Epoxy Stearic Acid, an Oxidative Product Derived from Oleic Acid

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Food Safety and Toxicology

Epoxy stearic acid, an oxidative product derived from oleic acid, induces cytotoxicity, oxidative stress and apoptosis in HepG2 cells Ying Liu, Yajun Cheng, Jinwei Li, Yuanpeng Wang, and Yuanfa Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01954 • Publication Date (Web): 06 May 2018 Downloaded from http://pubs.acs.org on May 6, 2018

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Journal of Agricultural and Food Chemistry

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Epoxy stearic acid, an oxidative product derived from oleic acid, induces

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cytotoxicity, oxidative stress and apoptosis in HepG2 cells

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Ying Liu, Yajun Cheng, Jinwei Li, Yuanpeng Wang, Yuanfa Liu*

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School of Food Science and Technology, Synergetic Innovation Center of Food

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Safety and Nutrition, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu

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Province 214122, People’s Republic of China

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*Corresponding author: Yuanfa Liu

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Phone: 0510-85876799; Fax: 0510-85876799; E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: In the present study, effects of cis-9,10-epoxy stearic acid (ESA)

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generated by the thermal oxidation of oleic acid on HepG2 cells including

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cytotoxicity, apoptosis and oxidative stress were investigated. Our results revealed

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that ESA decreased the cell viability and induced cell death. Cell cycle analysis with

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propidium iodide staining showed that ESA induced cell cycle arrest at the G0/G1

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phase in HepG2 cells. Cell apoptosis analysis with annexin V and propidium iodide

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staining demonstrated that ESA induced HepG2 cell apoptotic events in a dose- and

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time-dependent manner, the apoptosis of cells after treated with 500 µM of ESA for

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12 h, 24 h and 48 h was 32.16%, 38.70% and 65.80%, respectively. Furthermore,

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ESA-treatment to HepG2 cells resulted in an increase in ROS and MDA (from 0.84

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±0.02 nmol/mg protein to 8.90±0.50 nmol/mg protein) levels and a reduction in

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antioxidant enzyme activity including SOD (from 1.34±0.27 U/mg protein to

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0.10±0.007 U/mg protein), CAT (from 100.04±5.05 U/mg protein to 20.09±3.00

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U/mg protein) and GSH-Px (from 120.44±7.62 mU/mg protein to 35.84±5.99

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mU/mg protein). These findings provide critical information on the effects of ESA

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on HepG2 cells, particularly cytotoxicity and oxidative stress, which is important for

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the evaluation of the biosafety of oxidative product of oleic acid.

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KEYWORDS: cis-9,10-epoxy stearic acid, HepG2 cell, cytotoxicity, apoptosis,

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oxidative stress

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INTRODUCTION

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Lipid oxidation is a main cause of quality deterioration in lipid- and oil-containing

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foods. Lipid oxidation involves a wide variety of reactions including degradation,

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hydrolysis, polymerization and so on, which not only gives rise to nutrient loss and

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off-flavor generation but also causes formation of potentially toxic compounds, and

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thus decreases the product quality, sometimes even makes foods unsuitable for

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consumption.1-5 Meanwhile, oxidized products including triacylglycerol (TAG)

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polymers, TAG dimers, oxidized TAG monomers, diacylglycerols and free fatty

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acids are formed. Among them, epoxy groups linked to TAG molecules, one of

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oxidized TAG monomers have the highest content and are easily absorbed by both

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animals and humans.6-8 In general, epoxy fatty acids are produced by the reaction of

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corresponding fatty acids and the hydroperoxides9 and exert some adverse effects on

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health.10-12

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Cis-9,10-epoxystearic acid (ESA) was derived from thermal oxidation of oleic

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acid and has been widely found in many food matrices.13-15 Recently, our research

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group found ESA in frying oil and established an extraction method for ESA, and the

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content of ESA in frying oil samples reached up to 5900 mg/kg.16 In addition, based

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on the electron spin resonance spectroscopy method, the formation process of ESA

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may be that oleic acid loses hydrogen radicals to form alkyl radicals which could

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react with oxygen to form hydroperoxides, then the O-O bonds of hydroperoxides

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break to form alkoxyl radicals which could abstract hydrogen from other oleic acids

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to form ESA (Figure 1). Most of the research relevant to epoxy fatty acids focused 3



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on epoxy oleic acids. Fukushima et al.10 found 9,10-epoxy-12-octadecenoate could

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increase

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9,10-epoxy-12-octadecenoate induced pulmonary edema in rats. However, the effect

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of ESA derived from OA on animals or cells remains largely unknown.

the

risk

of

cardiovascular

diseases.

