Different Toxic Effects of Racemate, Enantiomers, and Metabolite of

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Agricultural and Environmental Chemistry

Different toxic effects of racemate, enantiomers, and metabolite of malathion on HepG2 cells using HPLC-QTOF based metabolomics Jin Yan, Wentao Zhu, Biao Xiang, DeZhen Wang, Shu Sheng Tang, Miaomiao Teng, Sen Yan, and Zhiqiang Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04536 • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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Journal of Agricultural and Food Chemistry

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Different toxic effects of racemate, enantiomers, and

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metabolite of malathion on HepG2 cells using HPLC-QTOF-

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based metabolomics

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Jin Yana, Wentao Zhua, Biao Xiangb, Dezhen Wanga, Shusheng Tangb, Miaomiao Tenga,

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Sen Yana, and Zhiqiang Zhoua*

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a. Beijing Advanced Innovation Center for Food Nutrition and Human Health,

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Department of Applied Chemistry, China Agricultural University, Beijing 100193,

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China.

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b. College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.

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*Correspondence to: Zhiqiang Zhou, Department of Applied Chemistry, China

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Agricultural University, Beijing 100193, P.R. China. E-mail: [email protected]

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Jin Yan and Wentao Zhu contributed equally in this study.

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Abstract

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Commercial malathion is a racemic mixture that contains two enantiomers, and

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malathion has adverse effects on mammals. However, whether these two enantiomers

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have different effects on animals remains unclear. In this study, we tested the effect of

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racemate, enantiomers, and metabolite of malathion on the metabolomics profile of

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HepG2 cells. HepG2 cells showed distinct metabolic profiles when treated with rac-

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malathion, malaoxon, R-(+)-malathion, and S-(-)-malathion, and these differences were


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attributed to pathways in amino acid metabolism, oxidative stress, and inflammatory

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response. In addition, malathion treatment caused changes in amino acid levels,

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antioxidants activity and expression of inflammatory genes in HepG2 cells. S-(-)-

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malathion exhibited stronger metabolic perturbation than its enantiomer and racemate,

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consistent with S-(-)malathion’s high level of cytotoxicity. R-(+)-malathion treatment

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caused significant oxidative stress in HepG2 cells, but induced a weaker disturbance in

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the amino acid metabolism and a pro-inflammatory response compared to S-(-)-

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malathion and rac-malathion. Malaoxon caused more significant perturbation on

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antioxidase and a stronger anti-apoptosis effect than its parent malathion. Our results

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provide insight into the risk assessment of malathion enantiomers and metabolites. We

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also demonstrate that a metabolomics approach can identify the discrepancy of the toxic

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effects and underlying mechanisms for enantiomers and metabolites of chiral pesticides.

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Keywords: metabolomics, malathion, metabolite, enantiomers

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Introduction

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Malathion, [O,O-dimethyl-S-(1,2-dicarcethoxyethyl) phosphorodithioate], is an

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organophosphate that is used as a pesticide to control insect pests on crops, and as an

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anti-parasitic drug in both animals and humans to eliminate ectoparasites1, 2. Like other

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organophosphate pesticides, malathion inhibits the enzyme cholinesterase to eliminate

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insect pests. Malathion is commonly used and there are several modes with which

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animals and humans may be exposed to malathion, including application in farms and

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gardens, consumption of contaminated food and water, and entering malathion treated



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areas3. While it is considered to have low toxicity on mammals, it still exhibits adverse

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effects on the plasma, muscle, and brain of mammals by inhibiting cholinesterase4, and

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can cause dysfunction of other organ systems, such as liver, kidney5, and testis6.

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Malathion has a chiral α-carbon atom on the succinyl ligand and has two enantiomers.

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Both enantiomers display the same physical and chemical properties in non-chiral

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environments7; however, they have different degradation rates in biological systems.

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For example, S-malathion degrades faster than R-malathion in soil, water, and several

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crops8, 9. R-malathion is more persistent in the environment and has stronger

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insecticidal activity than S-malathion. Malaoxon is the main metabolite of malathion

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and it is more toxic than malathion in insects. Malaoxon causes DNA damage by

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inducting oxidization and methylation DNA10, 11, and this may be the underlying reason

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why commercial malathion acts as a potential mutagen12. However, in the human

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choriocarcinoma (JAR) cell line, malathion causes cyto- and genotoxic effects before

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it is metabolized into malaoxon13. Despite this, very limited work has been done to

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investigate the potential toxic effects of malathion enantiomers and its metabolites on

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mammals.

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Metabolomics provides holistic information on endogenous metabolites, and it is a

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promising tool for toxicology, pharmacology, and oncology. Metabolites are involved

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in multiple biochemical processes and can reflect the biological status and functioning

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mechanism of living organs14. Thus, metabolomics can provide insights into the

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functional mechanisms of the toxic effects of compounds, and help to find biomarkers

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to indicate exposure to these chemicals15.


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Here, we sought to identify the consequences of low dose malathion exposure on

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human liver cells by taking a metabolomics approach. Our results provide insights into

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the underlying mechanisms of malathion cytotoxicity. We compared the metabolic

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profiles of liver cells treated with racemate, enantiomers, and metabolite of malathion

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and further verified findings from the metabolomics analysis to elucidate the different

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effects of the four malathion compounds on mammalian cells.

