Glutathione Reduction of Patulin-Evoked Cytotoxicity in HEK293 Cells

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Food Safety and Toxicology

Glutathione (GSH) reduces cytotoxicity evoked by patulin (PAT) in HEK293 cells by preventing oxidative damage and mitochondrial apoptotic pathway Xiaorui Wang, Chengni Jin, Yujie Zhong, Xuan Li, Jiahui Han, Wei Xue, Peng Wu, Xiaodong Xia, and Xiaoli Peng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01212 • Publication Date (Web): 20 Apr 2018 Downloaded from http://pubs.acs.org on April 20, 2018

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Journal of Agricultural and Food Chemistry

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Glutathione (GSH) reduces cytotoxicity evoked by patulin (PAT) in HEK293 cells by

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preventing oxidative damage and mitochondrial apoptotic pathway

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Xiaorui Wang a, b, 1, Chengni Jin a, b, 1, Yujie Zhong b, Xuan Li b, Jiahui Han b, Wei Xue

4

b

5

a

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Technology and Business University (BTBU), Beijing, 100048, China

7

b

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Shaanxi 712100, China

, Peng Wu b, Xiaodong Xia b, * and Xiaoli Peng a, b, * Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing

College of Food Science and Engineering, Northwest A&F University, Yangling,

9 1

These authors contributed equally to this work.

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*

Corresponding author: Xiaoli Peng

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College of Food Science and Engineering, Northwest A&F University

14

22 Xinong Road, Yangling, Shaanxi, China 712100

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E-mail address: [email protected]

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Fax: +86-29-87092817

17

*

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College of Food Science and Engineering, Northwest A&F University

19

22 Xinong Road, Yangling, Shaanxi, China 712100

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E-mail address: [email protected]

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Fax: +86-29-87091391

10 11

Additional corresponding author: Xiaodong Xia

22

ACS Paragon Plus Environment


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Abstract

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Patulin (PAT) is a mycotoxin which frequently detected in moldy fruits and fruit

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products. This study investigated the protective role of glutathione (GSH), a

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antioxidant agent, against PAT induced cytotoxicity and its potential mechanisms in

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HEK293 cells. The obtained results showed that addition of GSH significantly

28

reduced cell viability and apoptosis induced by PAT. Additionally, GSH decreased

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intracellular ROS and mitochondrial ROS overproduction, suppressed the decline of

30

mitochondrial membrane potential, and maintained the cellular ATP content. GSH

31

prevented the impairment of mitochondrial oxidative phosphorylation system,

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especially enhanced the amount of mRNA and protein expression of electron transport

33

chain complex III (UQCRC2), complex V (ATP5, ATP6 and ATP8). Furthermore,

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GSH increased endogenous GSH content, enhanced the antioxidant enzyme activities

35

of SOD, CAT, GR, GPx and modulated oxidative damage. These results suggest that

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GSH reduces PAT-induced cytotoxicity via inhibition of oxidative damage and

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mitochondrial apoptotic pathway in HEK293 cells.

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Keywords: Glutathione (GSH), Patulin (PAT), Oxidative stress, Apoptosis, HEK293

39

cell

40 41

Abbreviations Used

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ATP, adenosine triphosphate; CAT, catalase; DMEM, Dulbecco's minimal essential

43

medium; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; GPx, glutathione

44

peroxidase; GR, glutathione reductase; GSH, glutathione; GSSG, oxidized glutathione;


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LDH, lactate dehydrogenase; MMP, mitochondrial membrane potential; MRC,

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mitochondrial respiratory chain; mtROS, mitochondrial reactive oxygen species; MTT,

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PAT, patulin; PI,

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propidium iodide; PMSF, phenylmethylsulfonyl fluoride; ROS, reactive oxygen

49

species; SOD, superoxide dismutase.



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Introduction

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Mycotoxins are toxic secondary metabolites produced by molds in their

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contaminated food. Patulin (PAT) is a mycotoxin produced by genera Penicillium,

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Aspergillus and Bryssochamys.1, 2 This toxin primarily contaminates different foods

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including grains, moldy fruits and their by-products, especially in apples, hawthorn,

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pears, grapes, and strawberries.1, 3 It has reported that PAT has toxic effects including

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immunotoxicity, neurotoxicity, dermal toxicity and genotoxicity in certain

57

experimental animals.4, 5 Concerning mechanism of toxicity induced by PAT, previous

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research has found that PAT has obvious electrophilic reactivity, leading to the

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formation of adducts when it interacts with cysteine-containing tripeptide glutathione