Hu

et

al.17

found

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The HepG2 cell line which was selected because of its comparability to the

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normal hepatocytes in aspects of expression of specific enzymes and the enzyme

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activities,18-20 has been widely used as the human hepatoma model in the

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performance of lipid metabolic process.21-23 The present study investigated the effect

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of ESA formed by thermal oxidation of OA on HepG2 human hepatoma cells by

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considering cell viability, cytotoxicity, apoptosis, intracellular ROS level, activities

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of antioxidant enzymes and lipid peroxide level in order to explore cytotoxicity and

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oxidative stress of ESA, which is important for evaluating the biosafety of oxidative

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product of oleic acid.

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MATERIALS AND METHODS

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Chemicals. Cis-9,10-epoxystearic acid (99% purity) was purchased from Toronto

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Research Chemicals (Toronto, Canada). OA and dimethyl sulfoxide (DMSO) were

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purchased from J & K Chemical Technology (Shanghai, China). HepG-2 cells were

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purchased from the Institute of Biochemistry and Cell Biology, SIBS, CAS

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(Shanghai, China). Minimum Essential Medium (MEM) was obtained from

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Shanghai BasalMedia Technologies Co., LTD (Shanghai, China). MTT was

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purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Trypsin, fetal bovine

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serum (FBS), and other cell culture materials were purchased from Gibco BRL, Life 4


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Technologies

(USA).

The

Reactive

Oxygen

Species

(ROS)

Assay

Kit,

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Malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and

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glutathione peroxidase (GSH-Px) assay kits were all purchased from Beyotime

94

Biotechnology Co. Ltd (Shanghai, China). The Annexin V-FITC Apoptosis

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Detection Kit, Cell Cycle Analysis Kit were also obtained from Beyotime

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Biotechnology Co. Ltd (Shanghai, China). All chemicals and reagents were of

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analytical grade or higher.

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Cell culture and treatment. HepG2 human hepatocellular carcinoma cells were

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cultured in MEM medium containing 10% FBS, 100 U/mL penicillin and 75 U/mL

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streptomycin (Gibco BRL, Life Technologies, USA). Cells were incubated at 37 °C

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with 5% CO2 in humidified atmosphere. HepG2 cells were treated with OA and ESA

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at various concentrations (10, 20, 50, 100, 200 and 500 µM) for 12, 24 and 48 h.

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Cytotoxicity measurements by MTT assay. Undifferentiated HepG2 cells were

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plated into a 96-well plate (5 × 104 cells per mL) and preincubated for 24 h to

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ascertain cell attachment at 37 °C. The viability of cells was determined by the MTT

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assay. HepG2 cells were treated with fatty acids at various concentrations for

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different time. Following treatment, 10 µL MTT (5 mg/mL) reagent was added to the

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wells, and the cells were further incubated at 37 °C for 4 h. And then the medium

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was replaced with 150 µL DMSO and incubated for 15 min. The absorbance was

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measured at 490 nm using a microplate reader (Thermo, USA). The cell viability (%)

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was calculated using the following equation:

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Cell viability (%) = Atreated/Acontrol × 100% 5



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Cell cycle analysis. Cell cycle analysis was conducted using the Cell Cycle

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Analysis Kit (Beyotime, Shanghai, China), according to the manufacturer’s

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instructions. Briefly, HepG2 cells (1 × 106 cells/well) were seeded in six-well plates

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and treated with fatty acids at various concentrations for different time. Then the

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cells were harvested, washed with ice-cold PBS buffer and fixed with 70% alcohol at

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4 °C for 12 h. After that, DNA was stained with 10 µL propidium iodide (PI; 1

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mg/ml) and 10 µL RNase A (10 mg/ml) for 30 min at room temperature. Cells were

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then subjected to flow cytometry (BD FACSCalibur, San Jose, CA, USA). The

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percentage of cells in G1, S, and G2 phases of the cell cycle was calculated using

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Cell Lab Quanta SC software (Beckman Coulter Inc, Fullerton, CA).

