Metabonomics Indicates Inhibition of Fatty Acid Synthesis, Î²-Oxidation

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Metabonomics indicates inhibition of fatty acid synthesis, beta-oxidation and the TCA cycle in triclocarbaninduced cardiac metabolic alterations in male mice Wenping Xie, Wenpeng Zhang, Juan Ren, Wentao Li, Lili Zhou, Yuan Cui, Huiming Chen, Wenlian Yu, Xiaomei Zhuang, Zhenqing Zhang, Guolin Shen, and Haishan Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05220 • Publication Date (Web): 24 Jan 2018 Downloaded from http://pubs.acs.org on January 25, 2018

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Journal of Agricultural and Food Chemistry

1

Metabonomics

indicates

inhibition

of

fatty

acid

synthesis,

2

beta-oxidation and the TCA cycle in triclocarban-induced cardiac

3

metabolic alterations in male mice

4 5

Wenping Xie#1, Wenpeng Zhang#2, Juan Ren3, Wentao Li1, Lili Zhou1, Yuan Cui1, Huiming Chen1,

6

Wenlian Yu1, Xiaomei Zhuang2, Zhenqing Zhang2, Guolin Shen*1, Haishan Li*1

7 8

1

Chinese Academy of Inspection and Quarantine, Institute of Chemicals Safety, Beijing, China

9

2

State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of

10

Pharmacology and Toxicology, Beijing, China

11

3

Pneumology department, The rocket army general hospital of the PLA, Beijing, China

12 13 14

Corresponding to [email protected] and [email protected]

15 16 17 18

#

These two authors contributed equally to this work.

19 20 21 22 23 24 25 26 27 28

ACS Paragon Plus Environment


29

Abstract

30

Triclocarban (TCC) has been identified as a new environmental pollutant that is potentially

31

hazardous to human health; however, the effects of short-term TCC exposure on cardiac function

32

are not known. The aim of this study was to use metabonomics and molecular biology techniques

33

to systematically elucidate the molecular mechanisms of the TCC-induced effects on cardiac

34

function in mice. Our results show that TCC inhibited the uptake, synthesis and oxidation of fatty

35

acids, suppressed the TCA cycle, and increased aerobic glycolysis levels in heart tissue after

36

short-term TCC exposure. TCC also inhibited nuclear receptor PPARα, confirming its inhibitory

37

effects on fatty acid uptake and oxidation. Histopathology and other analyses further confirm that

38

TCC altered mouse cardiac physiology and pathology, ultimately affecting normal cardiac

39

metabolic function. We elucidate the molecular mechanisms of TCC-induced harmful effects on

40

mouse cardiac metabolism and function from a new perspective, using metabonomics and

41

bioinformatics analysis data.

42 43

Keywords: Triclocarban, metabonomics, PPARα, TCA cycle

44 45 46 47 48 49 50 51 52 53 54 55 56


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INTRODUCTION

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Triclocarban (TCC) is a commonly used high-efficiency broad-spectrum fungicide. As a

59

pharmaceuticals and personal care product, it is widely present in textiles (especially lingerie),

60

washing powders, deodorants, skin care products, toothpastes, and wound disinfectants for

61

sterilization, bacteriostasis, and deodorization.1 In addition, it is also widely used in toys and

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building materials.2 In the environment, TCC has been detected at concentrations of micrograms

63

per liter in the aquatic environments of United States, indicating that their aquatic ecosystem has

64

been extensively polluted.3 TCC is adsorbed on sludge and particulates, and only a small fraction

65

is biodegraded. In the activated sludge of a sewage treatment plant, the TCC content was reported

66

as 51±15 µg/g dry soil.4 Biosolids left after sewage treatment are often used as fertilizer for land,

67

providing a pathway for TCC to enter the water environment and soil, to accumulate in the plant,

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and to be absorbed by humans. While TCC is metabolized rapidly in animals and humans,5,6 the

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rate of degradation of TCC in the environment is significantly slower because TCC has strong

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lipophilic properties.7,8 TCC may thus accumulate in the environment and be absorbed by the body,

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potentially affecting health.

