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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

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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


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Abstract

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Propiconazole is a triazole fungicide that has been widely used in agriculture and has

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been detected in the aquatic environment. This study aimed to investigate the effects of

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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

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scientific questions is lower cost, more efficient and suitable for meeting current

7

legislation than other traditional fish species. Exposure to propiconazole significantly

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inhibited the development of zebrafish embryos and larvae. This exposure also caused

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reduced locomotor activities in zebrafish. Furthermore, total cholesterol levels,

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lipoprotein lipase and fatty acid synthase activities were significantly decreased. The

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expression levels of genes involved in lipid metabolism were significantly up-regulated

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in response to propiconazole exposure. GC-MS/MS analysis revealed that fatty acids

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were significantly decreased. Together, the findings indicate the potential

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environmental risk of propiconazole exposure in the aquatic ecosystem.

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Keywords: propiconazole, zebrafish embryo, lipid metabolism, fatty acids


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Introduction

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Propiconazole (PCZ) (1-((2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl)methyl-

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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

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propiconazole is a common fungicide used for crop farming and is easily transported to

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the ecosystem through spray drift, surface run-off, and rainfall, its residues have been

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detected in the aquatic environment (Table 1), as well as in vegetables, fruits, and

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human sera

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environment has a potentially adverse effect on some aquatic organisms such as

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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

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hepatic cells and in mouse liver after exposure to propiconazole, and the changes

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resulting from propiconazole were also revealed in mice via genomics and proteomics

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9, 10.

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induces hepatocarcinogenesis of mice 11. Juvenile rainbow trout (Oncorhynchus mykiss)

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exposed to propiconazole exhibit various physiological responses, including hepatic

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ethoxyresorufin-O-deethylase (EROD) activity, antioxidant indices, morphological

34

indices, and hematological parameters

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activity of lanosterol-14α-demethylase enzyme, the one that is essential for ergo sterol

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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


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lipid biosynthesis and metabolism pathways 14-17.

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Lipids play an important role in energy supply and maintaining normal metabolic

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activities 18. Lipid homeostasis requires precise control of lipid process, including lipid

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accumulation, lipogenesis, fatty acid β-oxidation, cholesterol synthesis and metabolism.

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Disordered homeostasis leads to obesity, malnutrition, endocrine disruption, or other

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metabolic-associated diseases

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syndrome in 21 century. The WHO has reported that there are at least 41 million

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children under 5 years old with obesity or overweight 23. One possibility is that a rise

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in environmental pollutants may contribute to obesity or other health problems

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Thus, the investigation of the effects of environmental chemical exposure on the lipid

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metabolism of organisms in the early stages is urgent. Data have shown seasonal

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variation in lipid metabolism of yellow perch (Perca flavescens) chronically exposed

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to sub-lethal levels of heavy metals (Cd, Zn, Cu) (Rouyn-Noranda, Quebec)26. Studies

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have also shown that nano-sized particles transported through the food chain affect

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behavior and lipid metabolism in Crucian carp (genus Carassius), Bleak (Alburnus

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alburnus), Rudd (Scardinius erythrophthalmus), Tench (Tinca tinca), Pike (Esox esox),

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and Atlantic salmon (Salmo salar)27. Carnevali., et al have reported that the mixtures

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of endocrine disruption chemicals (EDCs) affect lipogenesis and fat deposition in the

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Juvenile seabream 28. Different types of toxicant substances alter lipid metabolism in

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marine and fresh water fish. Additionally, previous studies demonstrate that

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propiconazole increases cell proliferation and Ras farnesylation in AML12 mouse

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hepatocytes via the dysregulation of the cholesterol biosynthesis pathway 29. Therefore,

19-22.

For instance, obesity is an epidemic metabolic


24, 25.

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it is important to investigate the potential mechanism of the effect of propiconazole

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exposure on lipid metabolism in the early stages of freshwater organisms.

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The zebrafish embryo, as an ideal aquatic vertebrate model, has a rapid life cycle, and

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is easy to observe its morphological process during the development because the

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embryo is transparent

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questions is more suitable for meeting current legislation

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genes in mammals have been identified in zebrafish

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used for investigating the toxic effects of environmental pollutants 17, 34, 35.

