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Effects of Short-Chain Chlorinated Paraffins Exposure on the Viability and Metabolism of Human Hepatoma HepG2 Cells Ningbo Geng, Haijun Zhang, Baoqin Zhang, Ping Wu, Feidi Wang, Zhengkun Yu, and Jiping Chen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es505802x • Publication Date (Web): 07 Feb 2015 Downloaded from http://pubs.acs.org on February 12, 2015
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Effects of Short-Chain Chlorinated Paraffins Exposure on the
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Viability and Metabolism of Human Hepatoma HepG2 Cells
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Ningbo Geng,†,‡ Haijun Zhang,†,* Baoqin Zhang,† Ping Wu,† Feidi Wang,†, ‡
4
Zhengkun Yu,† Jiping Chen†,*
5 6
†
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China
‡
Graduate School of Chinese Academy of Science, Beijing 100049, China
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TOC/Abstract Art Cl
Cl
14
Cl Cl Cl Cl Cl Cl Cl Cl
12
① Linoleic acid metabolism ② Alanine, aspartate and glutamate metabolism ③ Glycine, serine and threonine metabolism ④ Arginine and proline metabolism
10 -log(P)
Cl Cl Cl
cell viability
100 80 60
4 24 h 48 h
2
①
0
50
12
②
6
90 70
④ ③
8
10 100 control 1 C10-CPs dose (µg/L)
0.0
.2
.4 .6 Pathway impact
.8
13
14 15 16 17
*Corresponding Authors
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Phone: +86-411-8437-9972, fax: +86-411-8437-9562; e-mail:
[email protected].
19
Phone/fax: +86-411-8437-9562; e-mail:
[email protected].
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ABSTRACT
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Short-chain chlorinated paraffins (SCCPs) have attracted considerable attention for their
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characteristic of persistent organic pollutants. However, very limited information is
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available for their toxic effects at environmentally relevant doses, limiting the evaluation of
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their health risks. In this study, cell viability assay and targeted metabolomic approach was
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used to evaluate the environmental dose (< 100 µg/L) effect of SCCPs on HepG2 cells. Cell
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viability was found to be decreased with increases in exposure dose of SCCPs. Exposure
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for 48 h to C10-CPs resulted in a significant reduction in cell viability compared with 24 h,
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even at 1 µg/L. SCCPs exposure altered the intracellular redox status and caused significant
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metabolic disruptions. As a kind of peroxisome proliferator, SCCPs specifically stimulated
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the β-oxidation of unsaturated fatty acids and long-chain fatty acids. Meanwhile, SCCPs
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exposure disturbed glycolysis and amino acid metabolism, and led to the up-regulation of
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glutamate metabolism and urea cycle. The toxic effects of SCCPs might mainly involve the
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perturbation of energy production, protein biosynthesis, fatty acid metabolism and
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ammonia recycling.
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INTRODUCTION
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Short-chain chlorinated paraffins (SCCPs, C10−13) are a large and complex group of
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polychlorinated n-alkanes found ubiquitously in the environment.1−4 They have been
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extensively used in metalworking fluids, paints, sealants, lubricant additives, flame
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retardants, and plasticizers as industrial additives.5 In 2009, SCCPs were reviewed as
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potential persistent organic pollutants (POPs) by the Stockholm Convention on POPs,
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concerning their persistence in the environment, long-range transport potential, liability to
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bio-accumulate and high toxicity to aquatic organisms.3,
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environmental SCCPs concentrations have resulted in “significant adverse human health
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effects” is still unknown, and evidence on the toxic action mechanisms of SCCPs is limited
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and controversial.9, 10 ACS Paragon Plus Environment
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However, whether the
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Assessment of the available data clearly indicates that SCCPs are of low acute toxicity in
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animals, although their carcinogenic toxicity has been demonstrated by a 2-year oral
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exposure study in F344/N rats and B6C3F1 mice.11 The highest dose that can be given
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without showing an adverse effect (the no observed adverse effect level, NOAEL) is 10
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mg/kg/day for rats.10 In subchronic toxicity studies on laboratory animals, SCCPs produce
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toxic effects on the kidney, liver, thyroid, and parathyroid glands.10, 12 Studies in rats and
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mice exposed to SCCPs indicate that the liver damage is associated with peroxisome
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proliferation, whereas thyroid effects are correlated to altered thyroid hormone status and
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glucuronyl transferase induction.12 However, biological events involved in the cellular
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response are not completely elucidated. As a result, the underlying toxicity mechanisms of
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SCCPs remain in a “black box”.