60

(GSH), and then further causing quick depletion of GSH.6 In addition，several

61

research indicated that PAT induced cytotoxicity through reactive oxygen species

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(ROS)-mediated oxidative damage pathway, mitochondrial dysfunction and

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mitochondrial apoptotic signal pathway.7-10

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The dynamic balance between oxidative damage pathway and antioxidant defense

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system under normal physiological conditions is associated with ROS.11, 12 ROS is

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several oxidative substance, whose chemical property is active and oxidative capacity

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is strong, mainly including superoxide anion (O2·-), hydrogen peroxide (H2O2),

68

hydroxyl radical (HO ·).13 Intracellular ROS is primarily produced by certain oxidases

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such as NADPH oxidase, and generated as the by-products of mitochondrial

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respiratory chain (MRC) with the regulation of cytochrome-P450 enzymes,

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endoplamic reticulum and peroxisome.14 ROS widely exists in the body and regulates


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body's physiological and pathological process. Overproduction of ROS results in

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oxidative damage, thus causing the dysfunction of many physiological processes and

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cell death.15 Oxidative stress normally refers to oxidative and antioxidation imbalance

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in vivo, featured with overwhelming ROS. Oxidative damage may induce biological

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macromolecules oxidation and damage such as proteins, lipids, nucleic acids, causing

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DNA mutation or abnormal replication.16 Moreover, mitochondria are the original

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resource of the generation of ROS. Excessive ROS generation also contributes to

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mitochondrial dysfunction, mitochondrial-dependent apoptotic pathway, and then

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causes cell death.

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The use of natural compounds with antioxidant effects might provide a strategy to

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reduce PAT toxicity. Indeed, numerous studies showed that antioxidant substances,

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such as green tea polyphenols,17 Vitamin E and apigenin,7, 18 protect cells against

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deleterious the effects of PAT. GSH is a main intracellular antioxidant with the

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capacity of antioxidant activity and scavenging free radical. GSH, a tripeptide

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containing sulfhydryl, is a combination of glutamic acid, cysteine and glycine.19 It has

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been reported that its antioxidant effects mainly depended on catalyzing by

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glutathione S-transferases (GST) and glutathione peroxidases (GPx).20, 21 GSH has

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two forms, including reduced GSH and oxidized GSH, and exhibits specific

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conversion rate in cells, organs and individuals. Once oxidative stress occurs, cell

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exhibits a decreased GSH to GSSG ratio.22, 23 Therefore, GSH deficiency induced

92

intracellular redox imbalance and then caused all kinds of diseases. And the biological

93

control of cellular GSH is crucial in apoptosis and oxidative stress. Intracellular GSH



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homeostasis depends on de novo GSH synthesis, GSH redox cycling, and

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extracellular GSH across the plasma membranes.22 It has been reported that

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supplementation of a certain amount of GSH by dietary GSH or direct supplemental

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GSH effectively maintains the balance of circulating GSH and treats several

98

diseases.23-25 Recent research have found that orally ingested GSH was immediately

99

converted to GSSG, transported as a protein-bound form in the blood, followed by

100

accumulated in red cells, and carried into liver tissues.23, 26 The GSSG form can be

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used for detoxification in the body. Several studies have been carried out to explore

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the protective effects of GSH in different biological models of atherosclerosis,27

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rheumatoid arthritis,28 ageing diabetic29 and hyperoxaluria.30 GSH treatment could

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attenuate tubular cell apoptosis in kidney tissues of rats with myoglobinuric acute

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kidney injury.31 Furthermore, GSH monoester was found to be efficient in protection

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against buthionine sulfoximin induced skeletal muscle degeneration, rat erythrocyte

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antioxidant defense system.32, 33 However, no information is available concerning the

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protective effect of GSH against PAT-induced kidney cell damage and apoptosis.

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In current study, we aimed to (1) evaluate the nephrotoxic effects of PAT, (2)

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investigate the protective effect of GSH administration against patulin-induced

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cytotoxicity using HEK293 cell lines. The kidney is one of the target organs for the

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toxic effects of PAT.34 In addition, our present research have provides an in vitro cell

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culture model for studying the mechanism of patulin.10,

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theoretical support for the inhibition of GSH depletion protect cell against PAT toxin.