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2. Materials and methods

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2.1 Chemicals

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Rac-malathion (98.0% purity) was provided by the China Ministry of Agriculture

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Institute for Control of Agrochemicals. The enantiomers of malathion were prepared

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following published protocols9. Malaoxon was purchased from Sigma-Aldrich, 3-(4, 5-

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dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and fetal bovine serum

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(FBS) was purchased from Zhejiang Tianhang biotechnology Co., Ltd. 25%Trypsin-

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0.05%EDTA solution, Penicillin-streptomycin solution, non-essential amino acids

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solution (NEAA), and minimum essential medium (MEM/EBSS) culture medium were

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purchased from GE Healthcare Life Science. Reactive oxygen species assay kit,

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annexin V-FITC/PI double staining apoptosis detection kit, catalase (CAT) assay kit,

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total superoxide dismutase (T-SOD) assay kit, and reduced glutathione (GSH) assay kit

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were

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Dimethylsulfoxide (DMSO), sodium chloride (NaCl), disodium hydrogen phosphate

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(Na2HPO4), sodium bicarbonate (NaHCO3), and potassium dihydrogen phosphate

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(KH2PO4) were purchased from Sinopharm Chemical Reagent Co., Ltd.

purchased

from

the

Nanjing

Jiancheng


Bioengineering

Institute.


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2.2 Cell culture and treatment

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HepG2 cells were purchased from the National Infrastructure of Cell-line Resource

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(Beijing). Cells were cultured in MEM medium with 10% FBS, 1% NEAA, and 100

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U/mL Penicillin-streptomycin. Cells were placed in a humidified atmosphere with 5%

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CO2 and 95% air at 37 ℃. The compounds used in experiments were dissolved in

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DMSO, and the final concentration of DMSO was 1% (v/v) for each treatment.

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2.3 Cytotoxicity assay

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Cytotoxicity was measured by the MTT method as described previously16. HepG2

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cells (1.5×104 cells/well) were seeded on 96-well plates and cultured for 24 h. Fresh

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media containing increasing concentration of the treatment compound (100, 200, 300,

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400, 500, 600, 700, and 1000 μΜ of malathion racemate, enantiomers and malaoxon)

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were then placed on the cells. Six replicates were conducted per concentration and at

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least three independent experiments were performed. The vehicle control was standard

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culture media with 1% DMSO. After culturing cells with treatment media for 48 h, 10

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μl MTT solution (5 mg/ml in sterile water) was added in each well. Cells were placed

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in 4 h at 37 ℃, the media were removed by injection syringe, and the formazan crystals

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were dissolved in DMSO. Samples were agitated for 10 min, and the absorption was

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measured using a spectrophotometer (Multiskan MK3, Thermo Scientiﬁc, Pittsburgh,

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PA) at 492 nm. The cytotoxicity was expressed by EC50 values (50% effects

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concentration), which were determined using IBM SPSS 21.

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2.4 Cell viability

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HepG2 cells were exposed to 5, 50, and 100 μΜ malathion racemate, R-enantiomer,


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S-enantiomer, and malaoxon for 48 h as described above. Cell viability was measured

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using a spectrophotometer (Multiskan MK3, Thermo Scientiﬁc, Pittsburgh, PA) at 492

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nm via the MTT method, which was identical to the cytotoxicity assay.

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2.5 Metabolomics analysis

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HepG2 cells (6×105 cells/well) were seeded on 6-well plates in 2 ml of culture

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medium. After incubation for 24 h, media containing malathion racemate, R-(+)-

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malathion, S-(-)-malathion and malaoxon at 5, 50, and 100 μΜ were added. Each

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treatment was conducted in six replicates. Samples were cultured for 48 h, and cells

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were washed with PBS three times and harvested using a cell scraper in 1ml PBS. After

117

counting the number of cells using an automated cell counter (Counter Star, Shanghai,

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China), we transferred the cell pellet into tubes and added 1 ml cold extraction solvent

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methanol:choloroform (9:1) to quench cellular metabolism. The counted number of

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cells was used to normalize metabolomics data. Cells were ultrasonicated in an ice bath

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ultra-sonicator (Sonics Vibra-Cell™, Sonics&Materials Inc, USA) for 5 min, and

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subsequently centrifuged at 4 ℃ for 5 min at 12,000 g. The supernatant was transferred

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and divided into two parts, one of which was used for untargeted metabolomics analysis,

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while the other was used for targeted amino acid analysis.

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2.5.1 Untargeted metabolomics analysis

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700 μL supernatant of the cell extractions were collected and dried with a stream of

127

nitrogen. The residues were reconstituted in 500 μL solvent consisting of 60%

128

acetonitrile and 40% water and purified with 0.22 μm filters before HPLC-QTOF

129

analysis. Cell metabolic profiles were obtained using Agilent 1200 series HPLC system



130

coupled with Agilent 6510 QTOF mass spectrometry (Agilent, USA). The analysis was

131

performed in reversed-phase column (ACQUITY BEH C18, 150 mm × 2.1 mm, 1.7 μm,

132

Waters, Milford, CT, USA) equipped with a guard cartridge system and maintained at

133

40 ℃. The flow rate was 0.3 mL/min and the mobile phase contained solvents A (10%

134

acetonitrile/90% H2O containing 5 mM ammonium acetate and 0.2% acetic acid) and

135

B (90% acetonitrile/10% H2O containing 5 mM ammonium acetate and 0.2% acetic

136

acid). Samples were passed through a gradient of 10% A for 1.5 min, solvent with

137

linearly increasing concentration of A from 10% to 50% for 3.5 min and held for 7 min;

138

then, samples were returned to 10% A solution for 2 min, and held in 10% A for 16

139

min. Electrospray ionization (ESI) conditions were conducted in the positive and

140

negative electrospray modes in separate runs with full scan mode ranges from m/z 60

141

to 1000. The capillary voltages were 3800 and 4000 V with a scan rate of 1.03 scans

142

per second. The nebulizer gas flow rate was 10 L/min; the pressure was maintained at

143

45 psi and temperature was maintained at 325 ℃. Reference masses 121.0509,

144

922.0098 (positive mode) and 119.0363, 966.0007 (negative mode) were used for

145

continuous and online mass calibration throughout the analyses. Samples were injected

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in randomized sequence.