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In recent years, the increasing availability of information on the environmental stability,

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bioaccumulation and biological toxicity of TCC has led to it being identified as a new type of

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environmental pollutant that is potentially harmful to human health. According to the literature,9-11

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TCC has chronic toxicity in mammals, may interfere with mammalian reproduction, and may

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cause methemoglobinemia in humans. When present in soaps as an antibacterial agent, TCC may

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interact with low molecular weight non-steroidal molecules, thus affecting sterol receptor



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signaling and leading to a variety of disorders including cancer, reproductive dysfunction and

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developmental abnormalities.2 In rats, TCC affects fertility by decreasing birth weight and

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survival,12 and increases estrogen receptor- and androgen receptor-dependent gene expression,

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revealing a new mechanism of action for endocrine-disrupting compounds.13 A study on

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TCC-induced injury in the human liver cell line LO2 found that exposure to TCC at a low dose

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(2.38 µmol/L) caused DNA breakage.14 Studies also show that TCC is an effective inhibitor of

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soluble epoxide hydrolase (sEH) in mice and humans,15,16 sEH can hydrolyze the epoxides, such

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as serum epoxyeicosatrienoic acid (EET), thus reducing the chemical activity of surface oxide,

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increasing its water solubility and altering their biological functions.17 A previous study showed

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that after a single exposure for 24 hour, TCC inhibited sEH and produced anti-inflammatory and

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antihypertensive effects by increasing serum EET levels.16 However, the effects of short-term TCC

89

exposure (i.e. for several weeks) on the cardiac system have not been studied, especially the

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consequents from EET level interruption and potential energy metabolism disruption. Since heart

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is one of the most active metabolic organs (mitochondria are abundant in cardiac myocytes, and

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cytochrome oxidase system has high activity), any effects of TCC on mitochondrial function and

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oxidase activity is important to physiological function.

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The aim of this study was to investigate the effects and underlying mechanisms of TCC on

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sEH and cardiac function in mice after a continuous exposure of TCC for 35 days. As a previous

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study showed that TCC was detected in the 2686 urine samples from a general population and the

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highest concentration was up to 588µg/L.18 Since 27% of TCC intake excreted through urine,6 a

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total exposure as high as 3.0 mg per adult is possible, as a adult has an average 1.4 L urine volume


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per day.19 Using allometric scalling model,20 such a exposure could be converted to an oral dose of

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27.3 mg/kg for mice and TCC has been shown to have significant blood pressure lowering effects

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at 5 mg/kg in mice exposed for 24 h.16 Here, we trialed four different TCC doses for

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metabonomics research to elucidate the molecular mechanisms of TCC-induced effects on cardiac

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function in mice, and improve our understanding of the effects of TCC on humans.

104 105

Materials and methods

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Chemicals

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TCC and chromatographic grade acetonitrile were purchased from Sigma-Aldrich (St. Louis, MO,

108

USA). Mouse acyl coenzyme A synthetase (ACS) was sourced from JianglaiBio (Shanghai, China).

109

A citrate synthase assay kit and ELISA kits for mouse carnitine palmitoyl transferase-1 (CPT1),

110

ACS, acetyl coenzyme A carboxylase and fatty acid synthase were sourced from Nanjing Jiancheng

111

Bioengineering Institute (Nanjing, China). A murine sEH enzyme immunoassay kit was sourced

112

from Jiangsu Enzyme-labeling Biotechnology Co., Ltd (Jiangsu, China). The One Step RNA PCR

113

Kit was sourced from TaKaRa (Dalian, China), and 5,6-EET, 8,9-EET, 11,12-EET and 14,15-EET

114

were sourced from Cayman Chemical (Ann, Arbor, MI, USA).

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Animal experiments

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Male 5–6-week-old C57BL/6 mice were maintained on a 12-h light/dark cycle with free access to

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water and laboratory chow. All animal experiments were conducted at the Chinese Academy of

118

Inspection and Quarantine, Institute of Chemicals Safety. The animal experiments followed the

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protocol of the Institutional Animal Care and Use Committee of the center, in compliance with the



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guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care

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International. Mice were divided into five groups (n=12 per group): four TCC groups received TCC

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daily (3, 10, 30 or 90 mg/kg, intragastrically), and the control group received vehicle daily (10%

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PEG 400 in water, intragastrically) for 35 consecutive days. After these 35 days, plasma samples

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were obtained from each animal under anesthesia (4% isoflurane). Mice were then killed by

125

inhalation of carbon dioxide, and hearts were removed and weighed. Two hearts in each group were

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fixed in 10% buffered formalin for histology, and the remainder was flash frozen in liquid nitrogen

127

and stored at −80 °C until analysis. Plasma biochemical parameters were measured using an

128

automatic Hitachi Clinical Analyzer Model 7080 (Hitachi High-Technologies Corporation, Tokyo,

129

Japan).

