Ochratoxin A-Induced Apoptosis of IPEC-J2 Cells through ROS

Nov 14, 2017 - Miura , T.; Chiba , M.; Kasai , K.; Nozaka , H.; Nakamura , T.; Shoji , T.; Kanda , T.; Ohtake , Y.; Sato , T. Apple procyanidins induc...
0 downloads 6 Views 1MB Size
Subscriber access provided by READING UNIV

Article

Ochratoxin A induced apoptosis of IPEC-J2 cells through ROSmediated mitochondrial permeability transition pore opening pathway Hong Wang, Ying Chen, Nianhui Zhai, Xingxiang Chen, Fang Gan, Hu Li, and Kehe Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04434 • Publication Date (Web): 14 Nov 2017 Downloaded from http://pubs.acs.org on November 15, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Journal of Agricultural and Food Chemistry

Ochratoxin A induced apoptosis of IPEC-J2 cells through ROS-mediated mitochondrial permeability transition pore opening pathway Hong Wang

a, b

, Ying Chen a, b, Nianhui Zhai a, b, Xingxiang Chen

a, b

, Fang Gan a, b, Hu Li a, b,

Kehe Huang a, b * a

College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu

Province, China b

Institute of Nutritional and Metabolic Disorders in Domestic Animals and Fowls, Nanjing

Agricultural University, Nanjing 210095, Jiangsu Province, China

*Corresponding author: Prof. Kehe Huang Tel: +86-025-84395507; Fax: +86-25-84398669 E-mail address: [email protected]

1 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

1

ABSTRACT

2

With the purpose to explore the mechanisms associated with the intestinal toxicity of

3

Ochratoxin A (OTA), an intestinal porcine epithelial cell line (IPEC-J2) was applied in this

4

study as in vitro models for intestinal epithelium. The results confirmed that OTA induced

5

IPEC-J2 cell toxicity by MTT assay and apoptosis by Hoechst 33258 staining and flow

6

cytometer analysis. And then, we observed that OTA induced the mitochondrial reactive

7

oxygen species (ROS) production and mitochondrial permeability transition pore (mPTP)

8

opening by confocal microscopy. Western blot showed that OTA induced cytochrome c (cyt-c)

9

release and caspase-3 activation, which could be suppressed by inhibition of mPTP opening

10

with cyclosporin A. Treatment with Mito-TEMPO, the mitochondria-targeted ROS scavenger,

11

blocked OTA-induced mitochondrial ROS generation and mPTP opening and prevented cyt-c

12

release, caspase-3 activation and apoptosis in IPEC-J2 cells.

13 14 15 16 17 18 19 20

KEYWORDS: Ochratoxin A; IPEC-J2 cells; mitochondrial reactive oxygen species;

21

mitochondrial permeability transition pore opening; apoptosis.

2 ACS Paragon Plus Environment

Page 2 of 30

Page 3 of 30

Journal of Agricultural and Food Chemistry

22

INTRODUCTION

23

Ochratoxin A (OTA), a natural occurring mycotoxin produced by fungi of Aspergillus and

24

Penicillium genera, can contaminate feed and food and raw materials, such as forages,

25

cereals, fruits and pork1. Because of the wide distribution of Aspergillus and Penicillium,

26

animals or humans are at high risk of exposure to OTA through ingestion of contaminated

27

feed and foodstuff. OTA has been shown to have a diversity of toxic effects, such as

28

nephrotoxicity, immunotoxicity, hepatotoxicity, teratogenicity and carcinogenicity2, 3. OTA

29

contamination of feed and foodstuff are thus considered to affect animal and human health

30

and bring economic losses.

31

As known, the intestinal epithelium represents the first barrier to the access of microbial

32

pathogens, food contaminants and natural toxins to the whole body4. Due to its location and

33

function, the intestinal epithelium is delicate and vulnerable, and this constantly renewing

34

organ may be a potential target organ for food-associated mycotoxins5. OTA has been found

35

to damage the intestinal epithelium in vivo studies in chicken, rat6-8. The cytotoxicity of OTA

36

has also been reported in vitro studies with some transformed human intestinal epithelial cell

37

line, including Caco-2 and HT-29-D4 cells9, 10. However, the research on OTA toxicity in the

38

intestinal epithelium is still relatively lack and the mechanisms responsible for the intestinal

39

toxicity is still poorly understood.

40

It has been found that OTA could inhibit mitochondrial respiration chain and promotes

41

reactive oxygen species (ROS) generation11, 12. ROS-mediated oxidative stress is generally

42

considered be a potential common mediator of apoptosis13,

3 ACS Paragon Plus Environment

14

. The role of ROS in

Journal of Agricultural and Food Chemistry

43

OTA-mediated cytotoxicity has been reported in various cell types. Bhat et al. found OTA

44

elevated ROS generation and then induced apoptosis in Neuro-2a cells15. Palma et al. showed

45

that ochratoxin A could promotes the production of oxidative stress, which causes

46

mutagenesis in mammalian cells16. Thus we suppose that the intestinal toxicity of OTA may

47

be associated with its generation of ROS.