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The Molecular Feature Extractor (MFE) tool in the MassHunter Qualitative Analysis

148

Software (Agilent, USA) was used to clean unrelated ions and background noise from

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the data files. MFE is an untargeted feature-finding algorithm that can extract individual

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compound features from QTOF-MS chromatograms even when these are complicated

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and when compounds are not well resolved. The MFE outputs a list of all possible


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compound features extracted from full scan QTOF data. Then, the MassHunter Mass

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Profiler Professional Software B.02.00 (Agilent, USA) was used to align and filter

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extracted features. Absolute abundance above 5000 counts and with a minimum of two

155

ions were chosen for the analysis. Metabolites from different samples were aligned

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using a retention time window of 0.1% (0.15 min); multiple charge states were not

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selected. Common features present in > 80% of all samples were analyzed and corrected

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for individual bias.

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2.5.2 Analysis of the targeted amino acid metabolism

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180 μL supernatant of cell extraction was mixed with stable-isotope labeled internal

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standards (20 μL); then, the samples were derivatized with both reagent I (1-propyl

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alcohol/3-picoline = 77/23, 80 μL) and reagent II (chloroform/iso-octane/propyl

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carbonochloridate =71.6/11/17.4, 50 μL). The amino acid derivatives were extracted

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with ethyl acetate (250 mL), and the supernatant was dried by a stream of nitrogen and

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then re-dissolved in 200 μL 0.1% formic acid in water.

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The amino acid concentration was measured using an UltiMate 3000 system plus

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TSQ Quantum Access Max mass spectrometer (Thermo Fisher Scientific). An EZ:faast

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4u AAA-MS column (250 mm x 2.0 mm, 4 μm, Phenomenex, USA) was used to

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separate the amino acids using both mobile phase A (acetonitrile with 0.1v% formic

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acid) and mobile phase B (water with 0.1 v% formic acid) at an injection volume of 5.0

171

uL and a flow rate of 0.3 mL/min. The mobile phase constituent began with 62% A and

172

38% B, was then linearly increased to 79% A and 21% B for 12 min, and adjusted back

173

to 62% A and 38% B for 4 min. Experimental results were processed using Xcalibur



174

2.2 (Thermo Fisher Scientific).

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2.6 Antioxidant assays

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HepG2 cells were seeded in 6-well plates with 7×105 cells/well density and exposed

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to the four compounds for 48 h. Subsequently, the media was removed by vacuum

178

pump and cells were rinsed with phosphate buffered saline (PBS) three times. Cells

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solution were obtained by cell scraper with PBS, and the cell pellets were collected

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after centrifugation at 1000 × g for 5 min and stored in -80 ℃. Cells were suspended in

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1 ml PBS, sonicated for 2 min using an ice bath ultra-sonicator (Sonics Vibra-Cell™,

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Sonics&Materials Inc, USA), and centrifuged at 4000 × g for 5 min. Supernatants were

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obtained to measure the antioxidant GSH and antioxidant enzymes catalase (CAT),

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superoxide dismutase (SOD) using UV-2600 spectrophotometer (Shimadzu, Japan)

185

according to the instructions of the test kits from the Nanjing Jiancheng Bioengineering

186

Institute (Nanjing, China). All results represent three independent experiments.

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2.6. Gene expression analysis

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HepG2 cells were seeded in 6-well plates with 6×105 cells/well density and

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exposed to the four compounds for 48 h. The expression level of genes involved in the

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inflammatory response, cyclooxygenase-2 (COX-2) and nuclear factor-kappa B (NF-

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κB),

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lymphoma/leukemia-2gene (Bcl-2), and Bcl-2-associated X protein (Bax), were

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measured using qRT-PCR. Primer sequences are listed in Table S1. Total RNA was

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extracted from cells using RNA isolation kit (Tiangen, Beijing, China) following the

195

manufacturer’s protocol. NanoDrop 2000c spectrophotometer (Thermo Scientific,

and

genes

involved

in

apoptosis,

caspase-3,


caspase-9,

B-cell

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Wilmington, DE) was used to measure the concentration of the total extracted RNA.

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The quality of the total RNA was assessed by the ratio of absorbance (A260/A280) and

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the integrity of banding pattern on a 2% agarose gel. 1 μg of total RNA was reverse

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transcribed to cDNA using the FastQuant RT Kit (with gDNAase) (Tiangen, Beijing,

200

China). Quantitative real-time PCR was performed with 1 μL of the cDNA template

201

with the SuperReal premix Plus (SYBR Green) Kit on an ABI-7500 system (Advanced

202

Biosystems, Foster, California, USA). Experiments were performed at least three times.