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Analysis of heart tissue by high-resolution mass spectrometry

131

A UHPLC-Orbitrap-MS system with an ESI source was used to detect endogenous substances with

132

simultaneous scanning of positive and negative ions. Chromatographic conditions were achieved on

133

ultimate 3000 UHPLC system using a 2.1 mm × 50 mm i.d., 1.7 µm, Acquity BEH C18 column at

134

30 °C. The gradient elution procedure consisted of a 1 min isocratic gradient from 5% to 5%

135

acetonitrile in water, followed by linear gradient from 5% to 60% acetonitrile over 5 min, followed

136

by 60% to 100% acetonitrile over 3 min, and then followed by 100% to 60% acetonitrile over 3 min,

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at last, followed by 60% to 5% acetonitrile for the next 3 min. Each chromatography run was 18 min,

138

with a sample loading volume of 5 µL and total flow rate of 0.25 mL/min. The mass spectrometry

139

conditions were same as literature21 and the scan range was from 70 to 1050 m/z. Mouse heart

140

samples were homogenized in ultrapure water containing 50% methanol (1:10 m/v), then a 50 µL


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aliquot of each homogenate was mixed with 450 µL of precipitation agent containing the internal

142

standard (1:1 methanol:acetonitrile). The mixture was vortexed and centrifuged (14000×g, 10 min),

143

and 100 µL of each supernatant was removed for analysis.

144

Analysis of metabolic pathways

145

MetaboAnalyst3.0 websites were used to identify endogenous metabolites with Variable

146

Importance on the Projection (VIP) values greater than 1. An impact factor greater than 0.1 was

147

used as a criterion to identify the major metabolic pathways affected by TCC. The integrated

148

pathway analysis of MetaboAnalyst3.0 was used to analyze the correlation among related genes

149

with the effects of TCC on heart and the major endogenous metabolic pathways affected by these

150

genes.22 The Metscape model in Cytoscape was used to construct a metabolic network map of the

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major endogenous metabolites associated with TCC action.

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RT-PCR analysis

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Total RNA was extracted from frozen heart samples with Trizol reagent. The primers for sEH,

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Hexokinase-2 (HKII), Cytochrome P4502j6 (Cyp2j6), Cytochrome P4502b10 (Cyp2b10),

155

Cytochrome P4504a10 (Cyp4a10), Cytochrome P4504a14 (Cyp4a14), Cytochrome P4503a11

156

(Cyp3a11), Peroxisome proliferator-activated receptor α (PPARα) and β-actin are shown in Table

157

S1. An ABI 7500 was used for real-time PCR. The 2−∆∆Ct method was used for relative quantitative

158

analysis of differences in heart mRNA expression of mice from the control and TCC groups.

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Molecular docking

160

To investigate the effect of TCC on mouse and human PPARα, human PPARα (PDB ID: 1K7L)

161

was used as the template. The three-dimensional structure of mouse PPARα was constructed using



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the online modeling service SWISS-MOD (https://swissmodel.expasy.org/). Human PPARα

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protein

164

(http://www.rcsb.org/pdb/home/home.do). The ChemBio3D Ultra 14.0 software MMFF94 force

165

field was used to optimize the three-dimensional structure of TCC. A molecular docking test was

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performed in the Autodock Vina 1.1.2.23 The coordinates of active sites of mouse PPARα were set

167

as follows: center_x = −15.558, center_y = −15.003, center_z = −4.516, size_x = 15, size_y = 15,

168

size_z = 15. The coordinates of active sites of human PPARα were set as follows: center_x =

169

12.15, center_y = 5.367, center_z = -7.164, size_x = 15, size_y = 15, size_z = 15. To increase

170

calculation accuracy, we set the exhaustiveness parameter to 20. Finally, the conformation with the

171

highest score was selected for results analysis by PyMoL 1.7.6.