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Cell apoptosis analysis. Cell apoptosis was detected with an Annexin V-FITC

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Apoptosis Detection Kit (Beyotime, Shanghai, China) according to the

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manufacturer’s instructions. Briefly, HepG2 cells (1 × 106 cells/well) were seeded in

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six-well plates and treated with fatty acids at various concentrations for different

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time. Cells were then collected, washed with annexin-binding buffer, and stained

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with annexin V-fluorescein isothiocyanate (FITC) and PI for 15 min at room

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temperature. Finally, cells were analyzed by flow cytometry (BD FACSCalibur, San

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Jose, CA, USA).

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Measurement of ROS. The level of ROS was determined by measuring changes

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in 20, 70-dichlorofluorescein diacetate (DCFH-DA) fluorescence. After treated with

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fatty acids, cells were incubated with DCFH-DA according to manufacturer’s

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instructions. Subsequently, the formation of the fluorescent-oxidized derivative of 6


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DCF was measured by flow cytometer (BD FACSCalibur, San Jose, CA, USA) at

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emission wavelength of 525 nm and excitation wavelength of 488 nm.

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Measurements of SOD, GSH-Px, CAT and MDA. The assay for superoxide

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dismutase (SOD),

glutathione

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malondialdehyde (MDA) was carried out using commercial assay kits. Briefly, the

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SOD activity was measured using its ability to inhibit the reduction of WST-8

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according to the manufacturer’s instructions. SOD activity was monitored

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spectrophotometrically at 450 nm using a microplate reader (Thermo, USA).

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GSH-Px was detected by measuring the decreasing amount of NADPH and

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monitored at 340 nm according to the instructions of kit. CAT was detected by

145

measuring

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(N-(4-antipyryl)-3-chloro-5-sulfonate-p-benzoquinonemonoimine)

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hydrogen peroxide and oxygen at 520 nm. The content of MDA was determined by

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measuring the absorbance of MDA-TBA reacted by MDA and TBA at 532 nm.

the

peroxidase

absorbance

(GSH-Px), catalase (CAT) and

of

red

compound reacted

by

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Statistical analysis. Analytical determinations were performed in triplicate and

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the results were expressed as mean ± standard deviation of replicated measurements.

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Statistical comparisons were performed by one-way ANOVA combined with

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Duncan’s multiple-range test using the SPSS statistical package (Version 19.0, SPSS

153

Inc., Chicago, Illinois, USA). P < 0.05 was considered significant.

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RESULTS

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Effects of OA and ESA on HepG2 cell viability. Effects of OA and ESA on the

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viability of HepG2 cells are shown in Figure 2. The MTT assay demonstrated a 7



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gradual decrease in HepG2 cell viability with the increasing concentrations of fatty

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acids treated for 12-48 h. At the lower treatment dose (10-50 µM) of OA, the

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viability of cells was kept above 70%. When HepG2 cells were exposed to OA with

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concentration of 100-500 µM, the cell viability slightly decreased and maintained at

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a certain level ranging from 57-67%. Compared to OA, ESA significantly inhibited

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cell viability, and cell viability decreased with the increasing concentration and time

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of ESA treatment. ESA treatment at 10-20 µM for 12, 24 and 48 h caused a 65-71%,

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51-56% and 50-52% loss in cell viability, respectively. While after treatment of 500

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µM ESA for 12, 24 and 48h, the number of the alive cells decreased, with cell

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viability values of 30.01%, 25.69% and 24.41%, respectively. The maximum

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inhibition (75.59%) was observed in cells treated with 500 µM ESA for 48 h. Based

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on these results, it was clear that ESA caused a dose- and time-dependent decrease in

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HepG2 cell viability.

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Effects of OA and ESA on cell cycle arrest in HepG2 cells. To investigate

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whether OA and ESA could induce cell cycle distribution in HepG2 cells, flow

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cytometric analysis of propidium iodide-stained nuclei cells was performed after

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treatment with OA and ESA at various concentrations (10-500 µM) for 12-48 h. As

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shown in Figure 3A, results revealed that HepG2 cells treated with OA presented

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with a dose- and time-dependent increase in the cell population of G0/G1 phase.