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2.7 Western blotting analysis

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HepG2 cells were seeded on to 6-well culture plates at a density of 6×105 cells/well

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and treated with 100 μM four malathion compounds at 37 °C for 48 h. Cells were

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harvested and lysed in 100 μL ice-cold lysis buffer. The supernatants of lysates were

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collected following ultrasonication and centrifugation. The protein concentrations were

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measured with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Western

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blotting was conducted with 150 μg protein per lane. The primary antibodies of rabbit

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polyclonal antibodies against caspase 3 and COX-2 were employed. The following

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secondary antibodies were employed: goat anti-rabbit IgG or rabbit anti-mouse IgG

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(Zhongshan Golden Bridge Co., Beijing, China). The results were normalized to either

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the β-actin standard and analyzed using ImageJ (National Institute of Mental Health,

214

Bethesda, MD, USA).

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2.7 Statistical analysis

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SIMCA P+ (Version 13, Umetrics, Sweden) was used for multivariate statistical

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analysis of metabolomics data. Principal component analysis (PCA) was performed to



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investigate the intrinsic clusters of different groups and to search for possible outliers.

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Partial least-squares discriminant analysis (PLS-DA) was subsequently conducted on

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the Q-TOF datasets to uncover treatment-related differences. Metabolite differences in

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cells after exposure to DMSO, malathion racemate, enantiomers, and malaoxon were

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evaluated using student’s t-test. Variable importance in the project (VIP) > 1 and p
1 and student’s t test p < 0.05,

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and found 78, 42, 85, and 50 altered endogenous metabolites in HepG2 cells treated

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with rac-malathion, R-(+)-malathion, S-(-)-malathion, and malaoxon, respectively.

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Metabolites that were affected by malathion compounds included carbohydrates, amino

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acids, nucleosides, fatty acids, phospholipids, and carboxylic acids (Table S2).

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Metabolic profiles were most perturbed under S-(-)-malathion treatment, followed by

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rac-malathion, malaoxon, and R-(+)-malathion. These results were similar to the

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cytotoxicity assays, where S-(-)-malathion resulted in the most severe effects. A total

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of 26 metabolites were changed in cells exposed to malathion racemate and its two

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enantiomers (Fig. 3A). More metabolites were affected only by one of the three


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compounds. Moreover, rac-malathion and S-(-)-malathion caused similar changes in

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26 metabolites, while only one metabolite was shared in the metabolic changes induced

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by rac-malathion and R-(+)-malathion. These results indicate that metabolic profiling

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changes induced by R-(+)-malathion and S-(-)-malathion were enantio-selective, and

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the metabolome of S-(-)-malathion treatment was similar to that of rac-malathion

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treatment. Metabolic changes in the rac-malathion and malaoxon treatment conditions

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shared 35 metabolites; however, both compounds still induced changes that were

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unique to each compound in HepG2 cells (Fig. 3B).

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We next identified the metabolic pathways that were altered by the four compounds.

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HepG2 cells exposed to rac-malathion (Fig.4), showed significant changes in

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glycerophospholipid metabolism, arginine and proline metabolism, alanine, aspartate

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and glutamate metabolism, arachidonic acid metabolism, pantothenate and CoA

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biosynthesis, phenylalanine metabolism, lysine degradation, aminoacyl-tRNA

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biosynthesis, glutamine and glutamate metabolism, as well as lysine biosynthesis. S-(-)-

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malathion affected seven metabolic pathways, all of which were also affected by rac-

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malathion. Six metabolic pathways were affected by R-(+)-malathion treatment, and

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the most significant change occurred in taurine and hypotaurine metabolism. This is a

301

pathway that was not affected by exposure to rac-malathion and S-(-)-malathion. R-(+)-

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malathion exposure also affected glycine, serine, and threonine metabolism, a pathway

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that was significant only in this treatment. Only four metabolic pathways were

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significantly affected by malaoxon exposure, including the histidine metabolism, which

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was unique to this treatment. These results suggest that changes of the metabolic



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pathways caused by R-(+)-malathion and S-(-)-malathion were also enantio-selective.

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Changes in metabolic pathways that were induced by S-(-)-malathion exposure were

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similar to rac-malathion exposure. Moreover, malathion treatment resulted in the

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disruption of more pathways than malaoxon treatment. In addition, the metabolic

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pathways that were disrupted by exposure to the four malathion compounds were highly

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related to the amino acid metabolism, taurine and hypotaurine metabolism,

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glycerophospholipid metabolism, and arachidonic acid metabolism.

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3.3 Amino acid metabolism

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To further investigate the effects of malathions on amino acid metabolism, we

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quantified amino acid levels using a targeted HPLC-MS/MS approach with stable

316

isotope-labeled internal standard. We found that levels of serine, asparaginate, glycine,

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alanine, lysine, proline, glutamate, phenylalanine, valine, leucine, and glutamine were

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significantly affected by the four malathion compounds (Fig. 5). Moreover, rac-

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malathion exposure, which induced changes in eight metabolites, led to the strongest

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perturbation of amino acid levels, followed by S-(-)-malathion and malaoxon. Only

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levels of two amino acids were altered by R-(+)-malathion exposure; therefore, R-(+)-

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malathion showed the weakest effect on amino acids in HepG2 cells.

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3.4 Antioxidant activity

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The four tested compounds altered several endogenous metabolites that are related

325

to oxidative stress in cells. We tested for changes of oxidative stress by measuring

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antioxidant activity and differences in redox homeostasis in cells treated with the four

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compounds. SOD converts O2- to H2O2, and its activity levels were reduced in the R-


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(+)-malathion treatment for all dosages. SOD activity was not affected by rac-

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malathion treatment, and only 5 μM malaoxon treatment decreased SOD activity. All

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three doses of S-(-)-malathion altered SOD activity, and SOD activity levels were

331

decreased in low and medium dose treatments, but increased in the high dose treatment.