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Histopathological examinations

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Heart samples fixed in 10% neutral formalin were subjected to the following: tissue cutting,

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paraffin embedding, routine sectioning, hematoxylin eosin staining, graded ethanol dehydration,

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clearing and mounting for histopathological observation under a light microscope (Olympus

176

BX41).

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Enzyme activity assays

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Subsamples of 50 mg of frozen heart tissue were obtained and immersed in phosphate buffered

179

saline (pH 7.4) at a ratio of 1:10 (w/v). Tissues were homogenized while immersed in an ice bath,

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and supernatants were collected after centrifuging the homogenates at 4000×g for 20 min. Mouse

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ACS, sEH, carnitine acetyltransferase, acetyl coenzyme A carboxylase, fatty acid synthetase and

182

citrate synthase activities were determined using kits following manufacturer's instructions, with

(PDBID:3VI8)

was

downloaded

from

the


Protein

Data

Bank

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five samples assayed in parallel for each group.

184

Assays for TCC and EET in mouse heart tissue

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A UHPLC-Orbitrap-MS system with an ESI source was used to detect EET and TCC and

186

chromatographic conditions were achieved on ultimate 3000 UHPLC system using a Hypersil Gold

187

3 µm, 2.1 mm × 100 mm column and Acquity BEH C18 1.7 µm, 2.1 mm × 50 mm column at

188

30 °C for EET and TCC respectively. The gradient elution procedure for EET determination

189

consisted of a 0.5 min isocratic gradient from 5% to 5% acetonitrile in water, followed by linear

190

gradient from 5% to 10% acetonitrile over 0.5 min, followed by 10% to 30% acetonitrile over 1 min,

191

followed by 30% to 60% acetonitrile over 0.5 min, followed by 60% to 85% acetonitrile for 0.5 min,

192

followed by 85% to 85% acetonitrile for 1 min, and then followed by 85% to 5% acetonitrile for the

193

next 1 min. Each chromatographic run was 7 min, with a sample loading volume of 5 µL and flow

194

rate of 0.3 mL/min. The gradient elution procedure for TCC determination consisted of a 0.5 min

195

isocratic gradient from 30% to 30% acetonitrile in water, followed by linear gradient from 30% to

196

60% acetonitrile over 0.5 min, followed by 60% to 95% acetonitrile over 0.5 min, followed by 95%

197

to 95% acetonitrile for 1 min, and then followed by 95% to 30% acetonitrile for the next 1 min.

198

Each chromatographic run was 5 min, with a sample loading volume of 5 µL and flow rate of 0.25

199

mL/min. Mass spectrometry conditions and sample preparation protocols are described above, and

200

five samples were assayed in parallel for each group. The compound parameters were: data

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dependent MS2 (Targeted-SIM), 5,6-EET, 8,9-EET, 11,12-EET and 14,15-EET (−), 319.22786;

202

tolbutamide (−), 269.09654; TCC (+), 314.98532; propranolol (+), 260.16451.

203

Statistical Analysis



204

We used mzCloud (Thermo, USA) to obtain the exact mass of 5 decimal places per endogenous

205

substance and identify the endogenous substance.24 MetaboAnalyst3.0 websites were used to

206

calculate the differences in cardiac endogenous metabolite levels between treatment groups, and to

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map principle component analysis and partial least squares-discriminant analysis model diagrams.

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Endogenous metabolites with significant differences (VIP values >1) were selected to further

209

analyze the effects of TCC on cardiac metabolic pathways. Data were processed using SPSS 12.0

210

software. Data are expressed as means ± standard deviations (SD) and compared using one-way

211

ANOVA or paired t-tests.