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Meanwhile, the proportion of cells in S phase decreased. A similar concentration-

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and time-related increase in the fraction of cells in the G0/G1 phase and decrease in

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the fraction of cells in the S phase was observed in ESA-treated groups (Figure 3B). 8


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As shown in Figure 3, untreated cells had 51.39%, 57.61% and 59.51% of cells in

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G0/G1 phase and 41.90%, 35.10% and 33.94% of cells in S phase after 12 h, 24 h

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and 48 h, respectively. However, 60.50%, 66.94% and 67.02% of cells in G0/G1

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phase and 29.69%, 30.62% and 30.98% of those in S phase were detected when

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HepG2 cells were exposed to 500 µM OA for 12 h, 24 h and 48 h, respectively. In

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particular, when the concentrations of ESA reached 500 µM, the fraction of cells in

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the G0/G1 phase increased dramatically (68.34%, 78.11% and 82.83%, compared to

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351.39%, 57.61% and 59.51% in untreated cells), which suggested that HepG2 cells

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underwent DNA fractionation, one of the biochemical events leading to apoptosis.

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Taken together, these results indicated that ESA could inhibit HepG2 cell

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proliferation significantly by blocking the G0/G1 to S phase transition in the cell

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cycle in a dose- and time-dependent manner.

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Effects of OA and ESA on apoptosis in HepG2 cells. In the present work,

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combined analysis of Annexin V-FITC and PI based on flow cytometry was carried

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out to determinate the apoptotic rate of HepG2 cells induced by OA and ESA. In

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Figure 4, cells in the Q2 are described as advanced apoptotic or necrotic, normal

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cells are seen in the Q3 and cells in the Q4 are classified as early apoptotic. As

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shown in Figure 4, the cell apoptosis of HepG2 increased slowly by treated with

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different concentration of OA for 12 h and 24 h. In terms of ESA, increasing

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concentrations of ESA for 12-h treatment induced apoptotic events, especially early

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apoptosis, in a dose-dependent manner. While the percentage of late apoptosis and

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dead cells increased with the increased concentration of ESA for 24-h treatment. At 9



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500 µM of ESA for 12-h and 24-h treatment, the apoptosis was 32.16% and 38.70%,

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respectively. They were more than 8 and 6 times of that in control group,

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respectively. As the concentration of ESA increased, the apoptotic rate of the HepG2

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cells elevated dramatically (P < 0.05). Specially, compared with the control group,

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HepG2 cells treated with 500 µM OA and ESA for 48 h increased the apoptosis rate

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from 15.51% to 56.10% and 65.80% apoptosis, respectively. Interestingly, after

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treating HepG2 with 200-500 µM OA for 48 h, the apoptosis rate was above 40%,

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one possible reason may be that HepG2 cells occurred cell death after long-time

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OA-treatment and nutrient deficiency. These results suggested that ESA could induce

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dramatic apoptosis in HepG2 cells.

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Effect of OA and ESA on ROS accumulation in HepG2 cells. DCF

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fluorescence was used to measure the level of ROS that was induced by OA and

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ESA exposure. As shown in Figure 5, compared with the control group, the

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intracellular ROS level in HepG2 cells after different concentrations of

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OA-treatment for 12 h and 24 h showed a gradual and slight increase in a dose- and

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time-dependent manner. A significant increase in ROS generation was observed over

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time ranging from 12-24 h in the cells treated with different concentration of ESA (P

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< 0.05). After HepG2 cells were treated with 500 µM ESA for 12 h and 24 h, cellular

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fluorescence intensity were 2.6-fold and 3.0-fold of the non-ESA-treated control

220

HepG2 cells. However, in contrast to HepG2 cells treated for 12 h and 24 h, although

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intracellular ROS level increased after OA and ESA exposure for 48 h, it maintained

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at relatively low level after 48 h exposure to all concentrations of OA and ESA. 10


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Leakage of probe was not observed in cells in pre-tests. Therefore, the final

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fluorescence caused by extracellularly oxidized DCF could be excluded. The reason

225

for this may be long-time OA- and ESA-treatment and nutrient deficiency led to a

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high percentage of cell death, the level of intracellular ROS therefore relatively low.

