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New Insights into the Cytotoxic Mechanism of Hexabromocyclododecane (HBCD) from a Metabolomic Approach Feidi Wang, Haijun Zhang, Ningbo Geng, Baoqin Zhang, Xiaoqian Ren, and Jiping Chen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03678 • Publication Date (Web): 13 Feb 2016 Downloaded from http://pubs.acs.org on February 13, 2016
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New
Insights
into
the
Cytotoxic
Mechanism
2
Hexabromocyclododecane (HBCD) from a Metabolomic Approach
3
Feidi Wang,†,‡ Haijun Zhang,†,* Ningbo Geng,†,‡ Baoqin Zhang,† Xiaoqian
4
Ren,†,‡ Jiping Chen†,*
5
†
6
Chinese Academy of Sciences, Dalian, 116023, China
7
‡
Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics,
University of Chinese Academy of Sciences, Beijing 100049, China
8 9 10 11 12
of
TOC/Abstract Art
13 14 15 16 17 18
*Corresponding Authors
19
Phone: +86-411-8437-9972, fax: +86-411-8437-9562; e-mail:
[email protected].
20
Phone/fax: +86-411-8437-9562; e-mail:
[email protected].
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ABSTRACT
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The toxic effects of hexabromocyclododecane (HBCD) are complex, and the underlying
23
toxicological mechanisms are still not completely understood. In this study, a
24
pseudo-targeted metabolomic approach based on the UHPLC/Q-Trap MS system was
25
developed to assess the HBCD-intervention-related metabolic alteration in HepG2 cells. In
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addition, some physiologic indicators and relevant enzyme activities were measured. HBCD
27
exposure obviously impaired metabolic homeostasis and induced oxidative stress, even at
28
an environmentally relevant dose (0.05 mg/L). Metabolic profiling and multivariate
29
analysis indicated that the main metabolic pathways perturbed by HBCD included amino
30
acid metabolism, protein biosynthesis, fatty acid metabolism and phospholipid metabolism.
31
HBCD suppressed the cell uptake of amino acids, mainly through inhibition of the activity
32
of membrane transport protein Na+/K+-ATPase. HBCD down-regulated glycolysis and
33
β-oxidation of long-chain fatty acids, causing a large decrease of ATP production. As a
34
result, the across-membrane transport of amino acids was further inhibited. Meanwhile,
35
HBCD induced a significant increase of total phospholipids, mainly through the remodeling
36
of phospholipids from the increased free fatty acids. The obtained metabolomic results also
37
provided some new evidence and clues regarding the toxicological mechanisms of HBCD
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that contribute to obesity, diabetes, nervous system damage, and developmental disorders.
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INTRODUCTION
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Hexabromocyclododecane (HBCD) is a brominated cyclic alkane used primarily as an
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additive flame retardant in polystyrene-based materials including resins and fabrics.1, 2 It
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has been listed as a new persistent organic pollutant (POP) in Annex A of the Stockholm
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Convention in 2013,3 but with specific exemptions for expanded and extruded polystyrene
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foams in buildings until 2024.4 Because of its persistence and lipophilicity, HBCD can
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accumulate in the human body through a combination of diet, dust ingestion and indoor air
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inhalation.5 As a result, HBCD has been frequently detected in human blood and breast ACS Paragon Plus Environment
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milk.6, 7
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The acute toxicity of HBCD appears to be low,5 and it lacks significant genotoxic
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potential to an organism.1, 8 However, the subacute and subchronic effects of HBCD are
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manifest.9, 10 Hepatic toxicity and thyroid toxicity of HBCD were universally observed in in
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vivo animal studies.11,
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hyperplasia,14 cytochrome P450 enzyme induction,11 and thyroid hormone disruption.12
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Furthermore, potential reproductive effects,15 nervous system damage16 and developmental
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toxicity17 were also observed in response to HBCD exposure.
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The adverse effects include liver weight increase,13 thyroid
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Accumulating evidence shows that HBCD can induce endocrine disruption,13 metabolic
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dysfunction and obesity,18 even at environmentally relevant doses. HBCD has the ability to
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interact with the constitutive androstane receptor (CAR) and pregnane X receptor (PXR).19
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An in vivo study indicated that oral exposure to HBCD gave rise to a dose-dependent
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decrease of trabecular bone mineral density in female rats with a BenchMark Dose Lower
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confidence bound (BMDL) of 0.056 mg/kg body weight/day.20 Another in vivo study found
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that the body and liver weight were markedly increased in high-fat diet mice treated with
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HBCD at a medium dose of 35 and a high dose of 700 µg/kg body weight/week, paralleled
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by the disruption of lipid and glucose homeostasis.18 These low-dose effects raise serious
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concerns for human health based on human body burdens of HBCD, particularly for the
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occupational exposure setting.