332

The activity of CAT, a scavenger of H2O2, was reduced after exposure to 5 μM R-(+)-

333

malathion, as well as 50 μM R-(+)-malathion and malaoxon. GSH content, which also

334

converts O2- to H2O2, were decreased after exposure to 5 μM of rac-malathion, R-(+)-

335

enantiomer, S-(-)-enantiomer, and malaoxon. Surprisingly, the GSH content was not

336

affected in HepG2 cells treated with high doses of the four malathion compounds.

337

These results suggest that R-(+)-malathion caused stronger oxidative stress compared

338

to the other compounds, and the metabolite malaoxon perturbed antioxidants more than

339

its parent malathion.

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3.5 mRNA expression of inflammatory genes

341

Arachidonic acid metabolism disorder contributes to inflammatory response;

342

therefore, we investigated the expression of cyclooxygenases-2 (COX-2), which is an

343

immediate-early response gene for inflammation. Rac-malathion, R-(+)-malathion, S-

344

(-)-malathion, and malaoxon treatment induced a significant and dose-dependent up-

345

regulation of COX-2 gene in HepG2 cells (Fig. 7). In addition, expression levels of the

346

inflammatory transcription factor NF-κB were also higher in cells treated with the four

347

malathion compounds. These obtained results suggest that exposure to malathion

348

compounds led to pro-inflammatory activity in HepG2 cells. Furthermore, R-(+)-

349

malathion did not alter gene expression levels of COX-2 and NF-kB in low dose



350

treatment, and presented the lowest effect on inflammatory response.

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3.6 mRNA expression of apoptosis genes

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Exposure to cytotoxic compounds can induce apoptosis; therefore, we tested

353

whether treatment with malathion compounds altered gene expression of caspase-3,

354

caspase-9, bax, and bcl-2 (Fig. 8). Expressions of the pro-apoptotic genes caspase-3

355

caspase-9, and bax were not affected by exposure to the four malathion compounds.

356

However, the expression level of bcl-2, an anti-apoptotic gene that delays the

357

progression of mitochondrial pathways in apoptosis, was up-regulated in a dose

358

dependent manner upon treatment with the four malathion compounds. These results

359

suggested that malathion compounds had an anti-apoptosis effect on HepG2 cells, and

360

no significant differences in anti-apoptosis ability were observed among the four tested

361

compounds.

362

3.7 Protein content of caspase 3 and COX-2

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To confirm the effect of the four malathion compounds on inflammatory and

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apoptosis response, HepG2 cells were exposed to 100 μM rac-malathion, R-(+)-

365

malathion, S-(-)-malathion, and malaoxon. The results of Western blot revealed that all

366

the malathion compounds elicited an increase in COX-2 (Fig. 9). Rac-malathion

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induced the strongest expression of COX-2 compared to others. Similar to gene

368

expression, the induction of COX-2 protein in R-(+)-malathion exposure group which

369

presented the lowest effect on inflammatory response in gene expression was modest.

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Moreover, the caspase 3 protein was not changed in cells exposed to rac-malathion, R-

371

(+)-malathion, S-(-)-malathion, and malaoxon.


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4. Discussion

373

Most molecules within living organism are chiral compounds. Malathion is one of

374

the most commonly used chiral organophosphate pesticide, and has two enantiomers:

375

R-(+)-malathion and S-(-)-malathion. Thus, the enantiomers of malathion may interact

376

differently in living systems, making it stereo-selection, and several studies have

377

reported the stereo-selective acute toxicity of malathion enantiomers in living organism.

378

However, how different enantiomers of malathion cause toxicity in mammals are

379

unclear. In present study, we took a metabolomics approach to test how low doses of

380

rac-malathion, R-(+)-malathion, S-(-)-malathion, and malaoxon affect the metabolome

381

of HepG2 cells and clarify the underlying toxic mechanism of malathion compounds.

382

Exposure to rac-malathion, R-(+)-malathion, S-(-)-malathion, and malaoxon all

383

affected the survival of HepG2 cells. S-(-)-malathion showed highest cytotoxicity in

384

HepG2 cells in comparison to the other malathion compounds. However, these results

385

were inconsistent with previous studies that report R-(+)-malathion as having the

386

strongest toxic effect on Daphnia magna, earthworms and honeybees, followed by rac-

387

malathion, and S-(-)-malathion9,

388

difference in the micro-environment between tissues in vivo and in vitro. Similar toxic

389

differences were also observed in isomers of endosulfan, where β-endosulfan had

390

higher toxicity on HepG2 and SH-SY5Y cells than α-endosulfan19, 20; however, toxicity

391

of α-endosulfan was higher than that of β-endosulfan in Daphnia, rainbow trout, and

392

Hyalella in vivo21.

393

17, 18.

This inconsistency may be caused by the

The metabolomics profiles of HepG2 cells were significantly changed by exposure



394

to rac-malathion, R-(+)-malathion, S-(-)-malathion, and malaoxon at concentrations

395

that did not result in cell death. PCA and PLS-DA revealed that metabolic perturbations

396

were different and enantio-selective, and the patterns were similar to the results of the

397

cytotoxicity test. S-(-)-malathion treatment resulted in the strongest disturbance on the

398

cell metabolome, with 85 metabolites altered, followed by rac-malathion, malaoxon,

399

and R-(+)-malathion. Changes in metabolites were related to several metabolic

400

pathways, and these may shed light on the underlying mechanism for different toxicity

401

of malathion racemate, enantiomers, and metabolite.