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Our results indicated that ESA treatment could induce abnormal accumulation of

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intracellular ROS in HepG2 cells.

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Effects of OA and ESA on the activities of antioxidant enzymes and lipid

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peroxide levels in HepG2 cells. As ESA might induce accumulation of intracellular

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ROS and lead to oxidative damage to HepG2 cells, MDA, a product of lipid

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peroxides induced by reactive oxygen species, was also measured in HepG2 cells. As

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can be seen in Figure 6A, treatment for 12-48 h with different concentrations of OA

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could only slightly increase the content of MDA in HepG2 cells, indicating a

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relatively low level of lipid peroxidation in response to OA-treatment in cells. The

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increase concentration of MDA in HepG2 cells treated with ESA was found to be

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dose- and time-dependent. Meanwhile, statistically significant increase in MDA

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levels was observed after ESA-treatments for 12-24 h in cells when compared with

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the control group. Interestingly, after cells with OA-treatment for 48 h, levels of

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MDA showed a trend to be almost similar to those of control untreated cells, even

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after 48 h with 500 µM. Although the 48-h treatment of HepG2 with ESA evoked an

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increase in the cellular concentration of MDA, the levels of intracellular MDA were

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much lower than those treated for 12 h and 24 h. This could be attributed to the lack

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of nutrients and long-time treatment, which was in line with the results of 11



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intracellular ROS levels.

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Cellular damage caused by ROS depends not only on the intracellular ROS level

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but also on the balance between ROS and endogenous antioxidant species. The

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intracellular SOD, a biomarker for antioxidative status in the cell, was detected in

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HepG2 cells treated with OA and ESA. Results demonstrated that the SOD content

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decreased with the increasing concentration and time of OA- and ESA-treatment

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(Figure 6B). Treatment for 12-24 h with different concentrations of OA only slightly

252

decreased the content of SOD in HepG2 cells. While SOD levels in ESA-treatment

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groups decreased rapidly (P < 0.05), the activity of SOD in cells that were exposed

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to ESA at 500 µM for 12 h, 24 h and 48 h were reduced by 51.37%, 69.95% and

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84.64%, respectively, when compared to the control group.

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Furthermore, decreased activity of CAT in HepG2 cells treated with OA and ESA

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was also observed (Figure 6C). The activity of CAT in cells treated with different

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concentrations of OA for 12 h and 24 h induced a slight decrease when compared to

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the control group from 100.04 U/mg pro and 92.49 U/mg pro to 82.62 U/mg and

260

81.29 U/mg, respectively. However, a significant decrease (P < 0.05) in CAT levels

261

were shown after ESA-treatments for 12 h and 24 h in HepG2 cells, at 500 µM of

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ESA for 12-h and 24-h treatment, the activity of CAT in cells were reduced by 32.72%

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and 58.34%, respectively. Specially, in contrast to HepG2 cells treated for 12 h and

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24 h, intracellular CAT activity decreased dramatically (P < 0.05) after OA and ESA

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exposure for 48 h.

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GSH-Px could catalyze the reduction of lipid hydroperoxides to hydroxides using 12


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GSH, in which oxidized glutathione is produced by GSH and then be reduced back

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to GSH with the GR catalyst. As shown in Figure 6D, a reduction in the levels of

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GSH-Px was detected in HepG2 cells after treatment with OA and ESA, as

270

compared to the control group. Specially, ESA treatment significantly reduced

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GSH-Px levels in a dose- and time-dependent manner. The presence of 500 µM ESA

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in the culture medium for 12 h and 24 h induced a significant decrease in the activity

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of GSH-Px compared with control group from 120.44 mU/mg pro and 113.32

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mU/mg pro to 61.09 mU/mg pro and 50.73 mU/mg pro, respectively. Furthermore,

275

after pretreatment of cells with different concentrations of OA and ESA for 48 h,

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levels of GSH-Px showed a trend to be far below those of cells treated with OA and

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ESA for 12 h and 24 h.