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In the past decade, significant advances have been made toward understanding
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mechanisms underlying the adverse effects of HBCD. As a xenobiotic, HBCD is first
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metabolized by cytochrome P450 enzymes, and thereby reactive oxygen species (ROS) are
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generated.21 Overproduction of ROS likely results in oxidative damage to lipids, proteins
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and DNA.21 Meanwhile, HBCD-induced activation of the PI3K/Akt pathway and
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Nrf2-ARE pathway provide a cytoprotective process that responds to HBCD-induced ROS
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generation.22, 23 HBCD can induce CYP2B and CYP3A enzymes through interaction with ACS Paragon Plus Environment
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CAR/PXR receptors, which may account for the disruption of the thyroid hormone axis.19
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Several in vitro studies also indicated the inhibitory effect of HBCD on the
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sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, which is accompanied by an
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intracellular Ca2+ increase and mitochondrial dysfunction.24, 25 Gene expression profiles in
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rat livers revealed that HBCD exposure disrupted several specific pathways, such as
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PPAR-mediated regulation of lipid metabolism, triacylglycerol metabolism, cholesterol
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biosynthesis, and phase I and II pathways.9 Furthermore, untargeted metabolomics based on
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in vitro study revealed that exposure to HBCD for 72 h could significantly induce the
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metabolic changes.26 However, the metabolic pathways intervened by HBCD exposure
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require further confirmation, and there still remains a lack of metabolomic evidence
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supporting the underlying mechanisms of HBCD toxicity.
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The biological events induced by HBCD appear to be complex, and the underlying
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toxicological mechanisms of HBCD are not completely elucidated. To better understand the
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mode of action of HBCD, metabolomic evidence is required. Metabolomics aiming to
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systematically study small molecule metabolites is a method of understanding metabolic
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regulation.27 It can offer a perspective on how mechanistic biochemistry relates to the
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phenotypic state of an organism because metabolites serve as direct signatures of
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biochemical activity.28 The intrinsic physiological responses of an organism after HBCD
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exposure are reflected and propagated by its metabolism, and conversely, the variation in
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metabolic fingerprints can reveal and verify the mechanisms that underlie the toxicity of
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HBCD.29 In this study, the cytological effects of HBCD exposure on HepG2 cells were
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examined, and a pseudo-targeted metabolomic analysis was performed to investigate
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HBCD-intervention-related metabolic alteration. The obtained results are expected to
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provide a better understanding of metabolic disturbances induced by HBCD exposure and
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provide new evidence and clues concerning the toxicological mechanisms of HBCD from a
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metabolomic perspective. ACS Paragon Plus Environment
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EXPERIMENTAL SECTION
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Cell Culture, Exposure Conditions and Sample Collection. Human hepatoma
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HepG2 cell was adopted as an in vitro model for the study of cytotoxic mechanism. HepG2
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cell has high stability and unlimited life-span, and it retains a wide range of human
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liver-specific functions.30 In addition, its molecular expression and biological phenotypes
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have been extensively characterized.30 The test HepG2 cells were provided by China
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Infrastructure of Cell Line Resources (Shanghai, China), and these cells were maintained in
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Dulbecco’s Modified Eagle Medium (DMEM basic (1X), Gibco-BRL) supplemented with
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10% fetal bovine serum (FBS, Gibco-BRL) and 1% Penicillin–Streptomycin (Beyotime,
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China) under humidified air containing 5% CO2 at 37 °C. Cells in the logarithmic phase of
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growth were rinsed with phosphate-buffered saline, trypsinized, and then seeded in culture
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plates for exposure experiments.
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HepG2 cells were exposed to a reagent-grade HBCD formula (purity: > 95%, Aladdin
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Industrial Corp.) with varying concentrations in the culture medium. HBCD stock solutions
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were made up in ACS grade dimethyl sulfoxide (DMSO; Amresco, Solon, OH, USA).
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After cells were seeded and grown to 80% confluency, HBCD stock solution was
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incorporated into the cell culture medium. The final DMSO content in the culture medium
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was 0.05% (v/v), and background control cells were treated with only 0.05% DMSO.
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Cell viability was first tested using a thiazolyl blue tetrazolium bromide (MTT, Amresco,
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Solon, OH, USA) assay to ascertain the appropriate exposure conditions for metabolomic
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study. HepG2 cells were exposed to a series of HBCD concentrations with incubation
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durations of 1, 2 and 3 days. The details of the cell viability assays are shown in the
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Supporting Information. According to the results of MTT assay (see Supporting
122
Information), an exposure time of 1 day was adopted for metabolomic study; and three
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HBCD exposure doses relating to different effects on cell viability, the lowest dose with
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observable inhibition effect (0.05 mg/L), a middle dose without observable effect (1 mg/L) ACS Paragon Plus Environment
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and the highest dose with observable stimulation effect (10 mg/L), were selected. The
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exposure dose of 0.05 mg/L is comparable with the maximum serum concentration of
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HBCD (856 ng/g lipid) in individuals occupationally exposed.7
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In the metabolomic study, HepG2 cells were seeded in 6-well plates at about 3 × 105
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cells/well and incubated with HBCD at concentrations of 0.05, 1 and 10 mg/L for 24 h.
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After exposure, 200 µL of culture medium was transferred to an Eppendorf tube for
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analysis of extracellular metabolites, and then the remaining culture medium was removed.