402

Amino acid and their metabolites are important for building proteins and

403

polypeptides as well as regulating cell growth, reproduction, and immunity. Disordered

404

amino acid levels could cause neurological disorders, oxidative stress, cardiovascular

405

disease, and cell death22. According to the results of metabolic pathways analysis, many

406

pathways altered by the four malathion compounds were involved in the amino acid

407

metabolism. We quantified amino acid concentrations and found that rac-malathion,

408

malaoxon, R-(+)-malathion, and S-(-)-malathion caused significant changes in amino

409

acid levels, but to different extent.

410

Serine is a nonessential amino acid and plays an important role in cell proliferation.

411

It showed increase in levels after exposure to rac-malathion, malaoxon, and S-(-)-

412

malathion. In cells, serine can be derived from glycine, which is regarded as an indicator

413

of cancer cell proliferation23. Treatment of malaoxon and S-(-)-malathion induced a

414

significant increase in the glycine level. These results suggest that malathion

415

compounds may induce proliferation of HepG2 cells and these changes are stereo-


Page 20 of 32

Page 21 of 32


416

selective. HepG2 cells exposed to low levels of malathion showed increase in cell

417

proliferation24, and inductions of proliferation by malathion were also observed in rat

418

mammary gland25 and earthworms spermatogonia26. Changes in asparagine and

419

glutamine may be related to toxic effects of malathion on the immune function27, and

420

changes induced by rac-malathion differed to other malathion compounds. Moreover,

421

the amounts of glutamate, phenylalanine, leucine, proline, and lysine were also

422

decreased to different extent in HepG2 cells that were exposed to one or two malathion

423

compounds. The decreased amino acid levels observed in malathion treated cells may

424

account for the increase of protein synthesis, which was also observed in fish exposed

425

to malathion28-30. More importantly, this is the first investigation of the different effects

426

of racemate, enantiomers, and metabolite of malathion on amino acid metabolism, and

427

R-(+)-malathion, which only induced changes in two amino acids, showing the weakest

428

disturbance to amino acid metabolism compared to the other three compounds.

429

Several amino acid changes observed in the experiment above were related to

430

oxidative stress. The taurine and hypotaurine metabolism pathway, which was involved

431

in oxidative stress, was also affected by R-(+)-malathion and malaoxon. We also found

432

that levels of taurine, which have efficacy against chemical-induced oxidative stress31,

433

32,

434

product of glutathione formation and is involved in the antioxidant defense. S-(-)-

435

malathion treatment reduced spermidine levels in HepG2 cells. The metabolite of

436

spermidine (spermine) can prevent glutathione release33 and was increased after

437

exposure to rac-malathion and malaoxon. Changes in these metabolites suggest that the

were increased in HepG2 cells after exposure to malaoxon. Spermidine is a by-



438

redox balance in HepG2 cells was disordered. In accordance with this, we observed

439

decreased levels of glutathione in cells exposure to the four malathion compounds.

440

Except for glutathione, cells always express an elaborate array of antioxidant

441

enzymes to eliminate free radicals. SOD and CAT are enzymes that convert superoxide

442

anion (O2-) to hydrogen peroxide (H2O2) and reduce H2O2 to water34. R-(+)-malathion

443

and malaoxon treatment caused significant decreases in CAT and SOD activities;

444

moreover, SOD activity was affected by S-(-)-malathion exposure. Cells tend to

445

regulate their enzymatic system as well as their endogenous metabolites to remove the

446

ROS and maintain the redox homeostasis when exposed to xenobiotic compounds.

447

However, xenobiotics deplete antioxidants and disturb the redox state, thus resulting in

448

oxidative stress and oxidative damage35, 36.

449

Our results suggest that HepG2 cells regulated levels of antioxidative enzymes and

450

metabolites to resist oxidative stress when exposed to the four malathion compounds.

451

Rac-malathion exposure induced significant changes in antioxidants without altering

452

CAT and SOD activity levels. This implies that cells exposed to rac-malathion undergo

453

a different oxidative stress response compared to after exposure to the other three

454

compounds. In addition, changes in oxidative stress resistance induced by malathion

455

were not dose dependent in a predictable way, and the reaction was more sensitive when

456

cells were exposed to low dose of malathion compounds. Previous studies have also

457

shown that malathion induces ROS production and causes oxidative stress in diverse

458

organs24, 37-41, and exposure to low dose of malathion alters CAT and SOD activities in

459

liver, kidney, lung, diaphragm, quadriceps, and brain39, 42. Here, we firstly compared


Page 22 of 32

Page 23 of 32

460


the different effects of racemate, enantiomers, and metabolite of malathion.

461

Oxidative stress induces inflammation response and apoptosis43. In our study, we

462

found evidence that malathion treatment induces an inflammation response in HepG2

463

cells. This non-targeted metabolomics study showed that glycerophospholipid

464

metabolism (i.e. LysoPC and PC) was altered after exposure to the four malathion

465

compounds. LysoPC is involved in inflammatory diseases and regulates formation of

466

arachidonic acid44, and LysoPC(18:1) stimulates IL-1β production in human

467

monocytes45. LysoPC(18:1(9Z)) levels were altered in HepG2 cells exposed to S-(-)-

468

malathion and rac-malathion, and perturbation of arachidonic acid metabolic pathway

469

was observed in rac-malathion, S-(-)-malathion, and malaoxon treatments. We also

470

observed a significant increase in arachidonic acid in cells exposed to the four

471

compounds. Arachidonic acid mediates inflammation and organ function either directly

472

or upon conversion to eicosanoids. PGE2 is an eicosanoid synthesized from arachidonic

473

acid through cyclooxygenase-2 (COX-2), and it activates the secretion of IL-1α and IL-

474

246. We found elevated gene and protein expression of COX-2 after exposure to the

475

four malathion compounds. In summary, these results suggest that malathion compound

476

treatment triggers an inflammatory response in cells.