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In the present study, we investigated the developmental effects, locomotor activity,

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lipid synthesis and metabolism change, and free fatty acid alterations in the early stages

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of zebrafish with exposure to different concentrations of propiconazole (0, 0.5 mg/L,

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2.5 mg/, 4.5 mg/L). These results provide the insight into the underlying mechanism of

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the effects of propiconazole exposure on lipid metabolism, predicting the potential

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environmental risk of propiconazole to aquatic organisms.

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Material and methods

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Reagents. Propiconazole (CAS#: 60207-90-1; 95% purity) was obtained from China

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Ministry of Agriculture. The stock solutions were prepared in acetone (purity > 99%).

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Standard water was used in the lab, containing 0.5 mM Mg2+, 2 mM Ca2+, 0.074 mM

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K+ and 0.75 mM Na+. 36. All other chemicals were at the analytical grade.

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Experimental design and sample collection. Five-month old wild-type zebrafish (AB

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strain, Danio rerio) were obtained from Hongdagaofeng fish shop. The parental

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zebrafish were cultured in flow-through equipment (Esen Corp, Beijing, China) under

30.

In addition, using zebrafish embryos to address scientific

32, 33.


31.

Furthermore, most of

Therefore, the embryo was


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14h:10h light/dark at 28 oC for 14 days. Zebrafish were fed with Artemia nauplii twice

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daily. Zebrafish embryos were maintained according to our previous study 17. A range

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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,

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2.75 mg/L) of propiconazole exposed to zebrafish embryos on the basis of pre-

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experiment data to test acute toxicity of embryo. Six replicates were used for each test

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solution of propiconazole (0, 0.5, 2.5, and 4.5 mg/L) based on the results from pilot

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studies. Control and propiconazole treatments received 0.01% (v/v) acetone. Two hours

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post fertilization (hpf), normal embryos were randomly assigned into the test groups.

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Each replicate contained 200 embryos in 500 mL solution. During the exposure period,

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the test solution was kept at 28 oC and the day/night cycle was 14h/10h. The exposure

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media was changed daily. The embryos from each group were observed and recorded

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using light microscopy at 24, 48, 72, 96, and 120 hpf. After 120 h exposure, zebrafish

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larvae were collected and frozen in liquid nitrogen, and then stored at -80 oC for further

95

analysis.

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Embryonic developmental test. During the propiconazole exposure, 10 larvae were

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selected as one replicate (n = 6 replicates) and put into 24-well plates to observe the

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embryonic development. We observed the spontaneous movement of embryos in 20s

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at 24 hpf and counted the heartbeat number in 20s at 48 hpf. The hatching rate was

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recorded at 72, 96, and 120 hpf. After 120 hpf, we recorded teratogenic effects and

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measured the body length of hatched individual larvae using digital microscope (Aigo

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GE-5, Beijing, China).

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Determination of larval locomotor activity. Based on the previous study, the 6 ACS Paragon Plus Environment

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locomotor activity of 120 hpf larvae was measured using the Video-Track system (UI-

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3240CP-C-GL, IDS Imaging Development System GmbH, Obersulm, Germany)

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following the manufacture’s protocol37. Average velocity, distance movement, active

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and inactive time, average acceleration and deceleration were recorded from 10 larvae

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per concentration every 10 mins (n = 6 replicates) and further analyzed using uEye

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Cockpit software Loligo System, United Ststes).

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Measurement of triglyceride (TG) and total cholesterol (TCHO) content. 30 larvae

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(n = 6 replicates) were homogenized in 270 μL cell lysates (Applygen, Beijing, China)

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and centrifuged at 3000 g for 10 min at 4 oC. Thus the supernatant was used for

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determinations of total protein using a bicinchoninic acid (BCA) protein assay kit

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(Cwbiotech, Beijing, China)16. And then the remained samples were incubated for 10

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min in 70 oC water to detect TG and TCHO contents according to the enzymatic kits

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(Applygen, Beijing, China) according to previous studies16. TG and TCHO were

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normalized to the sample protein concentration. The content of was measured

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Absorbance of TCHO, TG and total protein were measured using the microplate reader

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(Multiscan MK3, Thermo Scientific) at 540, 540, 595 nm, respectively. The calibration

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of protein was shown in Figure S1.

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Lipoprotein lipase (LPL) and fatty acid synthase (FASN) activity assay.