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The increase in the MDA content and decrease in the SOD, CAT and GSH-Px

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activities of cells demonstrated that exposure to ESA induced oxidative stress, and

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cells treated with ESA may lose the ability to maintain the balance between ROS and

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antioxidants.

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DISCUSSION

283

Lipid oxidation products have been claimed to exert adverse effects on health in vivo

284

and vitro studies,5,24-27 including rising risks of nonalcoholic fatty liver disease,

285

atherosclerosis and diabetes mellitus. Moreover, cytotoxicity and oxidative stress

286

have been observed in subjects after the intake of oxidative products of lipid.28-31

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However, the adverse effects of ESA produced from oxidized oleic acid remain

288

unknown. In the present study, the effects of ESA on cytotoxicity and oxidative 13



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stress of HepG2 cells were investigated.

290

Our results showed that cell viability, as determined by the MTT assay reduced

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remarkably in a dose- and time-dependent manner after HepG2 cells were exposed

292

to ESA. The relatively little effect of OA on HepG2 cell survival rate was also

293

observed. This was consistent with the studies reported by Greene et al.32 and Cao et

294

al.33 in which epoxy fatty acids and triacylglycerol polymer (both derived from lipid

295

oxidation process) were used, respectively. In addition, cell cycle analysis were

296

carried out to illustrate ESA-induced cell cycle arrest. Results showed that the

297

percentage of cells in G0/G1 phase increased and those in S phase decreased in

298

HepG2 cells exposed to ESA, indicating that the inhibition of ESA on HepG2 cells

299

mainly occurred in G0/G1 phase. The apoptotic effect of ESA on HepG2 cells was

300

confirm by Annexin V-FITC/PI assay. Results indicated ESA-treatment for 12 h and

301

24 h mainly induced early apoptosis and late apoptosis in HepG2 cells based on a

302

dose-dependent manner, respectively. This finding was in agreement with other

303

studies, where the ingestion of oxidized products, containing polar compounds,

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oxidized phospholipids, triacylglycerol polymer and auto-oxidation products from

305

cholesterol, inhibited cell proliferation and induced apoptosis compared to those

306

untreated cells.28,32-34

307

Further study found that the intracellular ROS levels in HepG2 cells were

308

markedly elevated after ESA-treatment, suggesting that ESA-induced cell damage

309

may

310

9,10-epoxy-12-octadecenoate on rats was observed by Ozawa et al.35 Ozawa and

be

related

to

excessive

ROS

production.

14


A

similar

effect

of

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311

co-workers found that 9,10-epoxy-12-octadecenoate, which is biosynthesized by

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human neutrophil, led to stress response in lung. These results strongly suggested

313

that epoxy fatty acids could induce excessive generation of the intracellular ROS

314

level. It has been reported that ROS could perform normal functions when the

315

generation and elimination of ROS in normal cell systems is in equilibrium which

316

was maintained by the endogenous antioxidant system.33,36 However, excessive

317

accumulation of ROS causes injury to cellular components including nucleic acids,

318

cellular proteins and lipids, and activates cell apoptosis signaling pathways, leading

319

to a state known as oxidative stress.37 Moreover, oxidative stress is related to the

320

etiopathogenesis of several human chronic diseases such as many cardiovascular

321

diseases, aging, neurodegenerative diseases, diabetes and cancer.38-40

322

An elevated intracellular MDA level suggested that lipid peroxidation

323

significantly increased in HepG2 cells after ESA-treatment with the increase of ESA

324

concentration and exposure time. MDA which is the principal and most studied lipid

325

oxidation product of polyunsaturated fatty acid has been widely analyzed to assess

326

the level of oxidative stress. It has been reported that an increase in MDA value

327

indicated increased lipid peroxidation, which results in tissue injury and the failure

328

of antioxidant defense mechanisms for preventing excess ROS formation.41,42

329

Therefore, our results implied damage to the antioxidant defense system in cells. A

330

reduction of SOD activity, one of major components of the antioxidant capacity to

331

defense against ROS-mediated injury in tissue,43 was found in cells treated with ESA

332

compared to control group, indicating generation of oxidative stress was 15



333

accompanied by the reduced enzymatic antioxidant activity in HepG2 cells, which

334

was in accordance with the results reported by Bhor and colleagues,44 in which

335

intestinal oxidative stress occurred with a reduction of enzymatic antioxidant activity