212 213

Results and Discussion

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Effects of TCC on cardiac organ coefficients, biochemical markers, and histopathology in

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mice

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TCC was administered intragastrically to mice four doses for 35 consecutive days. Mice receiving

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a TCC dose of 30 or 90 mg/kg showed significant differences in body weight, heart weight, organ

218

coefficients (Table 1) and concentrations of some blood biochemical markers (ALB, CRE, ALP,

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GLU and Na+) compared with controls. In mice receiving a TCC dose of 3 mg/kg, concentrations

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of some biochemical markers (CRE and K+) were significantly different from those of controls

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(Table S2). Histopathological examination showed that cardiac fibers were normal in control

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group but in mice receiving a TCC dose of 30 or 90 mg/kg, some cardiac fibers became thicker,

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with relatively mild staining (Fig. 1). Therefore, exposure to TCC for 35 days affected the

224

structure of the heart in mice in a dose-dependent manner.

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Effects of TCC on endogenous metabolites in the mouse heart


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Endogenous metabolites were identified and the data processed (Fig. S1 and Table S3) as

227

described

228

squares-discriminant analysis were calculated using the MetaboAnalyst 3.0 website. The results

229

showed that endogenous levels of these compounds were significantly different between the

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control and TCC groups (Fig. 2(i) for PCA (principle component analysis) and Fig. 2(ii) for

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PLS-DA (partial least squares-discriminant analysis), the model quality parameters are: Accuracy

232

=0.8293, R2=0.9990, Q2=0.8541. Endogenous substances with VIP values >1 were selected to

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further analyze endogenous metabolites reflecting certain metabolic pathways. As shown in Table

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2, 23 endogenous metabolites in the heart had VIP values of >1, 15 of which were increased and 8

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decreased with TCC treatment. These results indicate that TCC affects secretion and metabolism

236

of endogenous substances in the mouse heart, especially those of amino acids, fatty acids and

237

carnitine substances.

238

Effects of TCC on the metabolic pathways of endogenous substances in the mouse heart

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As shown in Fig. 3A and Table S4, analysis using the MetaboAnalyst 3.0 website showed that

240

TCC primarily affected five metabolic pathways related to cardiac endogenous substances, with

241

impact values larger than 0.1. Fig. 3B shows the effects of these changes in cardiac endogenous

242

substances on metabolic pathways. Thus, network analyses reveal that TCC exposure for 35 days

243

affects the synthesis and metabolism of fatty acids and amino acids in the mouse heart, resulting in

244

changes in levels of endogenous substances in the cardiac tissue.

245

Construction of differential metabolite networks and correlation analysis

246

To further investigate the effects of TCC on metabolic pathways related to cardiac endogenous

in

previous

studies.21

Principle

component


analysis

and

partial

least


247

substances, we used the integrated pathway analysis of MetaboAnalyst3.0 to analyze correlations

248

between heart enzyme activities and endogenous substance metabolic pathways. As shown in Fig.

249

S2 and Table S5, TCC affected the normal metabolic function of the heart by affecting fatty acid

250

synthesis and metabolism in the heart. A metabolic network of major endogenous substances

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related to the actions of TCC was constructed using Metscape (Fig. S3). The network type

252

"Compound-Reaction-Enzyme-Gene" was selected, and the whole metabolic network consisted of

253

65 nodes and 66 edges. Fig. S3 shows that linoleic acid and 9,10-epoxyoctadecenoic acid were

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correlated with the activities of sEH, CYP2J6, CYP3a11, CYP2b10, CYP4a10 and CYP4a14.

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Expressed as a proportion of control group values, linoleic acid and 9,10-epoxyoctadecenoic acid

256

content and their relevant enzyme activities were decreased in mice treated with TCC (Table 3).

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The data in Table 3 and Fig. 4 validate the results of the metabolic network analysis. Expressed as

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a proportion of control group values, the content of other long-chain fatty acids, including palmitic

259

acid, docosahexaenoic acid, stearic acid and 9,10-epoxyoctadecenoic acid, showed decreasing

260

trends in the TCC groups (Table 2). ACS activity increased with higher TCC doses, indicating that

261

lower proportions of long-chain fatty acids were associated with increased ACS activity (Table

262

3).Our results also show that TCC inhibited PPARα mRNA expression (Fig. 4). Previous studies

263

show that PPARα regulates myocardial fatty acid uptake, and that decreased PPARα expression

264

lowers this uptake.25 This may explain the decreased levels of fatty acids (palmitic acid,

265

docosahexaenoic acid, stearic acid and 9,10-epoxyoctadecenoic acid) in our study. Compared with

266

the control group, the activities of acetyl coenzyme A carboxylase and fatty acid synthase, both

267

associated with fatty acid synthesis in the heart, tended to decrease in the TCC groups (Table 3).