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Materials and methods


18

This study provides a

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Materials and Reagents

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HEK293 cell lines were obtained from Zhongqiao Xinzhou Biotechnology Co., Ltd

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(Shanghai, China). L-Glutathione reduced (GSH, purity ≥ 98%) and PAT standard

119

products (purity ≥ 99%) were purchased from Sigma-Aldrich Chemical (St. Louis,

120

MO, USA). Dulbecco's minimal essential medium (DMEM) and fetal bovine serum

121

(FBS) were purchased from Thermo Fisher Scientific (USA). 1% Penicillin-

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Streptomycin solution, trypsin-EDTA solution, LDH assay kit, Hoechst 33342

123

fluorescent dyes, Annexin V-FITC apoptosis detection kit, ROS assay kit, GSH and

124

GSSG assay kit, total superoxide dismutase (SOD) assay kit, glutathion reductase

125

(GR) assay kit, total glutathione peroxidase (GPx) assay kit, mitochondrial membrane

126

potential (MMP) assay kit, ATP assay kit, BCA protein assay kit, caspase-3, -8 and -9

127

activity assay kits, cell lysis buffer for Western and IP and phenylmethylsulfonyl

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fluoride (PMSF) were obtained from Beyotime Institute of Biotechnology (Jiangsu,

129

China). Catalase (CAT) test kit was obtained from Jiancheng Bioengineering Institute

130

(Nanjing, China). MitoSOX red mitochondrial superoxide indicator was obtained

131

from Invitrogen Corporation (USA). Ultrapure RNA kit, Super RT cDNA kit and

132

UltraSYBR mixture were obtained from CWBIO (Beijing, China). Polyvinylidene

133

fluoride (PVDF) membranes with 0.45 µM mean pore size were obtained from

134

Milllipore Company (Bedford, MA, Germany). Acrylamide, Bis-Acrylamide, Tris,

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Glycine, Sodium Dodecyl Sulfate (SDS) and 10% ammonium persulfate (AP)

136

solution

137

Tetramethylethylenediamine (TEMED, purity ≥ 99%) and 3-(4,5-dimethylthiazol-2-

were

obtained

from

Biotopped

(Bejing,


China).

N,N,N',N'-


138

yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich

139

Chemical (St. Louis, MO, USA). ECL western blotting substrate was obtained from

140

Solarbio (Bejing, China).

141

Cell culture and treatment

142

HEK293 cells were cultured in DMEM medium supplemented with 10% FBS and

143

1% penicillin/streptomycin in a humidified incubator containing 5% CO2 and 95% air

144

at 37 ℃. For the agent treatment, PAT was dissolved in aseptic water to make a stock

145

solution of 10 mM and further diluted to a concentration of 7.5 µM with serum-free

146

medium. To evaluate the protective effect of GSH against PAT toxicity, GSH was

147

dissolved in aseptic water to make a stock solution of 50 mM and diluted in serum

148

free medium to the concentration needed, and then added to the cell plates for 3 h.

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After that, 7.5 µM PAT was added to the same cell plates for another 10 h and then

150

cell plates were used for detecting.

151

MTT and LDH assays

152

The effect of GSH on cell viability was evaluated by MTT and LDH assay.35

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Briefly, cells were seeded in a 96-well plate (BEAVER, USA) at a density of 1 × 105

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cells/well for 12 h, and then pretreated with different concentrations of GSH for 3 h

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before exposure to PAT for another 10 h. Then, 10 µl of MTT (5 mg/ml) dye was

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added to each well, the upper solution was removed after 4 hours of incubation. At the

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time point, 150 µl DMSO was added to each well to dissolve the formation of

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formazan crystals. Absorbance was measured at 570 nm by a microplate reader

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(Bio-Rad 680, USA). All experiments were performed in five times.


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LDH leakage was measured by LDH assay kit. Cells were treated as above method

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and then detected according to the kit instructions. Absorbance was measured at

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double-wavelengths of 490 nm and 630 nm with a microplate reader (Bio-Rad 680,

163

USA). All experiments were performed in three times.

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Hoechst 33342 staining assay

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Nuclear morphology was examined with Hoechst 33342 staining. After treatment

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as above method, cells were washed with PBS (pH 7.4) twice and stained with

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Hoechst 33342 (10 µg/ml) for 20 min at 37 ℃. The nuclear morphology was

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observed by fluorescence microscope immediately (Olympus, Japan).

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Measurement of apoptosis rate

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The apoptosis rate of cells was determined by Annexin V-FITC apoptosis detection

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kit. Briefly, collected cells were re-suspended with 195 µl Annexin V-FITC binding

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buffer, following by staining with 5 µl Anneixin V-FITC and 10 µl propidium iodide

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(PI). After that, cells were incubated at 25 ℃ for 20 min in the dark, and then the

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cells were immediately measured by flow cytometer (Becton Dickinson).

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Intracellular ROS and mitochondria ROS release assay

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Intracellular amount of ROS was measured by ROS assay kit. In brief, cells were

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treated with above-mentioned treatment and then incubated with a fluorometric assay

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with 2,7-dichlorofluorescein diacetate (DCFH-DA, 10 µM) at 37 ℃ for 20 min. The

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fluorescence intensity was immediately detected by multifunctional microplate reader

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in the excitation wavelength of 488 nm and emission wavelength of 525 nm (TECAN,

181

Switzerland).