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Further studies on the toxic mechanisms of SCCPs are, therefore, in high demand. We
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hypothesize that SCCPs can induce oxidative stress, and hence alter the cellular metabolism
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and cell viability; meanwhile SCCPs can act as a peroxisome proliferator and specifically
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disturb lipid metabolism. In order to address above hypothesis, a metabolomic approach
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was applied to explore the metabolic response of human hepatoma HepG2 cells to SCCPs
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exposure. Metabolomics is a method of understanding metabolic regulation by studying
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small molecule metabolites. It can provide a “closer” glimpse into the phenotypic state of
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an organism because it is downstream of both proteomics and transcriptomics compared
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with that of functional genomic analysis.13 Metabolomics has been widely applied in
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biomarker discovery and toxic risk assessment in pharmacology and toxicology, as well as
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to reveal active pathways and signaling metabolites.14, 15 In recent years, metabolomics has
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been applied for investigating metabolic changes in laboratory animals and cell lines
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exposed with high-toxicity organic pollutants, such as polychlorinated dibenzo-p-dioxins
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and furans,16 polycyclic aromatic hydrocarbons,17 polybrominated diphenyl ethers,18
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perfluorooctanoic acid,19 and bisphenol A.20 However, no metabolomic assessment for ACS Paragon Plus Environment
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SCCPs toxicity has been conducted.
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HepG2 cells can express a wide range of human liver-specific functions and have been
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widely used as a model for various omics studies concerning hepatotoxicity.21−23 The
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molecular expression and biological phenotypes of HepG2 cells have been extensively
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characterized, and thus, they can be used as metabolically relevant models for in vitro
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toxicology studies. In this study, the effects of SCCPs dose, chain length, and chlorine
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content on HepG2 cell viability were first performed to explore the potential toxicity of
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SCCPs, and then the oxidative stress induction, metabolic alteration and relevant metabolic
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enzyme activities were investigated. Furthermore, the physiological functions of
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metabolites that changed in response to SCCPs exposure were also considered. The
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obtained results were expected to provide a better understanding of the cellular disturbances
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induced by environmental dose-related SCCPs exposure and new toxicity evidence of
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SCCPs from a novel perspective at the molecular level.
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EXPERIMENTAL SECTION
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Chemicals and Materials. Phosphate-buffered saline (PBS), trypsin, fetal bovine serum
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(FBS), and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from
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Gibco-BRL
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tetrazolium bromide (MTT; Amresco, Solon, OH, USA) was purchased from
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Sigma–Aldrich. Penicillin–Streptomycin was obtained from Hyclone (Logan, UT, USA).
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Fatty acid, amino acid, lactic acid, urea, glucose, glycerol standards, and methyl tert-butyl
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ether (MTBE) were purchased from Aladdin Reagent Company. HPLC-grade ammonium
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acetate and formic acid were obtained from J&K Scientific (Beijing, China). HPLC-grade
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acetonitrile (ACN) and methanol were obtained from Fisher (Fair Lawn, NJ, USA). The
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water used in all experiments was ultrapure water from a Milli-Q system (Millipore,
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Billerica, MA, USA). A standard solution (mix-1) consisting of amino acids, lactic acid,
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urea, glucose, glycerol and a standard solution consisting of fatty acids (mix-2) were used
(Grand
Island,
NY,
USA).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
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as the quality control (QC) samples for the polar and apolar fractions, respectively.