336

in enterocytes. Moreover, a decrease in the CAT activity was seen in the HepG2 cells

337

pretreated with ESA. Similar results has been reported in previous studies with the

338

consumption of lipid oxidation products, in which the activities of SOD and CAT in

339

the liver were both significantly lowered.29 Catalase, an enzyme to catalyse the

340

oxidation of various hydrogen donors, could decompose hydrogen peroxide to

341

molecular oxygen and water. And reduced CAT activity in tissue causes oxygen

342

intolerance and triggers some deleterious reactions, especially DNA oxidation and

343

cell death.45 The GSH-Px activity, an essential antioxidant for the intracellular

344

quenching of cell damaging peroxide species, significantly decreased in cells treated

345

with ESA as compared to the control group. It is well known that the cellular

346

antioxidant enzyme system plays an important role in the defense against oxidative

347

stress, and the activity of antioxidant enzymes could be a biomarker of the

348

antioxidant response. Therefore, our results clearly demonstrated that ESA could

349

reduce the antioxidative capacity and induce oxidative stress in HepG2 cells, which

350

may aggravate the imbalance between oxidation and antioxidant in cells.

351

In conclusion, this is the first time to investigate the effect of ESA on the cytotoxic

352

and oxidative stress of human liver carcinoma cells. The current study clearly

353

demonstrated that administration of ESA to HepG2 cells for 12-48 h could induce

354

cytotoxicity, DNA damage, apoptosis and oxidative stress. ROS may play an 16


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essential role in DNA damage and oxidative stress induced by ESA in vitro. Our

356

results showed that ESA treatment induced apoptosis activity on HepG2 cells

357

including cell proliferation, apoptosis and possible genetic damage, and

358

accumulation of ROS level. In addition, ESA also enhanced the lipid peroxides and

359

caused the decrease of enzyme activities of SOD, CAT and GSH-Px which were

360

biomarkers of cellular oxidative status. Further studies should be carried out to

361

investigate the pathological mechanism of ESA derived from thermal oxidized oleic

362

acid on apoptosis and oxidative stress of HepG2 cells.

363

AUTHOR INFORMATION

364

Corresponding Author

365

*

366

[email protected] (Y. L.).

367

ORCID

368

Yuanfa Liu: 0000-0002-8259-8426

369

Funding

370

This work was supported by the Natural Science Foundation of China (31671786),

371

the Research Fund of National 13th Five-Year Plan of China (2016YFD0401404),

372

and Northern Jiangsu province science and technology projects (BN2016137), the

373

Fundamental Research Funds for the Central Universities (JUSRP51501).

374

Notes

375

The authors declare no competing financial interest.

376

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13.

508

FIGURE CAPTIONS

509

Figure 1

510

Structure formulas of formation of epoxy stearic acid derived from oleic acid during

511

thermal oxidation.

512

Figure 2

513

Effects of OA and ESA on the viability of HepG2 cells. Different lowercase letters in

514

the same column indicate significant differences (P < 0.05) for the same sample.

515

Figure 3

516

Effects of OA (A) and ESA (B) on the cell cycle arrest of HepG2 cells.

517

Figure 4

518

Flow cytometric analysis for apoptosis induction of HepG2 cells treated with OA

519

and ESA.

520

Figure 5

521

Effect of OA and ESA on the intracellular ROS level of HepG2 cells.

522

Figure 6

523

Effect of OA and ESA on the intracellular MDA (A) level and activity of antioxidant

524

enzymes including SOD (B), CAT (C) and GSH-Px (D) of HepG2 cells.

24


Page 24 of 34

Page 25 of 34


Figure 1. Structure formulas of formation of epoxy stearic acid derived from oleic acid during thermal oxidation.

25



100

a

a b

Cell viability (%)

c a a

d e

d e e

25

ESA

10 20 50 100 200 500 12 h

d d

c c

b

c f

0

e

c

c b

c

b b

d d

b

50

a

a b

a 75

Page 26 of 34

10 20 50 100 200 500 24 h

c d d d

10 20 50 100 200 500

OA

48 h

10 20 50 100 200 500

10 20 50 100 200 500

10 20 50 100 200 500

12 h

24 h

48 h

Concentration of ESA and OA (µM)

Figure 2. Effects of OA and ESA on the viability of HepG2 cells. Different lowercase letters in the same column indicate significant differences (P < 0.05) for the same sample.