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This indicates that TCC affects fatty acid synthesis and metabolic pathways in the heart, further

269

confirming the analytical results regarding metabolic pathways.

270

Effect of TCC on sEH enzyme activity in mouse heart

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Our results showed that TCC inhibited sEH activity (Fig. 4, Table 3). CYP2J is reported to be

272

distributed widely in myocardial cells, myocardial endothelial cells and coronary arterial

273

endothelial cells. Therefore, metabolism of arachidonic acid by CYP2J is regarded as an important

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source of EETs in the heart.26 CYP2J6 mRNA expression in the heart was decreased in all TCC

275

groups compared with the control group, leading to a decreased capability of CYP2J6 to

276

metabolize arachidonic acid and, thus, to increase arachidonic acid levels. In our experiment, the

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proportions of arachidonic acid in different TCC dose groups were increased to 128.4%±19.5%,

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104.9%±22.3%, 102.3%±18.7% and 116.9%±25.8%, respectively, from the lowest to the highest

279

dose groups. The heart EET content of TCC-treated mice was also increased compared with that

280

of control mice. These findings indicate that TCC not only decreased the capability of CYP2J6 to

281

metabolize arachidonic acid and produce EETs, but also increased EET levels because of its strong

282

inhibition of sEH (Table 4). Previous reports show that EETs maintain the stability of myocardial

283

physiology and energy metabolism, and prevent myocardial hypertrophy.27,28 In our study, 35 days

284

of TCC treatment led to decreased body and heart weights and reduced lipid deposition in the

285

heart and elsewhere in the body. Moreover, there may be a correlation between TCC exposure and

286

increased EETs levels.

287

Effects of TCC on PPARα in the mouse heart

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PPARα is reported to be a major transcriptional regulator of the fatty acid oxidase gene, playing a



289

key role in the regulation of energy and lipid metabolism in the heart.29,30 In our study, 35 days of

290

TCC exposure suppressed the heart mRNA levels of enzymes CYP4a10 and CYP4a14 and the

291

nuclear receptor PPARα, which corresponds to these enzymes (Fig. S3). We hypothesized that

292

TCC and PPARα may have a direct interaction and molecular docking showed that TCC was

293

bound to the active site of PPARα in mice (Fig.5A) and humans (Fig. 5B). Parts of the TCC

294

dichlorophenyl moiety formed stable hydrophobic interactions in hydrophobic pockets of the

295

amino acid residues Cys-276, Phe-318, Leu-321, Met-330, Ile-354 and Met-355 in mice, and

296

Cys-276, Phe-318, Leu-321, Met-330 and Ile-354 in humans. Conversely, the TCC chlorobenzene

297

group formed stable hydrophobic interactions in hydrophobic pockets of the amino acid residues

298

Ile-241, Leu-254, Phe-272, Cys-275, Leu-331, Ile-332, Ala-333 and Ile-339 in mice, and Ile-241,

299

Cys-275, Leu-331, Val-332, Ala-333 and Ile-339 in humans. The chlorine atom on the TCC

300

dichlorophenyl moiety can form Cl-π interactions with the amino acid residue Phe-318. The

301

presence of such specialized binding modes suggests that TCC will form stable complexes with

302

mouse and human PPARα. The results of these molecular biology and molecular docking studies

303

indicate that TCC can inhibit both mouse and human PPARα. Growing evidence suggests that

304

changes in energy metabolism substrates during the development of cardiac hypertrophy are

305

associated with decreased PPARα expression and its inactivation.31 In patients with severe

306

hypertension, especially those with left ventricular hypertrophy, a decrease in PPARα levels

307

greatly increased the risk of heart failure.32 Furthermore, long-chain fatty acids are endogenous

308

ligands for myocardial PPARα,33,34 the data about the proportion of long chain fatty acids in Table

309

2 further demonstrated the above results. In our study, 35-day TCC exposure may thus have


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affected normal cardiac metabolic function by suppressing PPARα expression.

