee,
484
K., A unified approach for high-throughput quantitative analysis of the residues of
485
multi-class veterinary drugs and pesticides in bovine milk using LC-MS/MS and GC–
486
MS/MS. Food Chemistry 2019, 272, 292-305.
487
43. Lehotay, S. J., Possibilities and Limitations of Isocratic Fast Liquid
488
Chromatography-Tandem Mass Spectrometry Analysis of Pesticide Residues in Fruits
489
and Vegetables. Chromatographia 2018.
490
44. Wang, W.; Zhang, X.; Wang, Z.; Qin, J.; Wang, W.; Tian, H.; Ru, S., Bisphenol S
491
induces obesogenic effects through deregulating lipid metabolism in zebrafish (Danio
492
rerio) larvae. Chemosphere 2018, 199, 286-296.
493
45. Simons, K.; Ikonen, E., Functional rafts in cell membranes. Nature 1997, 387, 569-
494
572.
495
46. Simons, K.; Toomre, D., Lipid rafts and signal transduction. Nature Reviews
496
Molecular Cell Biology 2000, 1, 31-39.
497
47. Sato, K.; Kamada, T., Regulation of bile acid, cholesterol, and fatty acid synthesis
498
in chicken primary hepatocytes by different concentrations of T0901317, an agonist of 24 ACS Paragon Plus Environment
Page 24 of 40
Page 25 of 40
Journal of Agricultural and Food Chemistry
499
liver X receptors. Comparative Biochemistry & Physiology Part A 2011, 158, 201-206.
500
48. Horling, A.; Müller, C.; Barthel, R.; Bracher, F.; Imming, P., A new class of
501
selective and potent 7-dehydrocholesterol reductase inhibitors. Journal of Medicinal
502
Chemistry 2012, 55, 7614.
503
49. Qi, Y.; Jiang, C.; Cheng, J.; Krausz, K. W.; Li, T.; Ferrell, J. M.; Gonzalez, F. J.;
504
Chiang, J. Y. L., Bile acid signaling in lipid metabolism: Metabolomic and lipidomic
505
analysis of lipid and bile acid markers linked to anti-obesity and anti-diabetes in mice
506
☆. Biochimica Et Biophysica Acta 2015, 1851, 19-29.
507
50. Skolness, S. Y.; Blanksma, C. A.; Cavallin, J. E.; Churchill, J. J.; Durhan, E. J.;
508
Jensen, K. M.; Johnson, R. D.; Kahl, M. D.; Makynen, E. A.; Villeneuve, D. L.,
509
Propiconazole Inhibits Steroidogenesis and Reproduction in the Fathead Minnow
510
(Pimephales promelas). Toxicology Science 2013, 132, 284-297.
511
51. Yang, Y.; Dong, F.; Liu, X.; Xu, J.; Wu, X.; Qi, S.; Liu, W.; Zheng, Y.,
512
Thifluzamide affects lipid metabolism in zebrafish (Danio reio). Sci Total Environ 2018,
513
633, 1227-1236.
514
52. Pandey, P. R.; Liu, W.; Xing, F.; Fukuda, K.; Watabe, K., Anti-cancer drugs
515
targeting fatty acid synthase (FAS). Recent Pat Anticancer Drug Discov 2012, 7, -.
516
53. Listenberger, L. L.; Han, X.; Lewis, S. E.; Cases, S.; Farese, R. V.; Ory, D. S.;
517
Schaffer, J. E., Triglyceride accumulation protects against fatty acid-induced
518
lipotoxicity. Proceedings of the National Academy of Sciences 2003, 100, 3077.
519
54. Wakil, S. J., Fatty acid synthase, a proficient multifunctional enzyme. Biochemistry
520
1989, 28, 4523-4530. 25 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
521
55. Hardie, D. G.; Pan, D. A., Regulation of fatty acid synthesis and oxidation by the
522
AMP-activated protein kinase. Biochemical Society Transactions 2002, 30, 1064.
523
56. Tugwood, J. D.; Issemann, I.; Anderson, R. G.; Bundell, K. R.; Mcpheat, W. L.;
524
Green, S., The mouse peroxisome proliferator activated receptor recognizes a response
525
element in the 5' flanking sequence of the rat acyl CoA oxidase gene. Embo Journal
526
1992, 11, 433-439.
527
57. Boukouvala, E.; Leaver, M. J.; Favrekrey, L.; Theodoridou, M.; Krey, G.,
528
Molecular characterization of a gilthead sea bream (Sparus aurata) muscle tissue cDNA
529
for carnitine palmitoyltransferase 1b (CPT1B). Comparative Biochemistry &
530
Physiology Part B Biochemistry & Molecular Biology 2010, 157, 189-197.
531
58. Suzuki, H.; Kawarabayasi, Y.; Kondo, J.; Abe, T.; Nishikawa, K.; Kimura, S.;
532
Hashimoto, T.; Yamamoto, T., Structure and regulation of rat long-chain acyl-CoA
533
synthetase. Journal of Biological Chemistry 1990, 265, 8681-8685.