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Lipoprotein lipase and fatty acid synthase were extracted from 120 hpf larvae samples

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(30 larvae, n = 6 replicates). Samples were homogenized in 270 μL saline and

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centrifuged at 3000 g for 10 min at 4 oC. The supernatant was used to enzymatic activity

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analysis. The LPL and FASN activity assays were determined using enzyme-linked 7 ACS Paragon Plus Environment


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immunosorbent assay (ELISA) kits (Fu life Industry Co., Ltd. Shanghai, China)38. The

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range suitability of LPL and FASN were 1.6-65 U/mL, 10-360 U/L, respectively. The

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standard calibration of LPL and FASN were shown in Figure S2.

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Quantitative real-time polymerase chain reaction (qRT-PCR) assay. After 120 h

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exposure, 30 embryos were collected and dissolved in Trizol reagent for total RNA

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extraction according to previous method

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using FastQuant RTase kit (Tiangen Biotech, Beijing, China). Quantitative real-time

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polymerase chain reaction (qRT-PCR) was conducted using SYBR Green PCR Master

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Mix reagent kit (Tiangen Biotech) and performed using an ABI 7500 PCR system

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(Advanced Biosystems, Foster City, CA, USA). Thermal cycling was set at 95 °C for

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15 min, followed by 40 cycles at 95 °C for 10 s, 60°C for 20s, and 72°C for 32s.

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Transcription of target genes was calculated using the 2−ΔΔCt method. β-actin was

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chosen as the house-keeping gene. All primers of target genes were designed using

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Primer 6.0 software and synthesized by Sangon Biotechology (Shanghai, China) (Table

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S1).

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Metabolomics analysis based Gas chromatography-mass spectrometry (GC-MS).

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Embryos (80 embryo, n = 6 replicates) were extracted as previously described

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Briefly, 80 embryos were collected and homogenized in 20 μL internal standard (50

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μg/L C15:0 fatty acid and C17:0 methyl ester) and 600 μL extracting solution

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(chloroform : methanol = 1:2, v/v). Then, the homogenization was added into 200 μL

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chloroform and 200 μL water, vortexed for 2 min, and centrifuged at 18001 g for 10

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min. Extraction was repeated again and evaporated to dryness under a stream of

22 39

The first-strand cDNA was synthesized


40, 41.

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nitrogen, following derivatization with 1 m L methanol/hydrochloric acid (41.5/9.7 mL)

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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

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transferred to an injection vial for analysis. Free fatty acids were measured using a

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Thermo Fisher Scientific Trace GC gas chromatograph coupled to a Quantum XLS

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mass spectrometer (Thermo Scientific, Waltham, MA, USA) using a DB-5 column (30

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m×0.31 mm×0.25 mm), running in full scan mode. Data analysis was conducted with

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Thermo Xcalibur software. Fatty acids were quantified by normalizing the integrated

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peak areas to the internal standards.

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Propiconazole in water analysis

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Water samples were collected at the beginning of exposure (0 h) and at 24 h. All

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exposure solutions were analyzed for all treatments. The experimental solutions were

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filtered with a 0.22 μm filtration membrane and then determined using ultra-high

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performance liqud chromatography – tandem mass spectrometry (UHPLC-MS/MS)

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(ultiMate 3000 system,

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described 42, 43.

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Statistical analysis. All statistical analyses were performed with SPSS 19.0 (IBM,

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USA). Significant differences were determined by one-way ANOVA analysis on the

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basis of Dunnett post hoc comparison (P < 0.05). All values were presented as the mean

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± standard deviation (SD).

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Results

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Assay validation

Thermo Scientific, Waltham, MA, USA) as previously



170

Spiked samples with 1, 50, and 2500 μ,/L of propiconazole were analyzed. The

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recovery of propiconazole in solutions ranged from 93.8% to 106.9%. (Table S2). An

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external standard calibration curve was used to calculate the amount of propiconazole

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(Figure S3). The range of propiconazole concentrations and linear regression equations

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was 1-1000 μg/L, R2=0.99, respectively. Concentrations of propiconazole in water (0,

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0.5, 2.5, 4.5mg/L) were detected. The measured propiconazole concentrations in water

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samples were 80-120% of nominal values (Table S3).

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Lethal effect of propiconazole

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According to the results, the 96 h half dose/lethal concentration (LD50/LC50) value of

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embryo (95% conference limit) was 12.90 (12.40-13.40) mg/L with linear equation Y=

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14.33X-15.92 (R2 = 0.97). The low observed effect concentration (LOEC) was 1.72

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mg/L and the no observed effect concentration (NOEC) was 1.07 mg/L at 96 hpf

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(Figure S4). There is no significance in other parameters.