311

Effects of TCC on cardiac energy supply in mice

312

To meet its high energy demands, the heart utilizes and metabolizes a variety of substrates.

313

Glucose conversion provides 20–30% of the heart’s energy, and fatty acid oxidation provides

314

50–70%.35-37 It has been reported that TCC provides precursors for oxidation by accelerating

315

long-chain fatty acid activation in the mouse heart. However, as shown in Table 5, CPT1 enzyme

316

activity showed a decreasing trend in the TCC groups compared with the control group. As CPT1

317

is a key enzyme for fatty acid oxidation,38 TCC thus decreased the rate of cardiac fatty acid

318

oxidation, affecting the normal energy supply to the heart. HKII activity showed an increasing

319

trend (Fig. 4). Compared with the control group, glucose levels in the hearts of mice from various

320

TCC treated groups (from lowest to highest doses) decreased to 75.2±11.5%, 77.9±18.9%,

321

80.0±20.6% and 50.9±8.7%, respectively. As shown in Table S2, glucose levels in mouse blood

322

were also decreased, suggesting that TCC increased aerobic glycolysis in the cardiac

323

microenvironment. This finding may also relate to suppressed oxidation and utilization of fatty

324

acids, because the myocardium requires sufficient ATP for its normal physiological function,

325

which is sometimes provided by increasing glycolysis. Also shown in Table S2, the plasma T4

326

levels in all the TCC groups were increased compared with the control group, though these

327

differences were not statistically significant. The heart is a key target organ of the thyroid hormone

328

T4. After being secreted, T4 enters the heart via the circulation. Increased blood T4 levels can

329

increase myocardial oxygen consumption and stimulate physiological hypertrophy of the

330

myocardium.39,40 In addition, an important metabolic change in the hypertrophic myocardium is



331

decreased oxidation and utilization of fatty acids and increased glucose utilization.41,42Therefore,

332

the risk of myocardial hypertrophy was higher in mice with increased TCC exposure. However,

333

TCC exposure changes the heart energy supply in mice from beta-oxidation of fatty acids to

334

aerobic glycolysis using glucose, an effect known as "the recapitulation of foetal energy

335

metabolism".43 In our histopathological analyses, some of the myocardial fibers were larger and

336

thicker in TCC-treated mice, consistent with our other findings.

337

In the present study, ACS activity was increased at higher TCC doses. This led to an increase in

338

acyl coenzyme A content, because acyl coenzyme A in cellular fluids is produced by

339

ACS-catalyzed activation of fatty acids. Furthermore, because of the decreased CPT1 activity,

340

acyl carnitine synthesis was decreased and acyl coenzyme A accumulated in the cellular fluid. The

341

proportion of carnitine in the mouse heart was also elevated compared with the control group

342

(Table 2). The heart cannot synthesize carnitine, instead obtaining it from the blood.44 In our

343

experiment, the proportion of carnitine in the blood was increased because decreased CPT1

344

activity led to decreased acyl carnitine synthesis, compared with the control group, carnitine

345

levels from TCC treated groups (from lowest to highest doses) increased to 148.6±29.7%,

346

115.0±10.7%, 155.2±17.2% and 116.2±14.5%, respectively. This decreased the carnitine

347

utilization rate, leading to a higher proportion of carnitine in the heart. It has been reported that

348

accumulation of acyl coenzyme A in myocardial cellular fluid results in decreased activities of

349

citrate synthase and adenine nucleotide transferase, hindering production and transport of ATP.45

350

In our study, citrate synthase activity (required for citric acid synthesis) was decreased with TCC

351

treatment compared with controls, an observation coinciding with the influence of acyl coenzyme


Page 16 of 32

Page 17 of 32


352

A accumulation (Table 3). The proportion of citric acid showed a downward trend in the various

353

TCC treated groups (Table 2). Citric acid is an important endogenous substance in the TCA cycle,

354

and a decrease in its concentration would affect the TCA cycle. In contrast, TCC treatment

355

increased the proportion of acetyl coenzyme A, a key endogenous substance in citric acid

356

synthesis. Compared with the control group, the proportions of acetyl coenzyme A in the TCC

357

treated groups (from lowest to highest dose) increased to 179.6%±12.3%, 138.1%±25.6%,