534
59. Pawar, A.; Jump, D. B., Unsaturated fatty acid regulation of peroxisome
535
proliferator-activated receptor alpha activity in rat primary hepatocytes. Journal of
536
Biological Chemistry 2003, 278, 35931-35939.
537
60. Patsouris, D.; Reddy, J. K.; Müller, M.; Kersten, S., Peroxisome proliferator-
538
activated receptor alpha mediates the effects of high-fat diet on hepatic gene expression.
539
Endocrinology 2006, 147, 1508-1516.
540
61. Abdul-Wahed, A.; Guilmeau, S.; Postic, C., Sweet Sixteenth for ChREBP:
541
Established Roles and Future Goals. Cell Metabolism 2017, 26, 324-341.
542
62. Yamashita, A.; Hayashi, Y.; Matsumoto, N.; Nemotosasaki, Y.; Oka, S.; Tanikawa, 26 ACS Paragon Plus Environment
Page 26 of 40
Page 27 of 40
Journal of Agricultural and Food Chemistry
543
T.; Sugiura, T., Glycerophosphate/Acylglycerophosphate acyltransferases. Biology
544
2014, 3, 801-830.
545
63. Kawano, Y.; Cohen, D. E., Mechanisms of hepatic triglyceride accumulation in
546
non-alcoholic fatty liver disease. Journal of Gastroenterology 2013, 48, 434-441.
547
64. Cisar, L. A.; Hoogewerf, A. J.; Cupp, M.; Rapport, C. A.; Bensadoun, A., Secretion
548
and degradation of lipoprotein lipase in cultured adipocytes. Binding of lipoprotein
549
lipase to membrane heparan sulfate proteoglycans is necessary for degradation. Journal
550
of Biological Chemistry 1989, 264, 1767-74.
551
65. Fraher, D.; Sanigorski, A.; Mellett, N.; Meikle, P.; Sinclair, A.; Gibert, Y.,
552
Zebrafish Embryonic Lipidomic Analysis Reveals that the Yolk Cell Is Metabolically
553
Active in Processing Lipid. Cell Reports 2016, 14, 1317-1329.
554
66. Haemmerle, G.; Lass, A.; Zimmermann, R.; Gorkiewicz, G.; Meyer, C.; Rozman,
555
J.; Heldmaier, G.; Maier, R.; Theussl, C.; Eder, S.; Kratky, D.; Wagner, E. F.;
556
Klingenspor, M.; Hoefler, G.; Zechner, R., Defective Lipolysis and Altered Energy
557
Metabolism in Mice Lacking Adipose Triglyceride Lipase. Science 2006, 312, 734.
558
67. den Besten, G.; van Eunen, K.; Groen, A. K.; Venema, K.; Reijngoud, D. J.; Bakker,
559
B. M., The role of short-chain fatty acids in the interplay between diet, gut microbiota,
560
and host energy metabolism. J Lipid Res 2013, 54, 2325-40.
561
68. Loftus, T. M.; Jaworsky, D. E.; Frehywot, G. L.; Townsend, C. A.; Ronnett, G. V.;
562
Lane, M. D.; Kuhajda, F. P., Reduced Food Intake and Body Weight in Mice Treated
563
with Fatty Acid Synthase Inhibitors. Science 2000, 288, 2379.
564
69. Itoh, T.; Fairall, L.; Amin, K.; Inaba, Y.; Szanto, A.; Balint, B. L.; Nagy, L.; 27 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
565
Yamamoto, K.; Schwabe, J. W. R., Structural basis for the activation of PPARγ by
566
oxidized fatty acids. Nature Structural &Amp; Molecular Biology 2008, 15, 924.
567
70. Bucher, H. C.; Hengstler, P.; Schindler, C.; Meier, G., N-3 polyunsaturated fatty
568
acids in coronary heart disease: a meta-analysis of randomized controlled trials. The
569
American Journal of Medicine 2002, 112, 298-304.
570
71. Lemaitre, R. N.; King, I. B.; Mozaffarian, D.; Kuller, L. H.; Tracy, R. P.; Siscovick,
571
D. S., n−3 Polyunsaturated fatty acids, fatal ischemic heart disease, and nonfatal
572
myocardial infarction in older adults: the Cardiovascular Health Study. The American
573
Journal of Clinical Nutrition 2003, 77, 319-325.
574
72. Klüver, N.; König, M.; Ortmann, J.; Massei, R.; Paschke, A.; Kühne, R.; Scholz,
575
S., Fish Embryo Toxicity Test: Identification of Compounds with Weak Toxicity and
576
Analysis of Behavioral Effects To Improve Prediction of Acute Toxicity for Neurotoxic
577
Compounds. Environmental Science & Technology 2015, 49, 7002-7011.