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Cell Culture and Exposure Experiments. Human hepatoma HepG2 cells were
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cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in
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a humidified atmosphere of 5% CO2. The cells were rinsed with PBS, trypsinized, and then
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transferred onto culture plates in the logarithmic phase of growth. HepG2 cells seeded in a
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96-well plate at a density of 2 × 104 cells/well were incubated for 24 h or 48 h and then
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subjected to the assay of cell viability. HepG2 cells seeded in 6-well plates at 3 × 105
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cells/well were cultured for 24 h and then submitted to the analysis of metabolites and the
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determination of oxidative stress markers and relevant metabolic enzyme activities,
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respectively.
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After cell seeding and growing to 80% confluency, the SCCPs dissolved in DMSO were
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added into the cell culture medium. The final DMSO content in culture medium was 0.05%
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(v/v), and background control wells were treated with only 0.05% DMSO. HepG2 cells
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were exposed to the SCCPs congeners with varying concentrations (1, 10, and 100 µg/L) in
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culture medium, carbon chain lengths (C10-, C11-, C12- and C13-CPs; 100 µg/L) and chlorine
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contents (C10-CPs, 100 µg/L; chlorine content: 40.4%, 57.9% and 64.4%). The exposure
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doses of 1 and 10 µg/L are comparable with SCCPs concentrations in surface water24 and
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human milk25, respectively. The SCCPs congeners with known carbon chain length and
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chlorine content used in this experiment were synthesized by chlorination of n-alkane in
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our laboratory.26
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Cell Viability Assay. Cell viability was tested using the MTT assay. When the SCCPs
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exposure experiment was finished, 20 µL of MTT (5 mg/mL, in PBS) was added directly to
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the culture medium, and then an additional 4-h incubation at 37°C and 5% CO2 was
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conducted. Violet crystals generated by viable cells were directly dissolved by 150 µL
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DMSO and well-mixed for analysis. The optical density of the sample was quantified at a
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wavelength of 492 nm with a multiwell microplate reader (Tecan Infinite F50). The cell ACS Paragon Plus Environment
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viability was calculated by setting the viability of the control cells as 100%.
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Determination of Oxidative Stress Markers and Relevant Metabolic Enzyme
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Activities. To further explore the toxicity mechanisms of SCCPs, several oxidative stress
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markers, together with activities of some crucial metabolic enzymes regulating glycolytic
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pathway, glutamine metabolism, β-oxidation of fatty acid and urea cycle, were determined.
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After exposure to C13-SCCPs (Cl, 55.0%) with varying concentrations (0, 1, 10, and 100
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µg/L) for 24 h, HepG2 cells were trypsinized, rinsed with PBS, and then resuspended in
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PBS for ultrasonic cell disruption. The supernatant was centrifuged and then subjected to
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the further analysis.
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The specific assay kits (Nanjing Jiancheng Bioengineering Institute, China) were
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adopted to determine the level of reduced glutathione (GSH) and total protein (TP), the
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activities of superoxide dismutase (SOD), catalase (CAT), pyruvate kinase (PK) and lactate
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dehydrogenase (LDH). The activities of other metabolic enzymes, phosphofructokinases
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(PFK), long-chain acyl-CoA dehydrogenase (ACADL), long-chain acyl-CoA synthetase
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(ACSL), hexokinase (HK), arginase (ARG), glutamate dehydrogenase (GDH) and
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glutaminase (GLM), were measured using the enzyme-linked immune sorbent assay
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(ELISA) kit (RapidBio, USA). The assay was performed according to the instructions of
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the manufacturer, and the detailed assay procedure is shown in Supporting Information (SI).
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All biochemical determinations were normalized to the protein content.
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Metabolism Quenching and Metabolites Extraction. Six replicates were performed
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for the control and each dose of treatment. When the SCCPs exposure experiment was
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finished, the culture medium was removed. Cells were rinsed by gently dispensing 2 mL
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ultrapure water to the surface, aspirated rapidly within 10 s, and then quenched by liquid
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nitrogen freezing. Approximately 5 mL of liquid nitrogen was used to completely fill the
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well. When liquid nitrogen was evaporated off, 2 mL of MeOH was immediately added to
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each well. Cells were scraped by a cell lifter and transferred to an Eppendorf tube. 27 The ACS Paragon Plus Environment
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obtained cells were freeze dried and stored in a −80°C freezer for further extraction and
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analysis.