358

131.3%±21.9%, and 171.2%±36.7%, respectively. A possible explanation is that the decrease in

359

citrate synthase activity hindered citrate synthesis and led to a decrease in acetyl coenzyme A

360

utilization. The effect may also relate to the large amounts of acetyl coenzyme A not entering

361

cellular fluids for fatty acid synthesis. To summarize, 35 days of TCC exposure suppressed the

362

TCA cycle in the mouse myocardium, thus preventing the complete oxidation of fatty acids and

363

glucose and the release of ATP in the myocardium, leading to an insufficient energy supply to the

364

heart tissue. These findings demonstrate that short-term (35-day) TCC exposure affects cardiac

365

metabolic function in mice.

366

Cells usually absorb a few heterologous substances, some of which are oxidized to epoxides

367

during their metabolism. Because epoxides are triatomic heterocyclic compounds that contain

368

polar carbon–oxygen bonds and have a variety of affinities, they can cause irreversible toxicity

369

when reacting with DNA, amino acids, purines and other cellular components. This can lead to

370

genetic mutations and carcinogenesis.46 Therefore, removal of intracellular epoxides is essential

371

for maintaining the normal physiological functions of cells. sEH plays a key role in this process,

372

hydrolyzing these substances into water-soluble diols, which can be easily metabolized or



373

excreted.47,48 When sEH is inhibited or the sEH protein is down-regulated, cells will be damaged

374

by excessive accumulation of toxic substances. In our study, although TCC increased cardiac

375

levels of ETTs by inhibiting sEH in the mouse heart, histopathological analyses showed that parts

376

of the myocardial fibers became thicker, suggesting that the increased EET levels did not

377

completely protect the heart. This indicates that TCC affected the normal physiological structure

378

of the heart. Moreover, metabonomics and other techniques reveal that short-term TCC exposure

379

inhibits the uptake, synthesis and oxidation of fatty acids, suppresses the TCA cycle, and increases

380

aerobic glycolysis in the mouse heart (Fig. 6). Thus, short-term TCC exposure had a toxic effect

381

on the mouse heart, potentially related to sEH inhibition.

382

In conclusion, the present study elucidates the molecular mechanisms of TCC-induced harmful

383

effects on cardiac metabolic function from a new perspective, using high resolution mass

384

spectrometry for metabonomics and other bioinformatics analysis methods.

385 386

ACKNOWLEDGMENTS

387

This work was supported by grants from the Natural Science Foundation of Beijing (No. 8164071),

388

the Chinese Academy of Inspection and Quarantine (No. 2017JK009 and 2017JK047), the Natural

389

Science Foundation of China (No. 81273125 and 81373472), and the General Administration of

390

Quality Supervision, Inspection and Quarantine (AQSIQ) of the People’s Republic of China (No.

391

201510024 and 201510203-02).

392

Competing financial interests statement

393

The authors declare no competing financial interests.


Page 18 of 32

Page 19 of 32


394

Supplementary Material

395

Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

396 397

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the

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Page 25 of 32


517

TABLES AND FIGURES

518

Table 1. Effect of different doses of TCC on body weight and viscera coefficient of heart in mice Group

35 days exposure

Body

Heart

Heart weight

TCC(ng/g)

weight(g)

weight(g)

index(%)

Control

0.7±0.2

25.06±1.63

0.15±0.02

0.59±0.08

3 mg/kg

72.1±15.4

24.51±1.12

0.13±0.01

0.53±0.04

10 mg/kg

75.6±9.8

23.04±1.11

0.13±0.02

0.54±0.02

30 mg/kg

105.6±22.3

23.24±1.08

0.12±0.02

0.50±0.05

90 mg/kg

120.9±23.9

22.98±1.04

0.12±0.01

0.51±0.03

3 mg/kg P value

0.0000**

0.5516

0.1475

0.0400*

10 mg/kg P value

0.0000**

0.0136*

0.0991

0.0147*

30 mg/kg P value

0.0000**

0.0216*

0.0074**

0.0056**

90 mg/kg P value

0.0000**

0.0096**

0.0019**

0.0012**

519

Values are presented as mean±SD. (n=10). * p

Metabonomics Indicates Inhibition of Fatty Acid Synthesis, Î²-Oxidation

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