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Mitochondria ROS was assayed by MitoSOX Red Mitochondrial Superoxide

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Indicator. Cells were treated as above method, and then reacted with working Probe

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fluid for 10 min at 37 ℃. After incubation, the cells were immediately observed and

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photographed using Laser confocal microscope (NIKON, A1, Japan).

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GSH and GSSG measurements

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GSH and GSSG were detected by GSH and GSSG assay kit. Following treatment,

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cells were washed with PBS, centrifuged, and collected to calculate cells precipitation

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weight. Then, protein removal regent was added into cells, and cells were frozen

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using liquid nitrogen and thawed at 37 ℃ twice. Cells were placed for 5 min in the

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ice and then centrifuged at 10,000 × g for 10 min at 4 ℃. The supernatant was used

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for GSH and GSSG assays according to the manufacturer's protocol. The content of

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total GSH was detected by DTNB-GSSG recycling assays. Before detected the

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content of GSSG, the supernatant was added scavenging auxiliary liquid to remove

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GSH, and then added corresponding GSH scavenging working solution provided with

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the kit. The amount of GSH was calculated by subtracting the amount of GSSG from

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that of total GSH. Absorbance was measured with a multifunctional microplate reader

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(Tecan) at 412 nm. GSH and GSSG content were expressed as the form of µmol/mg

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cells.

200

Determination of SOD, CAT, GR and GPx activities

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HEK293 cells were treated with above-mentioned treatment and lysed in cell lysis

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buffer. Then the cells were centrifuged and the supernatants were collected to measure.

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Concentration of protein was detected by BCA protein assay kit.


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SOD activity. SOD activity was measured by total superoxide dismutase assay kit

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with WST-8. The cell supernatants were incubated with mixture solutions including

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WST-8 and start working liquid at 37 ℃ for 30 min. Absorbance was measured with

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a multifunctional microplate reader (Tecan) at 450 nm. SOD activity was expressed as

208

the form of U/mg protein.

209 210

CAT activity. CAT activity was detected by catalase test kit. The method was based on color reaction and CAT activity was expressed as the form of U/mg protein.36

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GR activity. Cellular GR levels were detected by glutathion reductase assay kit.

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The detection principle of GR is GSSG change to GSH by the function of GR, while

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GSH can react with DTNB to produce yellow TNB and GSSG. GR activity can be

214

detected by detecting the amount of yellow TNB production. Absorbance was

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measured with a multifunctional microplate reader (Tecan) at 412 nm. GR activity

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was expressed as the form of U/mg protein.36

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GPx activity. GPx levels were detected by total glutathione peroxidase assay kit.

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GSH can be changed to GSSG under the function of GPx, while GR can catalyze

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GSH production by NADPH reacting with GSH. Thus, the amount of NADPH

220

reduction can reflect the activity of GPx. Absorbance was measured with a

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multifunctional microplate reader (Tecan) at 340 nm. GPx activity was expressed as

222

the form of U/mg protein.36

223

Measurement of Mitochondrial membrane potential

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The MMP was measured by mitochondrial membrane potential assay kit with JC-1

225

fluorescence dye. The cell samples were incubated with JC-1 dye at 37 ℃ for 20 min



226

and washed twice with JC-1 dyeing buffer (1 ×). The value of MMP was examined by

227

multifunctional microplate reader (TECAN, Switzerland) and observed by

228

fluorescence microscope (TECAN, Infinite M200 Pro, Switzerland). The values were

229

expressed as the red/green fluorescence ratio.

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ATP level assay

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The value of intracellular ATP was detected by ATP assay kit. Briefly, the new plate

232

was added 100 µl working solution/well provided with the kit and then placed 3 to 5

233

minutes at room temperature. After that, 20 µl cell samples were added in each well.

234

The result of ATP was measured by multifunctional microplate reader (Tecan). Protein

235

concentration was detected by BCA protein assay kit. ATP content was expressed as

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the form of nmol/mg protein.

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Caspases activity assay

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Caspases activity including caspase-3, -8 and -9 activities was measured by the

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corresponding caspase activity assay kits. Briefly, cells were treated as above method,

240

lysed in cell lysis buffer and centrifuged. Then, the supermatants were collected and

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incubated with 5 µl Ac-DEVD-pNA, Ac-IETD-pNA and Ac-LEHD-pNA respectively

242

as caspase-3, -8 and -9 reagents. After incubation for 2 h at room temperature in the

243

dark, luminescence was measured with a microplate reader (Bio-Rad 680, USA) at

244

405 nm. All samples were assayed in triplicate.