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Cells were rinsed by gently dispensing ultrapure water to the surface twice, aspirating
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rapidly, and then quenched by liquid nitrogen freezing. The quenched cells adhering to
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plates, together with the culture medium in Eppendorf tubes, were stored at –80 °C and
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then extracted within 7 days.
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Metabolomic Analysis. To analyze the intracellular metabolites, 1 mL of ultrapure
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water was added to each well, followed by ultrasonic disruption in an ice-water bath for 3
138
min, and then the suspension containing cell fragments was transferred into an Eppendorf
139
tube. The freeze-drying of the samples was then performed simultaneously for the disrupted
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cells containing intracellular metabolites and collected culture medium containing
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extracellular metabolites. Soon afterwards, metabolites in the freeze-dried samples were
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dissolved in 0.5 mL of 80% methanol, vortexed for 20 min, and centrifuged for 20 min at
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13,000 × g and 8 °C. Finally, the supernatant was filtered by an organic phase filter and
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transferred to a vial for metabolite analysis. Six internal standards (L-phenylalanine-d5,
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octanoyl
146
1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine,
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nonadecanoic acid) served as quality controls for sample preparation and instrumental
148
analysis.
(8,8,8-D3)-L-carnitine,
1-lauroyl-2-hydroxy-sn-glycero-3-phosphocholine, hendecanoic
acid
and
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Metabolomic analysis adopted a pseudo-targeted approach, which can display better
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metabolomic method.27, 31 In brief, the extracts from HepG2 cells and culture medium were
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analyzed by a Waters Acquity Ultra Performance liquid chromatography coupled online to
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an ABI Q-Trap 5500 (AB SCIEX, USA) system (UHPLC/Q-Trap MS) operated in multiple
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reaction monitoring (MRM) mode, for which the MRM ion pairs were acquired from the
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cell extracts through untargeted tandem MS using an Agilent 1290 Infinity Ultra
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Performance liquid chromatography system coupled online to an Agilent 6540 UHD
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Q-TOF MS (Agilent, Santa Clara, CA) system (UHPLC/ Q-TOF MS). The same condition
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of liquid chromatography was performed on both UHPLC/Q-Trap MS and UHPLC/Q-TOF
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MS. A total of 241 ion pairs with defined parameters were analyzed in the mode of positive
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electrospray ionization (ESI+) with an Acquity UPLC BEH C8 column (2.1 mm × 100 mm,
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1.7 µm, Waters, USA). In ESI– mode, an Acquity UPLC HSS T3 column (2.1 mm × 100
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mm, 1.8 µm, Waters, USA) was used for the chromatographic separation of 59 ion pairs.
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The details of instrumental analysis are shown in Supporting Information.
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Sequence analysis of intracellular metabolites and extracellular metabolites were
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performed. To ensure data quality for metabolic profiling, pooled quality control (QC)
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samples were prepared by mixing all of the samples. During analysis of the sample
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sequence, 7 replicates of the QC samples were inserted into the analytical sequence.
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Validations of the method, including precision, stability, and recovery, were carried out.
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The results indicated that this metabolomics method is reliable (see Supporting
170
Information).
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Data Processing. MultiQuant software (3.0.1, AB SCIEX) was used for data processing
172
by analyzing the extracted ion chromatograms of the Q-Trap MS data. After the peak
173
alignment and the removal of the missing values, ion peak areas across ESI+ and ESI–
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modes were normalized to internal standards and then merged into one data set. The
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merged data set of each sample was further normalized to the sum of peak areas to balance
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the difference in cell number. A one-way analysis of variance (ANOVA) was performed
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using SPSS PASW Statistics software (SPSS Inc., Chicago, IL), and P values of < 0.05
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were considered as significantly different from the control. Principal component analysis
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(PCA) and partial least squares discriminate analysis (PLS-DA) were applied with unit
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variance (UV) scaling using SIMCA-P11.5 software (Umetrics, Sweden), and hierarchical
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cluster analysis (HCA) was conducted using the MeV software package (version 4.8.1).
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The correlation network was constructed using the Cytoscape software package (version
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2.8.2). The metabolite set enrichment analysis and pathway analysis were based on
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MetaboAnalyst, a web service for metabolomics data analysis.
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Determination of Physiologic Indicators and Relevant Enzyme Activities. To
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further understand and verify the results of the metabolomic study, ROS generation, ATP
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level, several oxidative stress markers, together with activities of some crucial enzymes
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regulating glycolytic pathway, transport across cellular membranes, β-oxidation of fatty
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acid and phospholipid metabolism, were determined. The selected oxidative stress markers
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included the levels of reduced glutathione (GSH) and malondialdehyde (MDA) as well as
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the activities of superoxide dismutase (SOD) and catalase (CAT). Intracellular ROS levels
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in HepG2 cells were assayed using a fluorescence microscope with 2,7-dichlorofuorescin
193
diacetate (DCFH-DA), according to the instructions of the ROS detection kit manufacturer
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(Nanjing Jiancheng Bioengineering Institute, China). The specific assay kits (Nanjing
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Jiancheng Bioengineering Institute, China) and enzyme-linked immune sorbent assay
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(ELISA) were adopted to determine the oxidative stress biomarkers and the activities of
197
relevant metabolic enzymes, and the method details are shown in Supporting Information.