477

NF-κB is a transcription factor that regulates stress response47, and its activation

478

regulates the expression of COX-2 to promote inflammation48. Exposure to the four

479

malathion compounds caused a significant increase in NF-κB mRNA expression,

480

suggesting that changes in COX-2 expression may be mediated by the NF-κB signal

481

transduction pathway. These findings are consistent with previous studies that showed



Page 24 of 32

482

that malathion induced inflammatory responses and stimulated hepatic cytokines to

483

active NF-κB in the rat liver49. In addition, both arachidonic acid and LysoPC(18:1(9Z))

484

levels were not affected by R-(+)-malathion treatment, and changes in gene expression

485

of COX-2 and NF-kB were not as striking in low dose of R-(+)-malathion treatment

486

compared to the other compounds; therefore, R-(+)-malathion may result in a weaker

487

inflammatory response.

488

Apoptosis, known as a form of programmed cell death, can be triggered by

489

exogenous chemicals and mitochondria-generated ROS50,

51.

490

(DPA) reduces tumor growth by inhibiting bcl-252, 53 and its levels were increased in

491

cells exposed to the four malathion compounds. However, we observed enhanced

492

expression of bcl-2 while the expression of bax remained unchanged, which may tilt

493

the cells toward survival and render tumor cells more resistant to chemical-induced

494

apoptosis54. The increase in bcl-2 expression may be a result of the increased NF-κB

495

expression induced by these compounds, which can counteract cell death through

496

delivering anti-apoptotic signals55. The increase in DPA levels may be a stress response

497

in cells to resist toxic effects induced by these compounds. Further studies on

498

malathion-induced apoptosis will clarify the underlying mechanisms in the future.

Docosapentanoic acid

499

We used a metabolomics approach to identify the metabolic perturbations in HepG2

500

cells induced by rac-malathion, malaoxon, R-(+)-malathion, and S-(-)-malathion. We

501

aimed to explore the mechanisms underlying cytotoxicity in mammalian cells by the

502

chiral pesticides. The obtained results of the present study revealed that exposure to the

503

four malathion compounds caused significant changes in metabolic profiles of HepG2


Page 25 of 32


504

cells compared to control. Changes in metabolic profiles were involved in multiple

505

aetiologies and pathogeneses, including amino acid metabolism, oxidative stress, and

506

inflammatory response. Moreover, we verified results of the metabolomics analysis and

507

found that amino acid levels, antioxidant activity, and expression of inflammatory

508

genes were also changed after exposure to the four compounds. In addition, rac-

509

malathion, malaoxon, R-(+)-malathion, and S-(-)-malathion treatment resulted in a

510

unique profile of changes in HepG2 cells, suggesting that while they share several core

511

similarities, each compound has different effects on cells. For example, malathion

512

treatment resulted in a stronger disturbance of cell metabolic profile than malaoxon. R-

513

(+)-malathion showed weaker effects on amino acid metabolism and pro-inflammatory

514

response than S-(-)-malathion and rac-malathion. Our results illustrate that

515

metabolomics is a powerful tool for assessing the toxic effect of chiral pesticides and

516

can provide data for health risk assessment of malathion at holistic levels.

517

Acknowledgement

518

Funding was provided by the National Key Research and Development Program of

519

China (2016YFD0200202), the National Natural Science Foundation of China

520

(21337005), and the Young Elite Scientists Sponsorship Program by CAST.

521

References

522 523 524 525 526 527 528 529

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Page 28 of 32


Mortality rate%

100

R-(+)-MA S-(-)-MA rac-MA MO

80 60 40 20 0 0

200

400

600

800

1000

660 661 662 663 664

665 666 667 668

100

50

0 co

concentration/μM

150

nt r M ol A M -5 A M -5 A- 0 1 M 00 O M -5 O R- MO -50 R- (+)- -100 R- (+)- MA (+ M -5 )-M AS - A - 50 S- (-)-M100 S- (-)-M A(-) A 5 -M -5 A- 0 10 0

B

A

Viability(% of control condition)

Page 29 of 32

Fig. 1 A. Mortality rate of HepG2 cells induced by different concentrations of rac-malathion, R-(+)malathion, S-(-)-malathion, and malaoxon for 48 h. B. Effects of 5, 50, and 100 μM rac-malathion, R-(+)-malathion, S-(-)-malathion, and malaoxon on cell viability of HepG2 cells for 48 h.

Fig. 2. PLS-DA score plots based on Q-TOF data sets of HepG2 cells treated with three doses of malathion racemate, enantiomers, and malaoxon. (A) Positive mode of 5 μM exposure (R2X = 0.326, Q2 = 0.326); (B) negative mode of 5 μM exposure, (R2X = 0.614, Q2 = 0.544); (C) positive mode



669 670 671 672 673

674 675 676 677

of 50 μM exposure (R2X = 0.399, Q2 = 0.401); (D) negative mode of 50 μM exposure, (R2X = 0.597, Q2 = 0.495); (E) positive mode of 100 μM exposure (R2X = 0.435, Q2 = 0.543); (F) negative mode of 100 μM exposure, (R2X = 0.619, Q2 = 0.475).