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Real-time PCR analysis

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Total RNA was isolated from HEK293 cells using Ultrapure RNA Kit based on

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TRlzon cracking reaction according to the manufacturer’s instructions and RNA was


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reverse transcribed into cDNA according to the Super RT cDNA kit. For real-time

249

PCR, cDNA was amplified by the “2-step” UltraSYBR Mixture and the results were

250

detected by an IQ5 Multicolor Real-Time PCR Detection System (Bio-Rad). The

251

primers used in this research were synthesized by Invitrogen Corporation (Shanghai,

252

China) and the sequences were listed in table 1.

253

Western blotting

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To detect the protein expression levels of the MRC complexes, cells were lysed at 4

255

˚C in a buffer containing 1 mM PMSF solution. The supernatants were collected and

256

protein concentration was measured using the BCA protein assay kit and stored at

257

−80 ℃. For western blotting analyses, the protein samples were separated by 12%

258

SDS-PAGE, transferred to PVDF membrane and blocked with 5% non-fat milk in

259

TBST. Membranes were probed with the corresponding primary antibody, including

260

NDUFA4 (Abcam, ab133698), SDHA (CST, 11998), UQCRQ2 (Proteintech,

261

14742-1-AP), COX17 (Abcam, ab69611), ATP6V1B2 (CST, 14488), ATP8B2

262

(Abcam, ab104336) and β-actin (Proteintech, 20536-1-AP), followed by incubation

263

with horseradish peroxide-conjugated anti-rabbit IgG (CWBIO, China). Blots were

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developed with ECL system using a luminescent image analyzer (Bio-Rad, USA).

265

Statistical analysis

266

All values were presented as means ± standard deviation (S.D.). The data were

267

statistically analyzed by the means of one-way analysis of variance (ANOVA) using

268

SPSS 19.0 software. P < 0.05 was considered as statistical significant, and highly

269

significant at p < 0.01.

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Results



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Effect of GSH on PAT-induced cytotoxicity

272

The results of MTT and LDH were used to reveal the cytotoxicity. When the cells

273

were pretreated with 0.1, 0.5 and 1 mM GSH, respectively, MTT analysis indicated

274

cell viability was significantly increased by 9.61%, 30.37% and 46.23% respectively

275

when compared with 7.5 µM PAT group (Figure 1A). GSH alone did not affect cell

276

viability in the concentration range tested (0-1 mM), which means GSH has no

277

obvious cytotoxicity. Then we chose 1 mM GSH as an optimum dose to carry out the

278

following experiments. The results in Figure 1B showed PAT evoked a great increase

279

(4.34-fold of control group) in LDH activity while GSH appreciably moderated LDH

280

leakage induced by PAT.

281

Next, cell morphology was observed using a fluorescence microscope after stained

282

with Hoechst 33342. As illustrated in Figure 1C, there were minimal nuclear

283

morphological changes in the control group and GSH alone treatment group. However,

284

cells were shrinking, rounding and brightening after treatment with PAT, while GSH

285

exhibited an obvious meliorative effect, suggesting that GSH may attenuate

286

PAT-induced cytotoxicity.

287

Effect of GSH on PAT-induced apoptosis

288

Previous studies have been reported that apoptosis is one of the major mechanism

289

of PAT-evoked cytotoxicity. To further detect the effect of GSH on PAT-induced

290

apoptosis, we measured the apoptosis rate using flow cytometry. The results showed

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that the apoptosis rate was increased dramatically by 29% when cells were exposed to

292

PAT. However, GSH combined with PAT treatment decreased the value to 15.7%


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(Figure 2). All these observations indicated that GSH could protect HEK293 cells

294

against PAT-induced apoptosis.

295

GSH inhibits PAT-induced ROS overproduction and mitochondrial dysfunction

296

Mitochondria are called “cell-powered factory”. Mitochondria are the primary sites

297

for intracellular oxidative phosphorylation, the generation of intracellular ROS and

298

the synthesis of ATP. In addition, mitochondria are also involved in processes such as

299

cell apoptosis, cell growth and cell cycle.37 As indicated in Figure 3A, intracellular

300

ROS obviously increased by 3.48-fold of control when cells were treated with PAT

301

while GSH treatment reduced markedly the number of ROS generation. Then, we

302

examined the change in mtROS and the results revealed that the red fluorescence

303

brightened rapidly after the PAT treatment. However, GSH decreased observably the

304

red fluorescence compared with the PAT treatment group (Figure 3B).