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RESULTS AND DISCUSSION
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Basic Physiological Effects Induced by HBCD. Intracellular ROS plays an
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important role in cell signaling and homeostasis. In this study, HBCD exposure gave rise to
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a linear increase of intracellular ROS with increasing doses, and the increases were
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significant at all three exposure doses (0.05, 1 and 10 mg/L) compared with the control
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group (Figure S3). The over-production of ROS resulted in an observable oxidative stress
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response primarily characterized by the elevated activity of CAT (Figure S4). Moreover,
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exposure to a high-dose of HBCD caused a reduction in SOD activity and an increase of
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GSH levels (Figure S4). The MDA level, as a lipid peroxidation indicator, was also
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significantly altered by HBCD intervention (Figure S4).
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Cell viability was examined based on five exposure doses (0.05, 0.5, 1, 5 and 10 mg/L)
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for 24, 48 and 72 h. As shown in Figure S1, HBCD induced a dose- and time-dependent
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variation of cell viability. After 1 day exposure, cell viability was decreased at a dose of
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0.05 mg/L, whereas the viability of cells in the group with exposure dose of 1 mg/L was
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similar to that of control group. Subsequently, cell viability increased at doses of ≥ 1.0
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mg/L with a maximum at a dose of 10 mg/L. The increased cell viability was also observed
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for HepG2/C3A cell exposed to HBCD at concentration of 5 µmol/L (equivalent to 3.2
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mg/L) for 1 day.32 Exposure to HBCD at doses of ≤ 5 mg/L for 2 days significantly
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stimulated cell proliferation. However, HBCD produced statistically significant cell
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lethality at all doses after exposure for 3 days. Two previous in vitro studies observed an
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obvious inhibitory effect of HBCD on HepG2 cell at concentrations of 10–100 µmol/L
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(equivalent to 6.4–64.2 mg/L) after exposure for 3 days. 25, 33
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Metabolic Profiling and Multivariate Analysis. A pseudo-targeted metabolomic
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analysis was performed to profile the changes of intracellular metabolites in HepG2 cells
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induced by HBCD intervention. A total of 300 intracellular metabolites were detected.
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Among them, 133 metabolites were identified qualitatively using quasimolecular ions, and
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86 metabolites were further confirmed by authentic standard samples. PLS-DA was
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performed on the normalized data sets for all detected intracellular metabolites. The
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PLS-DA score plot showed a clear separation between HBCD-exposed groups and a
227
control group along the component 1 direction (Figure 1). Meanwhile, three
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HBCD-exposed groups were clearly separated from each other along the component 2
229
direction. This result indicated that HBCD caused a significant metabolic perturbation.
230 231 232 233 234
Component 2 (14.3%)
20
10
10 mg/L 0
control 1 mg/L -10
0.05 mg/L -20 -20
-15
-10 -5 0 5 Component 1 (32.8%)
10
15
235 236
Figure 1. PLS-DA score plot of metabolites in HepG2 cells exposed to HBCD at various doses
237
for 24 h. R2 = 0.977, Q2 = 0.920.
238 239
A one-way ANOVA was performed to find the differential metabolites among different
240
treatment settings. A total of 115 intracellular metabolites showed significant changes (P
0.76 were identified quantitatively and
243
are listed in Table S1. Subsequently, hierarchical clustering was used to arrange these
244
identified metabolites based on their relative contents across groups. A heat map showed
245
five dose-response trajectories (Figure S6). Compared with the control, almost all amino
246
acids presented a significant decrease in all three HBCD-exposed groups; whereas, most of
247
the phospholipids and fatty acids displayed a significant increase in low- and middle-dose
248
groups.
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Correlation Network of Differential Metabolites and Disturbance of Metabolic
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Pathway. The involved pathways of each differential intracellular metabolite were
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ascertained
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http://www.genome.jp/kegg/), and the results are listed in Table S2. To investigate the
253
latent relationships among the differential metabolites, a correlation network diagram was
254
constructed according to a comprehensive pair-wise computation of Pearson correlations
255
between the differential metabolites involved in the same metabolic pathway. As shown in
256
Figure 2, the differential metabolites were mainly bridged by fatty acids. All phospholipids,
257
including phosphatidyl choline (PC), lysophosphatidyl choline (LysoPC), phosphatidyl
258
ethanolamine (PE), lysophosphatidyl ethanolamine (LysoPE), sphingomyelin (SM) and
259
lysosphingomyelin (LysoSM), were highly up-regulated by HBCD exposure either at low-
260
and middle-doses or at a high dose. The similar up-regulation was also observed for fatty
261
acids. However, the levels of most amino acids were highly down-regulated by low- and
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middle-dose HBCD. Unlike long-chain acylcarnitine (C18:0) and medium-chain
263
acylcarnitine (C8:0), short-chain acylcarnitine (SC-AC, C2–6) presented a high
264
down-regulation at a high dose of HBCD.
by
an
online
database
of
metabolic
pathways
(KEGG,
265
Metabolic pathway perturbation induced by HBCD intervention was further studied
266
using MetaboAnalyst on the basis of the significantly different intracelluar metabolites.