578
73. Jumbe, T.; Comstock, S. S.; Harris, W. S.; Kinabo, J.; Pontifex, M. B.; Fenton, J.
579
I., Whole-blood fatty acids are associated with executive function in Tanzanian children
580
aged 4-6 years: a cross-sectional study. British Journal of Nutrition 2016, 116, 1537-
581
1545.
582
74. Theresia, J.; Comstock, S. S.; Hahn, S. L.; Harris, W. S.; Joyce, K.; Fenton, J. I.,
583
Whole Blood Levels of the n-6 Essential Fatty Acid Linoleic Acid Are Inversely
584
Associated with Stunting in 2-to-6 Year Old Tanzanian Children: A Cross-Sectional
585
Study. Plos One 2016, 11, e0154715.
586
75. Adjepong, M.; Pickens, C. A.; Jain, R.; Harris, W. S.; Annan, R. A.; Fenton, J. I., 28 ACS Paragon Plus Environment
Page 28 of 40
Page 29 of 40
Journal of Agricultural and Food Chemistry
587
Association of whole blood n-6 fatty acids with stunting in 2-to-6-year-old Northern
588
Ghanaian children: A cross-sectional study. Plos One 2018, 13, e0193301.
589
76. Pinedogil, J.; Tomásvidal, A.; Jovercerdá, M.; Tomásalmenar, C.; Sanzcalvo, M.
590
Á.; Martíndiana, A. B., Red beet and betaine as ingredients in diets of rainbow trout
591
(Oncorhynchus mykiss): effects on growth performance, nutrient retention and flesh
592
quality. Archives of Animal Nutrition 2017, 71, 486.
593
Acknowledgements
594
This work was supported by National Key Research and Development Program of
595
China (2016YFD0200504).
29 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure Captions Figure 1. Effects of propiconazole exposure on embryonic development in zebrafish. A. The number of spontaneous movements at 24 hpf. B. The number of heartbeat at 48 hpf. C. The hatching rate at 72, 96, and 120 hpf. D. The body length of hatched individual larvae at 120 hpf. * P < 0.05 compared with the control group, (n = 6 replicates, mean ± standard deviation). Figure 2. Embryos with malformations following exposure to propiconazole at 120 hpf. A. Embryo in the control group; B/C. Embryo with spinal deformation (Sd) in the 2.5 mg/L group. D/F/G. Embryo with spinal deformation in the 4.5mg/L group. E. Embryo with tail malformation (Tm) in the 4.5mg/L propiconazole group. H. Malformation rate of 120 hpf larvae exposed to propiconazole. * P < 0.05 compared with the control group, (n = 6 replicates, mean ± standard deviation). Figure 3. Locomotor activity of zebrafish larvae exposed to propiconazole at 120 hpf. A. The average velocity of zebrafish larvae at 120 hpf. B. The moving distance of zebrafish larvae at 120 hpf. C. Active and inactive time [s] of zebrafish larvae at 120 hpf. D. Average acceleration and deceleration of zebrafish larvae at 120 hpf. * P < 0.05 compared with the control group, (n = 6 replicates, mean ± standard deviation (SD)). Figure 4. Triglyceride (TG, A) and total cholesterol (TCHO, B) contents of zebrafish at 120 hpf following propiconazole exposure. * P < 0.05 compared with the control group, (n = 6 replicates, mean ±standard deviation). Figure 5. Lipoprtein lipase (LPL, A) and fatty acid synthase (FASN, B) activity of 30 ACS Paragon Plus Environment
Page 30 of 40
Page 31 of 40
Journal of Agricultural and Food Chemistry
zebrafish at 120 hpf following propiconazole exposure. * P < 0.05 compared with the control group, (n = 6 replicates, mean ± standard deviation). Figure 6. Propiconazole exposure induced the mRNA expression of genes involved in lipid metabolism in larvae at 120 hpf. A. The expression of genes involved in lipid accumulation. B. The expression of genes involved in lipogenesis. C. The expression of genes involved in fatty acid (FA) β-oxidation. D. The expression of genes involved in cholesterol metabolism. * P < 0.05 compared with the control group, (n = 6 replicates, mean ± standard deviation). Figure 7. Effects of propiconazole exposure on the contents of free fatty acids in larvae at 120 hpf. A: saturated fatty acids (SFA); B: monounsaturated fatty acids (MUFA). * P < 0.05 compared with the control group, (n = 6 replicates, mean ± standard deviation).
31 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 32 of 40
Tables Table 1. The Report of Propiconazole Concentration in the Water Environment in Different Areas. Sampled sites
Concentration
References
Banana plantation sites Costa Rica
0.15–13 μg L−1
(Castillo et al. 2006)
Influent of pharmaceutical company
(Van De Steene and Lambert 0.17–0.24 μg L−1
Belgium
2008)
Effluent of pharmaceutical company
(Van De Steene and Lambert 0.012–0.14μg L−1
Belgium
2008)
Paris sewer France
0.15–0.21μg L−1
(Gasperi et al.2008)