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Toxicological endpoints in embryos.

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The effects of propiconazole exposure on the spontaneous movement, heartbeat,

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hatching rate, and body length of embryos and larvae were shown in Figure 1.

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Propiconazole exposure resulted in significant increases in the spontaneous movement

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at 24 hpf and significant decreases in the number of heartbeat at 48 hpf in the 2.5 mg/L

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and 4.5 mg/L propiconazole groups. Compared with the control group, the hatching

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rate was inhibited at 72, 96, and 120 hpf by 4.5 mg/L propiconazole exposure, but was

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not affected by other doses. We also observed reductions of body length in larvae

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exposed to 2.5 and 4.5 mg/L propiconazole for 120 h. Larvae with malformation 10 ACS Paragon Plus Environment

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following exposure to propiconazole at 120 hpf were shown in Figure 2. In the

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propiconazole-exposed groups, the spine deformation (Sd) and tail malformation (Tm)

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were observed, indicating that propiconazole induced the developmental toxicity of

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zebrafish embryos. Although the malformation rate was observed in the groups treated

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with high doses, there were no statistical significances (Figure 2H).

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Locomotor activity analysis.

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Locomotor activity was conducted in 120 hpf larvae (Figure 3). Under the light, average

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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

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were significantly increased and inactive times were significantly decreased. Therefore,

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compared with the control group, the average acceleration was significantly decreased

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and the average deceleration was significantly increased in a dose-dependent manner.

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TG and TCHO contents.

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The effects of propiconazole exposure on TG and TCHO contents in larvae were shown

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in Figure 4. 120 h exposure to 2.5 mg/L propiconazole significantly reduced the levels

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of TCHO, while no significant changes of TG contents were observed in larvae exposed

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to propiconazole.

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LPL and FASN activity.

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After 120 h exposure, LPL enzyme activity of zebrafish larvae was significantly

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decreased in the 2.5 and 4.5 mg/L propiconazole groups (Figure 5A). The FASN

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activity was also significantly decreased in the larvae following propiconazole exposure

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(Figure 5B). 11 ACS Paragon Plus Environment


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Gene expression analysis.

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To further identify the effects of propiconazole on lipid metabolism in larvae, the

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transcription levels of genes associated with lipid metabolism were determined in the

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larvae with propiconazole exposure (Figure 6). As described in previous literatures,

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these genes are involved in lipid accumulation, lipogenesis, fatty acid (FA) β-oxidation,

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and cholesterol metabolism 16, 33, 44. At 120 hpf, compared with the control group, the

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transcription

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palmitoyltransferase 1 (cpt1), and hydroxymethyl glutaryl coenzyme A reductase b

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(HMGCRb) were significantly increased in larvae exposed to 0.5 and 4.5 mg/L

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propiconazole.

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acyltransferase (apgat4), carbohydrate response element binding protein (chrebp),

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acetyl-CoA carboxylase 1(acc1), peroxisome proliferator-activated receptor-α (pparα),

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sterol 14α-demethylase – cytochrome P51(CYP51), and cytochrome P7A1 (CYP7A1)

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were significantly upregulated in all three groups of larvae after propiconazole exposure.

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The expression levels of sterol regulatory element-binding protein 1(screbf1), acyl-

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CoA oxidase 1 (acox1), and hydroxymethyl glutaryl coenzyme A reductase a

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(HMGCRa) were significantly upregulated in the 4.5 mg/L propiconazole group.

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Upregulation of 7-dehydrocholesterol reductase (DHCR7) was also observed in the 2.5

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mg/L propiconazole group.

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Fatty acids analysis

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We further semi-quantitatively analyzed the composition of free fatty acids in larvae

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(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


o-

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treatment (B) were showed in Figure S4. Compared with the control group, most of

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saturated fatty acids (C16:0, C18:0, C20:0, Figure 7A) and monounsaturated fatty acids

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(C20:1N9, C20:1n11, Figure 7B) were significantly decreased in zebrafish following

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propiconazole exposure for 120 hpf. In contrast, the content of C18:1N9 was increased

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in 2.5, and 4.5 mg/l propiconazole-treated groups. We did not detect polyunsaturated

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fatty acids.