267
Pathway analysis was conducted to reveal the most relevant pathways influenced by
268
different exposure
269
(www.hmdb.ca/) ID that changed significantly upon HBCD intervention, considered to be
270
highly responsible for the perturbation of amino acid metabolism (Figure S7). The most
271
relevant pathways influenced by HBCD were Alanine, aspartate and glutamate metabolism;
272
glycine, serine and threonine metabolism; and arginine and proline metabolism.
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Furthermore, enrichment analysis indicated that protein biosynthesis, the urea cycle,
274
pantothenate and COA biosynthesis, glutathione metabolism and ammonia recycling were
275
also disturbed (Figure S8).
doses. There were 88 metabolites with identified
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Figure 2. Metabolic correlation networks of the differential metabolites and related pathways.
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FA:
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lysosphingomyelin; PC: phosphatidyl choline; LysoPC: lysophosphatidyl choline; PE:
281
phosphatidyl ethanolamine; LysoPE: lysophosphatidyl ethanolamine.
fatty
acid;
SC-AC:
short
chain
acylcarnitine;
SM:
sphingomyelin;
LysoSM:
282 283
Perturbation in Amino Acid Transport and Metabolism. Amino acids are the
284
essential building blocks of proteins, energy sources, metabolite precursors and signaling
285
molecules in all living cells.34 In this study, 15 types of amino acids were supplied in
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DMEM culture medium for HepG2 cell growth. Exposure to HBCD for 1 day did not give
287
rise to a significant variation in the extracellular total contents of amino acids (Figure 3).
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Only histidine, isoleucine, leucine and threonine in culture medium of low- or middle-dose
289
groups showed slight but significant differences from that of control group (Figure S10).
290
However, the intracellular total contents of amino acids in the three exposure groups were
291
all down-regulated significantly (P < 0.01), and the maximum down-regulation (45.2% of
292
control) was observed at a low exposure dose (Figure 3). Except tryptophan in high-dose
293
group, all intracellular amino acid nutrients in three exposure groups were largely and
294
significantly decreased when compared with a control group (Figure S11). These results
295
suggested a possible inhibitory effect of HBCD on amino acid transport.
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Membrane transport proteins, mainly the SLC-family, mediate the transfer of amino
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acids across cell membranes.34 The transport via most of SLC-family is dependent on the
298
Na+-gradient, which is driven by Na+/K+-ATPase and ATP.34 The activity of
299
Na+/K+-ATPase and ATP levels in HepG2 cells were further determined. As shown in
300
Figure 3, HBCD exposure led to a significant reduction in Na+/K+-ATPase activity in a
301
dose-dependent manner, and the high-dose exposure group showed a reduction of 65.7% in
302
Na+/K+-ATPase activity compared with the control group. This result suggested a direct
303
inhibitory effect. Meanwhile, the levels of ATP were down-regulated significantly by
304
HBCD exposure (Figure 3). The maximum down-regulation (73.8% of control) was
305
observed at a middle exposure dose (Figure 3). Evidently, the significant inhibition of
306
Na+/K+-ATPase activity and the large decrease of ATP levels both caused the suppressed
307
transport of amino acids across the membrane, as indicated by the large down-regulation of
308
intracellular amino acids.
309
A
previous
study
found
that
HBCD
had
the
potential
to
inhibit
the
310
sarcoplasmic-endoplasmic reticulum Ca2+-ATPase by affecting ATP binding and the E2 to
311
E1 transition step.35 In this study, a HBCD-induced decrease of Ca2+-ATPase was also
312
observed (Figure S5). Ca2+-ATPase and Na+/K+-ATPase are both P-type ATPases, which
313
can catalyze the decomposition of ATP into ADP and a free phosphate ion. Therefore, the
314
molecular mechanisms by which HBCD inhibits the Na+/K+-ATPase should be similar to
315
that of Ca2+-ATPase, i.e., altering ATP binding and affecting the E2 to E1 transition step of
316
Na+/K+-ATPase.