305

Previous studies demonstrated that the ability of PAT to induce accumulation of

306

ROS andO2·-. Indeed, superoxide anion has been reported to be mainly produced by

307

NADPH oxidases of the plasma membrane.38 To further explore the mechanism of

308

GSH inhibiting PAT-induced ROS overexpression, the mRNA expression of NADPH

309

oxidases was detected. The real-time PCR analysis showed that PAT up-regulated

310

ROS-generating NOX family numbers including subunit NOX2 and P47phox.

311

However, GSH exerted no obvious effects on reducing mRNA expressions of NOX2

312

and P47phox induced by PAT (Figure 3C and D). The data suggested that the

313

attenuation effect on ROS production of GSH might not through NADPH oxidases at

314

least at mRNA level.



315

Mitochondrial membrane potential and ATP levels are important indicators to

316

reflect mitochondrial function. Mitochondrial membrane damage directly influence

317

the oxidative phosphorylation to produce ATP, and lacking of energy supply lead to

318

cell injury and apoptosis. Our data showed that PAT appreciably induced the raise in

319

green fluorescence, and the decrease in red fluorescence and the level of MMP was

320

significantly decreased by 31% .However, the value of MMP was enhanced from 69%

321

to 91.5% after treatment with GSH (Figure 4A and B). Next, we measured the ATP

322

level and the results found that PAT caused the decline of ATP production while GSH

323

alleviated PAT-induced decrease of ATP level (Figure 4C). Taken together, these

324

results demonstrated that GSH can inhibit total ROS content as well as mtROS

325

production and mitochondrial dysfunction induced by PAT.

326

GSH remits GSH depletion evoked by PAT and improves antioxidant enzyme

327

effects

328

It's known that ROS overproduction results in oxidative damage, causing the

329

dysfunction of many physiological processes and cell death.15 We have reported that

330

PAT caused oxidative damage in vitro. To further examine the protective effect of

331

GSH on PAT-induced oxidative damage, the contents of GSH and GSSG were

332

measured and the results showed that PAT reduced GSH level and increased GSSG

333

content significantly. However, when cells were pretreated with GSH before treatment

334

with PAT, the content of GSH was increased and the content of GSSG was decreased

335

markedly (Figure 5A and B). Moreover, our data demonstrated that the ratio of GSH

336

to GSSG was remarkably increased from 68% in the PAT group to 408% in the GSH


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combined treatment group (Figure 5C). Next, the activities of SOD, CAT, GR and

338

GPx were measured. Results suggested that SOD, CAT, GR and GPx activities were

339

remarkably decreased by 30.53%, 28.85%, 55.55% and 32.35% respectively after

340

treatment with PAT. GSH pretreatment remarkably enabled four enzyme activities to

341

increase (Figure 5D-G). These results indicated that GSH can remit GSH depletion

342

evoked by PAT and then improved the activities of antioxidant enzyme.

343

GSH protects against PAT-induced mitochondrial respiratory chain complexes

344

disorders

345

Considering that the MRC complexes, which are located on the mitochondrial inner

346

membrane, are related to the oxidative phosphorylation process, the mRNA

347

expression and protein expression of the MRC complexes were measured by real-time

348

PCR and western blotting, respectively. As shown in Figure 6, mRNA expression of

349

Complex II (SDHA) and complex IV (COX17) were a significant increase by 38.03%

350

and 227.16% after treatment with PAT. In addition, GSH totally inhibited SDHA and

351

COX17 gene expression induced by PAT. By contrast, PAT reduced the mRNA

352

expression of complex III (UQCRC2) and complex V (ATP5, ATP6 and ATP8) by

353

37.59%, 31.46%, 51.54% and 56.47%, respectively, while the gene expression of two

354

complexes were markedly increased in the GSH combined group. Meanwhile,

355

western blotting results showed that the protein expression of Complex II and

356

complex IV were decreased from 130.76% and 134.83% in the PAT group to 82.14%

357

and 93.60% in the GSH combined treatment group. However, addition of GSH greatly

358

increased the protein level of complex III (UQCRC2) and complex V (ATP5, ATP6



359

and ATP8) (Figure 7). The results of ATP expression were in consisted with the ATP

360

content. There are no obvious changes in the mRNA and protein expression of

361

complex I (NDUFA4) between the PAT group and GSH-combined treatment group

362

(Figure 6A and 7B). These results suggested that GSH could attenuate mitochondrial

363

function disorders induced by PAT through regulating the gene and protein expression

364

of the MRC complexes.