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The extraction of intracellular metabolites was performed according to a method reported
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by Whiley et al..28 MTBE was used to extract apolar metabolites from HepG2 cells. The
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Eppendorf tube containing HepG2 cells from each well was added with 400 µL of MTBE
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and then vortexed for 20 min at room temperature. After adding 260 µL of ultrapure water,
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the sample was mixed and centrifuged for 15 min at 10,000 g, 4°C. Finally, 200 µL of the
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upper MTBE phases were transferred to new glass vials, dried with N2, and reconstituted in
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200 µL ACN for the analysis of the apolar fractions. Prior to extraction, 10 µL of the
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internal standards solution (norleucine, undecanoic acid and nonadecanoic acid in
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acetonitrile, 10 µg/L) was added as recovery correction for quantitative analysis. Methanol
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was used to extract the polar metabolites from the remaining part after MTBE extraction.
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The extraction was conducted using the mixture of 100 µL MTBE and 130 µL methanol.
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The sample was vortexed for 20 min and then centrifuged for 15 min at 10,000 g. A 200-µL
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aliquot of the lower methanol−water phase was used directly for the analysis of the polar
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fractions. For the extraction of extracellular metabolites, the culture medium from each well
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was transferred into a 2-ml sample tube and centrifuged at 6000 g for 10 min. 200 µL
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supernatant were freeze dried respectively and further extraction and analysis follow the
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same process with intercellular metabolites.
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LC–MS/MS Analysis. Extracts from the HepG2 cells and culture medium were analyzed
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by an Accela High Speed LC System (Thermo Fisher Scientific, San Jose, CA, USA)
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equipped with a TSQ Quantum Access MAX triple quadrupole (Thermo Fisher Scientific).
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The analyses of the polar fraction and apolar fractions were performed in two analytical
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modes. A C8 HPLC column (2.1 mm × 150 mm × 3 µm) was used for the chromatographic
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separation of the apolar fraction, with injection volume of 10 µL and total analysis time
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ammonium acetate; correspondingly, a reverse-phase HPLC C18 column (2.1 mm × 150
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mm × 3 µm; Waters, Milford, MA, USA) was used for the chromatographic separation of
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the polar fraction, mobile phase was H2O containing 0.5% formic acid and methanol, the
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injection volume was 2 µL and total time taken was 18 min. N2 was used as the auxiliary
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gas and sheath gas. The MS was operated in a positive ion mode with dynamic multiple
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reaction monitoring (MRM) for the detection of the amino acids profiles, lactic acid, urea,
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and glycerol, whereas negative ion modes with MRM was used for the detection of glucose
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and free fatty acid profiles. The details on operating parameters of LC–MS/MS are given in
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SI (Tables S1 and S2).
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Data Processing. Thermo Fisher Xcalibur 2.1 was used for data acquisition and
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qualitative analysis. Before statistical analysis, the concentration of each detected
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metabolite across all samples was normalized to the total concentrations of the
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corresponding samples (sum of all the detected metabolites in one sample) to balance their
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concentration differences resulted from the discrepancies in the number of cells for each
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sample. Multivariate analysis was performed using SIMCA-P11.5 software (Umetrics,
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Sweden). P values of < 0.05 were considered as significantly different from the control.
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Partial least squares discriminate analysis (PLS-DA) was applied with unit variance (UV)
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scaling, and hierarchical cluster analysis (HCA) was conducted using MeV software
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package (version 4.8.1). The metabolite set enrichment analysis and pathway analysis were
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based on MetaboAnalyst,29 a web service for metabolomics data analysis.
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RESULTS AND DISCUSSION
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Cell Viability. The HepG2 cells were first incubated with different concentrations of
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C10-CPs (1, 10, 100 µg/L; Cl, 60.9%) for 24 h and 48 h, respectively. As shown in Figure
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1A, C10-CPs produced an obvious impairment of cell viability. After 24-h exposure, middle-
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and high-dose C10-CPs produced statistically significant cell lethality compared with the
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negative control (P < 0.05). Exposure for 48 h with C10-CPs resulted in more reduction in
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cell viability, showing a significant decrease at all three concentrations of C10-CPs.