317
A map of amino acid-related metabolic pathways was constructed according to KEGG
318
pathways (Figure S9). The levels of all intracellular amino acids were down-regulated
319
significantly after exposure to HBCD at three doses, with the exception of aspartate. The
320
significant down-regulation of amino acid metabolism inevitably affected protein
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biosynthesis, urea cycle, pantothenate and CoA biosynthesis, glutathione metabolism and
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ammonia recycling, as indicated by the enrichment analysis (Figure S8). ACS Paragon Plus Environment
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327
40
SLC
100
Na+
80
110
Extracellular glucose
100
328
Na+
Glucose
60 120
50
Amino acids
90
Glucose SGLT +
Na+
Glucose
Na
GLUT
Glucose
Relative abundance (%)
326
Relative content (%)
325
120
Cell outside
324
Amino acids
∑ Extracellular amino acids
Cell intside
140
∑ Intracellular amino acids
30 20
* *
* *
* *
10 0.0015 0.0010
Intracellular glucose
0.0005
330 331 332
150 100 50
Na+/K+-ATPase *
* *
* *
+
Na+
Na
ATP
Na+/K+ -ATPase +
K
0 10 control 0.05 1 HBCD dose (mg/L)
ADP + Pi
Relative content (%)
329
Relative activity (%)
0.00000
150 100 50
ATP * *
* *
* *
0 10 control 0.05 1 HBCD dose (mg/L)
333
Figure 3. Effects of HBCD exposure on the transport of amino acids and glucose across the
334
membrane of HepG2 cells. SLC: amino acid transporter SLC-family; SGLT: glucose transporter
335
SGLT-family; GLUT: glucose transporter GLUT-family. Significant differences were indicated in
336
comparison of the control by T-test. *, P < 0.05; **, P < 0.01.
337 338
Cysteine is an important precursor of cystine, GSH and redox-sensitive proteins, which
339
are necessary antioxidants in cells.36 In this study, the contents of cysteine in three exposure
340
groups were significantly decreased by more than 50% compared with that of a control
341
group. The large down-regulation of cysteine inevitably inhibited the biosynthesis of these
342
antioxidants; otherwise, their content in cells might remarkably increase, responding to the
343
physiological need to reduce the H2O2 to H2O and increase the cellular capability to
344
eliminate the redundant ROS. In addition, the large decrease of neurotransmitters (aspartate,
345
taurine, glycine and alanine) and neurotransmitters-related metabolites (e.g., tryptophan,
346
tyrosine, valine and serotonin) might induce noticeable effects of HBCD on the functioning
347
of the nervous system.
348
Perturbation in Glucose Transport and Glycolysis. The transport of glucose across
349
the plasma membrane is mediated by two groups of glucose transporters: (I) the
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Na+-dependent glucose transporter SGLT members37 and (II) the Na+-independent glucose
351
transporter GLUT family.38 These two groups of transporters are both partly expressed in
352
the liver.37, 38 Therefore, the HBCD-induced decrease of Na+/K+-ATPase activity would
353
inevitably affect the glucose transport. In this study, the intracellular glucose content in the
354
three exposure groups only presented a slight decrease, but did not show a significant
355
difference from that of the control (Figure 3). Meanwhile, the extracellular contents of
356
glucose in the three exposure groups also showed no significant differences from those of
357
the control group (Figure 3). This implies that the Na+-independent transport mediated by
358
the GLUT family should be predominant. A previous study has indicated that GLUT1 and
359
GLUT9 proteins are the major contributors to glucose influx in HepG2 cells.39
360
The slight decrease of intracellular glucose content might partly result from the
361
glycolysis inhibition produced by HBCD exposure. In order to test the hypothesis, we
362
further determined the activities of two rate-limiting enzymes of glycolysis, hexokinase
363
(HK) and phosphofructokinase (PFK), which catalyze the conversion of glucose to and the
364
conversion of fructose-6-phosphate to fructose-1,6-bisphosphate, respectively. It was found
365
that exposure to a middle-dose or high-dose of HBCD led to significant reductions of
366
49.4% and 82.1%, respectively, in PFK activity when compared with the control group
367
(Figure S5). However, only slight increases of HK activity were induced by exposure to a
368
middle-dose and high-dose of HBCD (Figure S5). On the whole, glycolysis was inhibited.
369
The inhibitory effect can be further verified by the large down-regulation of lactate content
370
(above 45% of control) in the three exposure groups, though the activities of lactate
371
dehydrogenase (LDH) were only slightly changed (Figure S5). The inhibition of glycolysis
372
partly accounted for the large reduction of ATP levels in the HepG2 cells exposed to
373
HBCD (Figure 3).
374
Perturbation in β-oxidation of Free Fatty Acids. The free fatty acids in the
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cytoplasm are first activated to acylcarnitines before they are carried into the mitochondria
376
and peroxisome where fatty acid β-oxidation occurs.40 As shown in Figure 4, HBCD
377
exposure significantly increased the total content of intracellular long-chain fatty acids
378
(LC-FA, C14–22) by more than 24%, and the total content of LC-AC were also up-regulated
379
significantly in the low- and middle-dose exposure groups. This result clearly indicated an
380
inhibitory effect of HBCD on the metabolism of LC-FA. The activities of two rate-limiting
381
enzymes for β-oxidation, long-chain acyl-CoA synthetase (LCACS) and long-chain
382
acyl-CoA dehydrogenase (LCACD), were further determined. Compared with control
383
group, the activities of LCACD in the three exposure groups were not changed significantly
384
(Figure S5), while the activities of LCACS showed significant decreases of above 45% in
385
three exposure groups (Figure 4). The large reduction in the activity of LCACS could be
386
responsible for the significant suppression of LC-FA β-oxidation. The decreased activity of
387
LCACS could mainly result from the HBCD-induced down-regulation in the expression of
388
genes relevant to LCACS, such as ACSL1, ACSL3, ACSL4 and ACSL5, which has been
389
indicated by the hepatic gene expression profiles in rats exposed to HBCD.9
390
Mitochondria are responsible for the oxidation of the major portion of fatty acids, while
391
very long-chain fatty acids (VLC-FA, C>22) are exclusively oxidized in the peroxisome.41 In
392
this study, the level of intracellular VLC-FA was increased significantly (P < 0.01) only in
393
HepG2 cells exposed to a low-dose of HBCD (Figure 4). Furthermore, MC-AC, as an
394
activated product of medium-chain fatty acids, also showed slightly increased in the three
395
exposure groups compared with the control group (Figure 4). However, the total contents of
396
the SC-AC were largely down-regulated by the low-dose and high-dose HBCD, suggesting
397
an accelerated oxidation of SC-FA (Figure 4). On the whole, the β-oxidation of fatty acids
398
was down-regulated mainly due to the inhibited oxidation of LC-FA, which predominates
399
over free fatty acids in HepG2 cells. The down-regulation of fatty acid β-oxidation is
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another important reason for the energy deficiency in cells, as indicated by the lower ATP
401
levels.