365

GSH mitigated PAT-induced increase in the enzymatic activities and mRNA

366

expression of caspases

367

Caspases signal activates in the early stage of apoptosis and plays a key role in cell

368

apoptosis signal pathway. To investigate whether GSH exerts its protective effect via

369

caspase cascade-dependent apoptosis pathway, the enzymatic activities and mRNA

370

expression of initiator caspase-9, caspase-8 and executor caspase-3 were detected

371

after cells were treated as above method. As shown in Figure 8A, the activities of

372

caspase-3, caspase-8 and caspase-9 were increased from 118864.6, 626376.43 and

373

265775.93 U/mg protein in the control group to 1287074.88, 1547864.79 and

374

1440480.71 U/mg protein in the treatment of PAT, respectively. However, addition of

375

GSH caused a decrease by 708256.85, 679007.22, and 1126481.39 U/mg protein. The

376

mRNA expression of caspase-3, caspase-8 and caspase-9 by real-time PCR analysis

377

were also increased by 27.46%, 101.39% and 43.40%, respectively in the PAT group

378

when compared with control group (Figure 8B). GSH combined treatment reduced

379

this increasing trends. Our data showed that PAT caused a significant increase in the

380

caspases activities and mRNA expression while GSH reduced notably the enzymatic


Page 18 of 43

Page 19 of 43


381

activities and mRNA expression of caspases. The results of mRNA expression of

382

caspases were in keeping with the changes of caspases activities. Altogether, GSH

383

mitigated PAT-induced apoptosis via regulating caspases activities.

384

Discussion

385

PAT contaminates a variety of fruits and their by-product, and has potent toxic

386

effects on kidney, liver, intestinal, and immune systems.8,

387

Organization (WHO) and US Food and Drug Administration (FDA) regulated the

388

recommended limit level of PAT to 50 ppb in apple juice.8, 41, 42 Previous studies have

389

shown that PAT decreased quickly of GSH, increased intracellular ROS

390

overproduction, then resulted in oxidative stress and mitochondrial injury, and further

391

caused cell apoptosis and cell death,6,

392

renoprotective effect of GSH against this toxin. HEK293 cells were selected as the

393

cell model in this current study.

10, 43

39, 40

World Health

which provides a direction for

394

GSH can effectively remove excess oxidative products and free radicals in cells to

395

maintain normal cellular physiology. However, oxidation reduces GSH generation,

396

which is produced by cell synthesis. Reduced GSH is the biological active form that is

397

oxidized to GSSG.44 The decrease of GSH is an early apoptotic signal, and ROS

398

promotes the occurrence and development of apoptosis.38, 45 In our research, we found

399

that PAT induced intracellular ROS overproduction, reduced GSH level and increased

400

GSSG content significantly. However, when cells pretreated with GSH before

401

treatment with PAT, the ratio of GSH/GSSG was increased markedly. NADPH

402

oxidases are composed by membrane-bound subunits and can catalyze the reduction



403

of molecular oxygen. NOX2 and P47phox are the catalytic subunits and regulatory

404

subunits of NADPH oxidase, respectively. NADPH oxidase (NOX)-derived ROS at

405

the plasma membrane function in cellular signaling.37,

406

showed that PAT up-regulated NADPH enzyme catalytic subunit NOX2 and P47phox

407

mRNA expression. However, GSH exerted no obvious effects on reducing mRNA

408

expressions of NOX2 and P47phox induced by PAT. The results confirmed the theory

409

described above and suggested the inhibition of GSH depletion could attenuate

410

PAT-induced apoptosis and oxidative damage.

46

Real-time PCR analysis

411

Simultaneous treatment by GSH and PAT caused a significant reduction of cell

412

mortality as compared to cells treated with PAT alone. The observed protective effect

413

of GSH against PAT cytotoxicity can be explained by its efficient inhibition of

414

mitochondrial apoptotic pathway induced by this mycotoxin. Mitochondria are energy

415

factory, and mitochondrial oxidative phosphorylation is one of the main ways both the

416

generation of ROS and the synthesis of ATP.44,

417

synthesize GSH. GSH synthesis mainly depended on catalyzing by γ-glutamate-

418

cysteine ligase and glutathione synthase in the cytosolic compartments or obtained

419

from extracellular GSH. Extracellular GSH could cross plasma membrane via specific

420

plasma membrane transporters.22 Additionally, GSH hydrolyzate can be recycled for

421

GSH synthesis in the lumen of kidney.48 After synthesis, GSH may act on various

422

organs in cells. Among these organs, mitochondria play a key role. In our study, PAT

423

increased obviously mitochondria ROS while GSH administration reduced markedly

424

the number of ROS generation. We have demonstrated that PAT lead to the content of

47

However, mitochondria do not


Page 20 of 43

Page 21 of 43


425

ATP and the value of MMP decreasing, and disordered the mRNA levels and protein

426

expression of the MRC complexes, in particular, up-regulating mRNA and protein

427

expression of complex II (SDHA) and complex IV (COX17) and down-regulating the

428

mRNA and protein expression of complex III (UQCRC2) and complex V (ATP5,

429

ATP6 and ATP8). These results suggested that ROS-mediated mitochondrial oxidative

430

phosphorylation and mitochondrial dysfunction are the key points of this toxin. GSH

431

combined treatment inhibited ROS overproduction, regulated mitochondrial oxidative

432

phosphorylation via modulating gene expression of the MRC complexes and

433

protected mitochondria function through recovering ATP level and improving MMP.