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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

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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

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related to lipid raft45. In addition, TCHO was the tetracyclic hydrocarbon chemicals to

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be essential for the component of cell members and signal transduction between cells46.

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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


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demethylation in the process of cholesterol synthesis 16. The DHCR7 gene is the last

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step in cholesterol synthesis, which catalyzes the conversion of 7-dehydrocholesterol

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into cholesterol 48. In our study, the mRNA expression levels of four critical cholesterol

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synthesis transcripts (HMGCRa, HMGCRb, CYP51, and DHCR7) were significantly

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up-regulated, leading to increased content of TCHO. In contrast, CYP7A1, as a key

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rate-limiting enzyme gene, plays an important role in maintaining the homeostasis of

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lipids and participates in the cholesterol metabolism, through which process bile acids

265

are generated

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resulted in significantly decreased levels of TCHO following exposure to propiconazole,

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suggesting that propiconazole affects lipid metabolism in the zebrafish larvae. Similarly,

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the expression levels of HMGCRa, HMGCRb, CYP51, CYP7A1, and DHCR7 were

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significantly increased in female zebrafish exposed to triazole fungicide difenoconazole

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at environmentally relative concentrations for 15 days influenced the content of

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TCHO16. Skolness et al. also demonstrated that the reduction of TCHO content was

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found in the fathead minnow (Pimephales promelas) after three weeks exposure of

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propiconazole

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TCHO and affected lipid metabolism in adult zebrafish liver, due to the lack of energy

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supply 51. It would be of interest to examine any alterations in glucose metabolism in

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zebrafish following propiconazole exposure.

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FASN, as a multifunctional single-chain protein, catalyzes saturated fatty acids

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synthesis in cells 52, 53. Palmitate, a 16-carbon long-chain fatty acid (C16:0), is the major

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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


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Acox1 encodes a key lipogenic enzyme that catalyzes the conversion of acetyl-CoA to

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malnoyl-CoA and further converted by FASN to fatty acids 55, 56. The reduced activity

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of FASN led to decreased levels of fatty acids in zebrafish following exposure to

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propiconazole. Furthermore, the expression of genes associated with fatty acids β-

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oxidation process were altered by propiconazole. The first step of acyl-CoA was

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conversed to acyl-carnitine by cpt1 catalysis that transports fatty acids from the external

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membrane into the mitochondrial, and the pparα induces these oxidations

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pparα could be activated by free fatty acids or lipids, which reduces the rate of lipid

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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.

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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

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catalyzes the decomposition of TG into fatty acids and monoglycerides in very low-

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density lipoprotein (VLDL) to be used for tissue oxidation energy supply and storage

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64.

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the up-regulation of screbf1, chrebp, apgat4, dgat2 expression and decreased LPL

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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,



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was markedly decreased, suggesting that such changes resulted in reduced contents of

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fatty acids and disrupted lipid metabolism.

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Lipids are associated with biomembrane composition, energy metabolism, and other

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physiological processes, which plays a crucial role in zebrafish development

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According to the previous study, triazole fungicide difenoconazole could induce the

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disruption of lipid metabolism and impair embryonic development on the basis of

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transcriptomics and metabolomics by showing changes in expression levels of genes

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involved in lipogenesis and lipolysis 17. Similarly, in this work, we observed that the

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inhibition of developmental parameters were related to the disturbance of lipids

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metabolism.

312

Fatty acids are aliphatic acids and key components of cells membranes that play a key

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role in energy transport and storage, cell structure, and intermediates that provide

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hormone synthesis 66-69. Fatty acid metabolism usually takes place in mitochondria and

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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,

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is used for measuring testing behavioral toxicity in aquatic organism72. The reduction

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of fatty acids affected the energy metabolism and then further influenced locomotor

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activity of embryo and heartbeat number, which may cause decreased heartbeat,

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lowered average velocity, short in moving distance, more inactive time and less active

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time on larvae after propiconazole exposure for 120 hpf, compared to the control group.

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Previous studies have shown that fatty acids are involved in growth, cognition, and 16 ACS Paragon Plus Environment

65.

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stunting in the early development of organisms 73-75. In general, embryos have abundant

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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



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


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


to

Propiconazole-Induced


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


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


and

Overweight.


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


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


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


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


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


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


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


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).



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

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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).



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)

Danio rerio

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