0.10
VLC-FA
0.08
40
* *
30
0.06
405 406 407
* *
* *
* *
20
0.003 0.002
* *
*
0.001 0.000
LC-acyl-CoA
SC-AC
LC-AC
VLC-acyl--CoA VLC-AC
80 60 40
LC-FA
LCACS
* *
* *
* *
20
SC-AC
LC-acyl-CoA
MC-FA
Cytosol
0.2
VLC-FA
VLC-AC MC-AC VLC-acyl-CoA MC-acyl-CoA
LC-AC
0
O.M.
Inter-membrane space
0.6
0.05
0.4
MC-AC
I.M.
0.04
* *
* *
SC-AC
0.03
SC-acyl-CoA
0.02
409
Peroxisome lumen
SC-acyl-CoA
100 LC- AC
SC-AC
408
VLC-acyl--CoA β-oxidation
0.02
Relative activity (%)
404
Relative abundance (%)
0.04
LC-FA
VLCACS
403
b
0.12
MCACS
a
LCACS
402
0.0 control 0.05
1
10
control 0.05
1
LC-AC
MC-AC
LC-acyl-CoA β-oxidation
Mitochondrial matrix
MC-acyl-CoA
10
TAC
Acetyl-CoA
HBCD dose (mg/L)
410
Figure 4. Effects of HBCD exposure on fatty acid (FA) metabolism. a) Relative abundances; b)
411 412
Metabolic pathways. AC: acylcarnitine; VLC: very long-chain; MC: medium-chain; SC:
413
long-chain acyl-CoA synthetase; MCACS: medium-chain acyl-CoA synthetase. O.M.: out
414
membrane; I.M.: inner membrane. Significant differences were indicated in comparison of the
415
control by T-test. *, P < 0.05; **, P < 0.01.
short-chain;
VLCACS:
very
long-chain
acyl-CoA synthetase;
LCACS:
416 417
Perturbation in Phospholipid Metabolism. Phospholipids are a major component of
418
all cell membranes and are also involved in key regulatory functions within cells.42 As
419
shown in Figure 5, the metabolism of intracellular phospholipid was obviously disturbed. In
420
the glycerophospholipid pathway, the major membrane component PC and its hydrolysis
421
product lysoPC were significantly up-regulated at all three exposure doses, and the total
422
contents of lysoPC metabolites, glycerophosphocholine and choline, also showed
423
significant increases in the three exposure groups compared with the control group (Figure
424
5). Moreover, HBCD elevated lysoPE levels at low and middle exposure doses, but PE
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425
levels were only slightly increased in the HBCD exposure groups without significant
426
differences when compared to the control group (Figure 5). The slight increase of PE could
427
be attributed to the inhibited biosynthesis of its direct precursor, phosphatidylserine,
428
resulting from the large decrease of L-serine in the cells exposed to HBCD. Furthermore,
429
SM and lysoSM in the sphingolipid pathway were both significantly increased in the cells
430
exposed to a low-dose and middle-dose of HBCD (Figure 5).
433 434
30 20
3.0 LysoPC * * * *
2 * *
10
GP-C
* *
1
Choline (C) * * * *
2.0 1.5
0
14 PC * *
0.8 * *
8
* *
CDP-C
Phospho-C
0.6
GP: Glycerophosphate
PC: Phosphatidyl choline
PS: Phosphatidyl serine
CDP: Cytidine diphosphate
PLA1: Phospholipase A1
PE: Phosphatidyl ethanolamine
PLA2: Phospholipase A2
S1P: Sphingosine-1-phosphate
AA: Arachidonic acid
SM
LysoSM * *
10 * *
* *
8
0.4
6
SM: Sphingomyelin
12
1.0
12 10
2.5
* *
4
1
435 PE
150 Phospho-E
S1P
Sphingosine
0.4
436 0.3
437
CDP-E
1.0
3 LysoPE
2
*
0.8
* *
0.6 GP-E
438
1 control 0.05 1 10 HBCD dose (mg/L)
Ethanolamine (E)
0.4
L-serine
* *
* *
* *
Ceramide
0.2 control 0.05 1 10 HBCD dose (mg/L)
* *
Phospholipids containing AA
PLA1
100 Relative activity (%)
PS
* *
* *
2
6
0.2
0.5
3 * *
PLA2
432
Relative abundance (%)
431
50
150
8
PLA2
100
AA
*
6
* *
*
50
4
0 control 0.05 1 10 HBCD dose (mg/L)
control 0.05 1 10 HBCD dose (mg/L)
2
439 440
Figure 5. Metabolic changes of phospholipids after exposure to HBCD. Significant differences
441
were indicated in comparison to the control by T-test. *, P < 0.05; **, P < 0.01.