434

As cellular caspases are the main players in the execution phase of apoptosis

435

signaling pathways, and further demonstrate whether GSH prevents PAT-induced

436

HEK293 cells apoptosis via the cascade cascade pathway, we detected the enzyme

437

activities and mRNA expression of initiator caspase-8, initiator caspase-9 and

438

execution caspase-3. When the initiator caspases are activated, they produce a chain

439

reaction, and then activate executioner caspase-3,49 which indicated that cells

440

apoptosis have occurred. In this research, PAT increased the enzyme activity of

441

caspases while the trends can be reduced by GSH. At the same time, caspases mRNA

442

expressions, including caspase-3, caspase-8 and caspase-9, were increased when cells

443

were treated with PAT, confirming the involvement of caspase-dependent pathway in

444

apoptosis. However, GSH decreased the mRNA expression of caspases. Our data

445

indicated that GSH could protect HEK 293 cells from apoptosis through restraining

446

caspases activity.



447

To confirm the cellular and protective effect of GSH, acting as an antioxidant, on

448

oxidative damage induced by PAT, we measured antioxidants enzyme activities

449

including SOD, CAT, GR and GPx. Enzymes are biocatalysts that are produced by

450

living cells in the body, and antioxidant enzymes can slow the rate of oxidation.10, 44

451

When the contents of antioxidant enzyme are decreasing, oxidation damage cannot be

452

effectively inhibited since the body suffers external stimuli or produces excess free

453

radical. SOD primarily eliminate superoxide anion through disproportionation

454

reaction, and further turn it into H2O2 and O2.13 H2O2 and SOD catalytic by-products

455

can be effectively removed by CAT and turned into water. GR can maintain sufficient

456

cellular levels of reduced GSH. However, GPx can eliminate peroxides in cells, and

457

play a key role in protecting cells from the damage of free radicals.17 Our data

458

suggested that SOD, CAT, GR and GPx activities were remarkably decreased after

459

treatment with PAT. However, GSH pretreatment significantly enabled four enzyme

460

activities to increase. These results indicated that GSH can remit GSH depletion and

461

then attenuate oxidative damage evoked by PAT.

462

In conclusion, our research provides a new direction for the effects of GSH on

463

PAT-induced renal cytotoxicity. We found that GSH attenuated HEK293 cells viability,

464

LDH leakage and apoptosis evoked by PAT. Meanwhile, GSH remitted GSH

465

depletion and then inhibited oxidative damage induced by PAT. In addition, GSH

466

reduced ROS overproduction, regulated mitochondrial respiratory chain complexes,

467

and further attenuated mitochondrial dysfunction. These results suggest that GSH can

468

protect HEK293 cells from cytotoxicity induced by PAT via ROS-mediated oxidative


Page 22 of 43

Page 23 of 43


469

damage and mitochondrial-dependent apoptotic pathway, which will provide a

470

stronger evidence for a protective role of GSH against mycotoxin-caused toxicity in

471

mammalian cells.

472

Conflict of interest The authors declare that there is no conflict of interest.

473 474

Acknowledgment

475

This work was supported by grants from the Open Project Foundation of Beijing

476

Advanced Innovation Center for Food Nutrition and Human Health (20181028) and

477

the National Natural Science Foundation of China (NSFC, 31571928).

478

479

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480

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Figure captions

629

Figure 1. Effect of GSH on PAT-induced cytotoxicity in HEK293 cells. HEK293 cells

630

were pretreated with GSH for 3h before treated with 7.5 µM PAT for 10 h. Cell

631

viability was detected by MTT assay (A), LDH leakage was detected by LDH assay

632

(B). Cells were stained with Hoechst 33342 and photographed using a fluorescence

633

microscope (C). The results are expressed as mean ± S.D. from at least three

634

independent repeats. * p < 0.05, ** p < 0.01 versus control treatment; # p < 0.05, ## p

Glutathione Reduction of Patulin-Evoked Cytotoxicity in HEK293 Cells

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