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Moreover, 24-h incubation with different concentrations of C13-CPs (1, 10, 100 µg/L; Cl,
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55.0%) clearly indicated a decreasing tendency in cell viability with increasing doses, and a
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significant effect on cell viability was observed at concentrations of 10 µg/L and 100 µg/L
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(Figure 1B).
207 *
* *
* *
*
* *
*
120 48 h
24 h
208
100
* *
* * *
*
* *
10
100
B
* *
80
209
A
* *
211 212 213 214
cell viability (percentage of control)
60
210
40 20 0 control
1
10
100
control
1
C13-CPs dose (µg/L)
C10-CPs dose (µg/L)
120
* *
100
C
D
* * *
80 60
215 40
216
20 0
217
control 40.4 57.9 64.4 chlorine content (%)
control 10 11 12 13 carbon chain length
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Figure 1. Viability of HepG2 cells exposed to SCCPs. (A) exposure to C10-CPs (Cl, 60.9%) with
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different concentrations, (B) exposure to C13-CPs (Cl, 55.0%) with different concentrations, (C)
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exposure to C10-CPs (100 µg/L) with different chlorine contents, (D) exposure to SCCPs (100
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µg/L) with the similar chlorine contents (Cl, 55.0%−57.9%) and different carbon chain lengths.
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Significant differences were indicated in comparison of the control by Student t-test. *, P < 0.05;
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**, P < 0.01.
224 225
The effects of carbon chain length and chlorine content of SCCPs on HepG2 cell
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viability were further investigated. Cells were incubated with different SCCPs formulas at
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the same concentration of 100 µg/L for 24 h. As shown in Figure 1C, two formulas of ACS Paragon Plus Environment
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C10-CPs with chlorine contents of 40.4% and 57.9% did not exhibit an inhibitory effect on
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cell viability; only C10-CPs with the highest chlorine content (64.4%) led to a significant
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decrease of 15% (P < 0.01) in cell viability. When HepG2 cells were exposed to SCCPs
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formulas with the similar chlorine contents (Cl, 55.0%−57.9%), cell viabilities were not
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significantly altered by C10-CPs and C11-CPs; whereas cells treated with C12-CPs and
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C13-CPs exhibited significant cytotoxicity, with reductions of 7% and 22% in cell viability,
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respectively (Figure 1D).
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An earlier toxicity study that examined the toxicological mode of action of chlorinated
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paraffins indicated that C10-CPs induced more severe histopathologies in the liver of
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juvenile rainbow trout (Oncorhynchus mykiss) than did C11-, C12-, and C14-CPs, and the
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toxicity of SCCPs was inversely related to carbon chain length.30 However, our study
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suggests that SCCPs with a longer carbon chain and higher chlorine content are prone to
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producing higher in vitro cytotoxicity. SCCPs with a longer carbon chain and higher
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chlorine content had a higher octanol-water partition coefficient (KOW).31 The increased
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cytotoxicity induced by SCCPs with higher KOW values might result from their larger
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partition into HepG2 cells from the culture medium. In a previous in vitro cytotoxicity
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study of chlorinated alkanes, Zvinavashe et al. demonstrated a strong correlation between
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the acute in vitro toxicity of chlorinated alkanes and their log KOW values in Chinese
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hamster ovary cells.32
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Oxidative Stress Responses. The loss of cell viability should be mainly resulted from
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cell energy deficiency and metabolic disorder, all of which can be primarily driven by
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oxidative stress.33 In order to counteract the oxidative stress induced by SCCPs exposure,
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the antioxidant defense system in HepG2 cells would be inevitably altered. We observed
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that CAT activity significantly increased with the increase of SCCPs doses from 0 to 100
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ng/L, and the SOD activities were slightly higher at middle-dose exposure (10 ng/L) and
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high-dose exposure (100 ng/L) when compared to the control (Figure S2). Meanwhile, ACS Paragon Plus Environment
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GSH contents in HepG2 cells were significantly decreased after exposure to middle- and
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high-dose SCCPs. These results clearly indicated a dose-dependent variation of cellular
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oxidative status.