442 443
Phospholipids can be hydrolyzed by phospholipase A1 (PLA1) and phospholipase A2
444
(PLA2) to form free fatty acids and lysophospholipids, which maintain the homeostasis
445
between phospholipids and free fatty acids. The activity of PLA1 was determined to be
446
almost stable in different treatments, and the activity of PLA2 was only decreased
447
significantly in cells exposed to a high-dose HBCD (Figure 5). These results implied that
448
HBCD seems not to have the potential to disturb the mass balance between phospholipids
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449
and free fatty acids at low and middle exposure doses. Therefore, the elevated
450
phospholipids levels in cells should mainly result from the inversion of increased free fatty
451
acids due to the significant inhibition of HBCD on the β-oxidation of fatty acids. A
452
previous in vitro study found that H2O2-induced neuronal cell injury led to the remodeling
453
of phospholipids mediated by PLA2, and the arachidonic acid (AA) level increased prior to
454
the increase in phospholipids.43,
455
phospholipids containing AA were also observed in cells exposed to HBCD (Figure 5). The
456
up-regulation of AA could further inhibit the production of ATP through uncoupling the
457
oxidative phosphorylation in mitochondria.45 In addition, AA is a key inflammatory
458
intermediate. The elavated AA could provide an evidence for the liver weight increase
459
indcued by HBCD exposure, which was observed in in vivo study using Wistar rats.13
460
Proposed Mechanism for HBCD Toxicity. Our data suggest that the basic cytotoxic
461
mechanism of HBCD could be related to an excessive suppression of energy metabolism
462
(Figure 6). First, HBCD adsorbed on the cell membrane directly inhibits the activities of
463
membrane transport protein P-type ATPases, mainly the Na+/K+-ATPase and Ca2+-ATPase.
464
As a result, the cell uptake of amino acids and glucose is suppressed, and the intracellular
465
concentrations of Na+ and Ca2+ would be enhanced. The suppression of amino acid
466
transport across the membrane led to the down-regulation of intracellular amino acids and
467
further inhibits protein biosynthesis. The biosynthesis inhibition of GSH and
468
redox-sensitive protein, resulting from the large down-regulation of intracellular cysteine,
469
will restrict the ability of cells to eliminate the redundant ROS. When HBCD enters into
470
cells, it not only suppresses glycolysis by inhibiting the activity of enzyme PFK, but also
471
decreases the β-oxidation of LC-FA mainly by down-regulating the gene expression
472
relevant to enzyme LCACS.9 The down-regulation of both glycolysis and fatty acid
473
β-oxidation result in a large decrease of ATP production, which further inhibits the
44
In this study, the proportional increases of AA and
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474
across-membrane transport of amino acids and glucose. Moreover, the significant inhibitory
475
effect of HBCD on the β-oxidation of LC-FA induces the overall up-regulation of free fatty
476
acids in cells, which further increases the total phospholipids through the remodeling of
477
phospholipids from free fatty acids, mainly mediated by the enzyme PLA2.
478
479 480
Figure 6. Proposed mechanism for HBCD-induced cytotoxicity based on the metabolomic
481
approach. LCACS: long-chain acyl-CoA synthetase; PFK: phosphofructokinase; HK:
482
hexokinase;
483
adenosine-diphosphate; ROS: reactive oxygen species; GSH: reduced glutathione.
LDH:
lactate
dehydrogenase;
ATP:
adenosine-triphosphate;
ADP:
484 485
ASSOCIATED CONTENT
486
Supporting Information
487
Detailed description on cell viability assay, instrumental analysis, the repeatability of
488
metabolic profiling analysis, ROS measurement, oxidative stress marker determination,
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relevant metabolic enzyme activities and total protein; results of data processing;
490
HBCD-induced perturbation of extracellular and intracellular nutrients.
491
AUTHOR INFORMATION
492
Corresponding Authors
493
* Phone: +86-411-8437-9972, fax: +86-411-8437-9562; e-mail:
[email protected].
494
* Phone/fax: +86-411-8437-9562; e-mail:
[email protected].
495
Notes
496
The authors declare no competing financial interest
497
ACKNOWLEDGMENTS
498
The authors thank the National Natural Science Foundation of China (Grant No. 21337002)
499
and the National Basic Research Program (Grant No. 2015CB453100) for the financial
500
support.
501
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