26


Page 27 of 34


S

G0/G1

G2/M

Cell cycle distribution (%)

(A) 100

75

50

25

0 0

10

20

50 100 200 500 12 h

50 100 200 500 24 h Concentration of OA (µM)

0

10

20

S

G0/G1

0

10

20

50 100 200 500 48 h

0

10

20

50 100 200 500 48 h

G2/M

Cell cycle distribution (%)

(B) 100

75

50

25

0 0

10

20

50 100 200 500 12 h

0

10

20

50 100 200 500 24 h

Concentration of ESA (µM)

Figure 3. Effects of OA (A) and ESA (B) on the cell cycle arrest of HepG2 cells.

27



Page 28 of 34

12 h

OA

ESA

24 h

OA

ESA

48 h

OA

ESA

28

Control

10

20

50

100

Concentration of OA and ESA (µM) ACS Paragon Plus Environment

200

500

Page 29 of 34


Figure 4. Flow cytometric analysis for apoptosis induction of HepG2 cells treated with OA and ESA.

29



Page 30 of 34

12 h

OA

ESA

24 h

OA

ESA

48 h

OA

(A) ESA

30

Control

10

20

50

100

Concentration of OA and ESA (µM) ACS Paragon Plus Environment

200

500

Page 31 of 34


(B) 350 300

Relative levels of ROS (%)

c

c

250

c

e

d

e

e

d

f b

e d

b

Control OA ESA

f

f

d g

200 b

e 150 a

b

b

b

c

f

c c

a aa

100

e

d

c

b

d

d

e

f

b

50 0

Con 10

20

50 100 200 500 12 h

Con 10 20

50 100 200 500 24 h

Con 10 20

50 100 200 500 48 h

Concentration of OA and ESA (µM)

Figure 5. Effect of OA and ESA on the intracellular ROS level of HepG2 cells.

31



(A) 10

Control OA ESA

g

8

MDA (nmol/mg protein)

Page 32 of 34

f e

f 6 d

e d

4

c c

b bc a ab Con 10

20

f

d de

cd

e

g

b

e

2

0

g f

d

ab

50 100 200 500 12 h

e

d

c

Con 10 20

50 100 200 500 24 h

g e

d

c

ab

d

c

b

Con 10 20

f

50 100 200 500 48 h


(B) 2.0

SOD (U/mg protein)

1.5

Control OA ESA a b b bc

a c

b

d d e

1.0

e

e

c

f b

g

c

d

d

e

c d

a b

e f

0.5

b c

g

c

d d

e

g

f e

g

f 0.0

Con 10

20

50 100 200 500 12 h

Con 10 20

50 100 200 500 24 h

Con 10 20

50 100 200 500 48 h


(C) 120 a ab

b

c

b c

CAT (U/mg protein)

90

aa

c d d

Control OA ESA

c

b

d d

e e

f

e

b

a

g

b c

60

d

e

c b

f

c

d

d 30

0

Con 10

20

50 100 200 500 12 h

Con 10 20

f

e

g

g

50 100 200 500 24 h

Con 10 20


32


e

f

g

50 100 200 500 48 h

Page 33 of 34


(D) 150

GSH-Px (mU/mg protein)

125

100

a b

Control OA ESA

a c b

b c d d

75

c

e

a

e e

f

b

f g

b

d

bd c

e c

f

c b

d

c d

e

e d

50

f

g

e f

g

25

0

Con 10

20

50 100 200 500 12 h

Con 10 20

50 100 200 500 24 h

Con 10 20

50 100 200 500 48 h


Figure 6. Effect of OA and ESA on the intracellular MDA (A) level and activity of antioxidant enzymes including SOD (B), CAT (C) and GSH-Px (D) of HepG2 cells.

33



GRAPHIC FOR TABLE OF CONTENTS

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Page 34 of 34

Epoxy Stearic Acid, an Oxidative Product Derived from Oleic Acid

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