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Metabolic Profiling. The established analytical method for targeted metabolites was
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applied to profile the metabolic changes in HepG2 cells in response to SCCPs exposure. A
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total of 43 metabolites were quantified, and most of these metabolites showed significant
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concentration differences among different treatment settings. A one-way analysis of
261
variance was calculated to test the robust differences among different treatment settings,
262
and the results are showed in Table S4.
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For the set of control and dose-dependent groups, 39 out of 43 metabolites were
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differentially produced (P < 0.05), with a false discovery rate (FDR) of < 0.05. Likewise,
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for the set of control and chlorine content-dependent groups, 40 metabolites were
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differentially produced (P < 0.05), with an FDR of < 0.05; correspondingly, all quantified
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metabolites showed significant differences for the set of control and carbon chain
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length-dependent groups. Different SCCPs treatments resulted in more or less different
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alterations of metabolic profiles in the HepG2 cells. Nevertheless, it was found that all
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SCCPs treatments induced larger fold changes (FC) in concentrations of adrenic acid
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(log2FC > 3.38) and proline (log2FC < −0.67) (Table S5). This result suggests that adrenic
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acid and proline may serve as two potential biomarkers for the diagnosis of SCCPs
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exposure.
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Glucose and 15 amino acids were supplied as nutrients for HepG2 cell growth in the
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culture medium. It was found that the concentrations of glucose and 13 amino acid
276
nutrients in extracellular culture medium were slightly decreased after the HepG2 cells
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were exposed to the low-dose C13-CPs (Figure S3). On the contrary, the intracellular
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concentrations of glucose and 11 amino acid nutrients in the low-dose group were enhanced
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slightly compared with the control (Figure S4). These results indicated that exposure to ACS Paragon Plus Environment
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low-dose SCCPs promoted the import of extracellular nutrients into the HepG2 cells.
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Multivariate Pattern Recognition Analysis. To further determine the metabolic
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alterations related to SCCPs intervention, PLS-DA was performed to the normalized data
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sets for all detected 43 metabolites. The PLS-DA score plots showed that all SCCPs
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exposure groups were clearly distinct from the control group (Figure 2). Even the low-dose
285
group (1 µg/L) exhibited significant metabolic perturbation induced by SCCPs (Figure 2A).
286
The difference between the low-dose and control groups was displayed along the PC2
287
direction, whereas the high-dose (100 µg/L) and middle-dose groups (10 µg/L) better
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separated from the control group along the PC1 direction.
289 10
6 PC2 (33.7%)
10 µg/L
2
-2
control
B
40.4%Cl
4
2
-2
100 µg/L
-6
-6
control
C12-CPs
0
-8
-4
0 PC1 (27.6%)
4
8
-10 -10
C13-CPs
C10-CPs
-4
64.4%Cl
control
1 µg/L
-10
C
C11-CPs
57.9%Cl PC2 (23.8%)
6 PC2 (32.7%)
8
10
A
-8
-6
-2 2 PC1 (22.5%)
6
10
-8
-4
0 PC1 (27.1%)
4
8
290 291
Figure 2. PLS-DA score plots of metabolites in HepG2 cells exposed to (A) C13-CPs (Cl, 60.9%)
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with different doses, (B) C10-CPs (100 µg/L) with different chlorine contents, and (C) SCCPs
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(100 µg/L) with the similar chlorine contents (Cl, 55.0%−57.9%) and different carbon chain
294
lengths.
295 296
As shown in Figure 2B, SCCPs exposure group with higher chlorine content showed a
297
greater separation from the control group mainly along PC1 direction. This result suggested
298
that SCCPs with higher chlorine content caused more pronounced metabolic perturbation.
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In the PLS-DA score plot for the data set of the control and carbon chain length-dependent
300
groups (Figure 2C), SCCPs exposure groups showed a clear separation from the control
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group mainly along PC2 direction, and the different carbon chain length groups also
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exhibited a separation trend.
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Hierarchical Clustering Analysis. To visualize the relationship between these
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metabolites, hierarchical clustering was used to arrange the metabolites based on their
305
relative levels across groups. Metabolites with significantly altered concentrations (P