Fig. 3. Numbers of changed HepG2 cells endogenous metabolites after exposure to (A) racmalathion, R-(+)-malathion, and S-(-)-malathion; (B) rac-malathion and malaoxon.

678 679 680 681 682

Fig. 4. Global metabolic pathways affected by the four malathion compounds based on MetaboAnalyst 3.6. (A) rac-malathion; (B) R-(+)-malathion; (C) S-(-)-malathion; (D) malaoxon. The following abbreviations were used: a. Glycerophospholipid metabolism; b. Arginine and proline metabolism; c. Alanine, aspartate, and glutamate metabolism; d. Arachidonic acid


Page 30 of 32

Page 31 of 32

metabolism; e. Pantothenate and CoA biosynthesis; f. Phenylalanine metabolism; g. Lysine degradation; h. Aminoacyl-tRNA biosynthesis; i. Glutamine and glutamate metabolism; j. Lysine biosynthesis; k. Taurine and hypotaurine metabolism; l. Glycine, serine, and threonine metabolism; m. Histidine metabolism. MA

MO ck MA-5 MA-50 MA-100

15000

* ** *

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** ** *

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M

10000 ** ** **

0

* *

R-(+)-MA

8000

M

*

et

Se r Th r A sn

M

0 Le u G ln M et

2000

0

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2000

Se r Th r A sn G ly A la H is Ly s Pr o A sp G lu Va l Ph e

** **

4000

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*

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ly A la H is Ly s Pr o A sp G lu Va l Ph e

*

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G

*

6000

ck R-(+)-MA-5 R-(+)-MA-50 R-(+)-MA-100

10000

ck S-(-)-MA-5 S-(-)-MA-50 S-(-)-MA-100

*

Le u G ln M et

sn G ly A la H is Ly s Pr o A sp G lu Va l Ph e

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Le

sn G ly A la H is Ly s Pr o A sp G lu Va l Ph e

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5000

*

*

ck MO-5 MO-50 MO-100

** **

A

M

10000

15000

Le u

683 684 685 686


687 Fig. 5 Effects on HepG2 cells amino acid levels induced by 5, 50, and 100 μM rac-malathion, R(+)-malathion, S-(-)-malathion, and malaoxon for 48 h. (* P < 0.05, **P < 0.01 compared to the control group).

SOD

CAT

** * 50

** **

** 0

5

0

10 0

50

5

0

*

5

**

** **

0

concentration/μM

concentration/μM

concentration/μM

Fig. 6 Effects of 5, 50, and 100 μM rac-malathion, R-(+)-malathion, S-(-)-malathion, and malaoxon on the activity of antioxidant enzymes and intracellular concentration of reduced glutathione. (* P < 0.05, **P < 0.01 compared to the control group). NF-KB

COX-2 2.0

2.5

*

*

*

**

** * *

1.0 0.5

** *

1.5

*

** ** * *

** ** ** ** **

1.0

rac-MA MO R-(+)-MA S-(-)-MA

0.5

0 10

50

10 0

50

5

0

concentration/μM

5

0.0

0.0

0

fold change

* *

1.5

**

fold change

**

2.0

697

10

10 0

0

692

696

**

**

10 0

5

100


15

50

**

**

5

*

20

mgGSH/gprot

*

U/mgprot

U/mgprot

10

0

693 694 695

GSH

150

15

50

688 689 690 691

concentration/μM

Fig. 7 Effects on mRNA levels of inflammatory cascade gene in HepG2 cells treated with 5, 50, and



100 μM rac-malathion, R-(+)-malathion, S-(-)-malathion, and malaoxon for 48 h. (* P < 0.05, **P < 0.01 compared to the control group). Caspase 3

Caspase 9

705 706 707 708 709 710 711 712 713

1.0

0.5

fold change

0.5

fold change

1.0

1.0

** **

0.5

**

1.5

** **

** **

**


1.0 0.5

concentration/μM

10 0

50

0

10 0

50

5

0

0 10

50

concentration/μM

5

0.0

0.0 0

0 10

50

5

0.0 0

701 702 703 704

2.5

1.5

2.0

1.5

0.0

700

bcl-2

bax

1.5

fold change

fold change

2.0

5

698 699

Page 32 of 32

concentration/μM

concentration/μM

Fig. 8 Effects on mRNA levels of apoptotic genes in HepG2 cells treated with 5, 50, and 100 μM rac-malathion, R-(+)-malathion, S-(-)-malathion, and malaoxon for 48 h. (* P < 0.05, **P < 0.01 compared to the control group).

Fig. 9 Effects on protein content in HepG2 cells treated with 100 μM rac-malathion, R-(+)malathion, S-(-)-malathion, and malaoxon for 48 h. β-actin was used as the internal standard. (* P < 0.05, **P < 0.01 compared to the control group).

Table 1. Cytotoxicity of rac-malathion, R-(+)-malathion, S-(-)-malathion, and malaoxon to HepG2 cells for 48 h.

Compounds

EC50

R2

rac-Malathion R-(+)-Malathion S-(-)-Malathion Malaoxon

694.8+65.2 815.9+91.4 547.6+47.5 832.7+80.6

0.950 0.920 0.953

714 715


0.971

Different Toxic Effects of Racemate, Enantiomers, and Metabolite of

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