48

It is known that excessive production of ROS could directly induce mitochondrial

49

permeability transition pore (MPTP) opening17. Specially, the opening of mPTP leads to the

50

increase in mitochondrial permeability and the release of cytochrome c (cyt-c) from the

51

mitochondria to the cytosol18. In general, cyt-c is stably located in the mitochondrial

52

intermembrane/intercristae spaces, which works as an electron donor for cyt-c oxidase.

53

However, during early stage of apoptosis, it also releases from from the mitochondria to the

54

cytosol, where it helps to activate the caspase-319. Caspase-3 is one of the most commonly

55

shared downstream executioner in extrinsic and intrinsic apoptotic pathways20, 21. Normally,

56

it is an inactive, exclusively cytosolic homodimer. While, during apoptosis, it is activated

57

through cleavage into active fragment (cleaved caspase-3)22.

58

The IPEC-J2 cell line, a non-transformed intestinal cell line originally derived from jejunal

59

epithelia isolated from a neonatal, unsuckled piglet23, 24, was applied as an in vitro model of

60

jejunal epithelial cells in this study. The aim of this study was to determine the toxicity of

61

OTA on IPEC-J2 cells and provide mechanistic explanations for its toxic effects on the

62

jejunal epithelial cells.

63

MATERIALS AND METHODS 4 ACS Paragon Plus Environment

Page 4 of 30

Page 5 of 30

Journal of Agricultural and Food Chemistry

64

Chemicals

65

EGTA, Mito-TEMPO, Ochratoxin A (OTA), trypsin, cyclosporin A (CsA), CoCl2 were

66

obtained from Sigma-Aldrich, USA. Annexin V-FITC apoptosis detection kit was purchased

67

from Becton Dickinson Company, USA. Enhanced chemiluminescence (ECL) kit, Dimethyl

68

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kit, Sulphoxide

69

(DMSO), Bicinchoninic acid (BCA) protein assay kit and Hoechst 33258 were obtained from

70

Beyotime Institute of Biotechnology (Haimen, China). Mito-tracker Red, MitoSOX Red and

71

calcein AM were purchased from molecular probes (Eugene, OR, USA). USA. DMEM/F-12

72

medium and fetal bovine serum were purchased from Thermo Fisher Scientific (MA, USA).

73

Cell culture and OTA treatment

74

The IPEC-J2 cells (kindly provided by Professor. Zhanyong Wei, Henan Agricultural

75

University, Zhengzhou, China) were cultured in an incubator at 37 °C with 5% CO2 using

76

DMEM/F12 medium, which contained HEPES (25 mM), Streptomycin (100 µg/ml),

77

Penicillin (100 U/ml) and 10% fetal bovine serum.

78

Differentiated IPEC-J2 cells are less susceptible to mycotoxins compared to proliferating

79

cells25 26. In this study, we used the proliferating IPEC-J2 cells to investigate the cytotoxicity

80

of OTA. More specifically, IPEC-J2 cells were seeded on 6- or 96-well plates in DMEM/F12

81

supplemented with 10% FBS and then exposed to different doses of OTA when cells

82

proliferated to 70 - 80% confluence. Purified OTA purchased from Sigma-Aldrich was

83

diluted in DMSO to make a stock solution and further diluted in the culture media at the

84

indicated concentrations. IPEC-J2 cells were seeded on 96-wells plates and then exposed to

5 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

85

OTA to increasing concentrations of OTA (0-32 µM) for 6, 12 and 24 h when the confluence

86

approached 70-80%. According the MTT results, IPEC-J2 cells were exposed to 2, 4 and 8

87

µM OTA for 12h in the subsequent experiments.

88

Cell viability assay

89

IPEC-J2 cells were plated in 96-well plates and the exposed to different doses of OTA when

90

the confluence approached 70-80%. After treatments, cell viability was determined by MTT

91

assay. The cells were incubated with MTT solution for 4 h, and then solubilized in DMSO.

92

Absorbance was read by a spectrophotometer at 570 nm.

93

Analysis of apoptosis by Hoechst 33258 staining and flow cytometry

94

Hoechst 33258 was applied to detect the apoptotic morphological changes of IPEC-J2 cells.

95

Cells were stained with Hoechst 33258 solution for 10 min and then washed. After staining,

96

apoptotic morphological changes were detected under a fluorescence microscope (Zeiss).

97

The quantitative analysis of apoptosis was determined by flow cytometry analysis. After

98

being washed with cold-PBS, IPEC-J2 cells were incubated with FITC-annexin V and PI

99

following the manufacturer’s instructions. The percent of apoptosis were analyzed using

100

FACSCalibur flow cytometry (BD Biosciences).

101

Measurement of ROS production

102

H2DCF-DA probe was applied to detect the intracellular ROS levels. Cells were incubated

103

with H2DCF-DA (10 µM) for 30min at 37 °C in dark. H2DCFDA could be oxidized into

104

green fluorescent DCF by ROS generation27. To confirm the primary site of ROS production,

105

cells were also staining with 50 nM Mito-tracker Red. The mitochondrial ROS levels were

6 ACS Paragon Plus Environment

Page 6 of 30

Page 7 of 30

Journal of Agricultural and Food Chemistry

106

detected using MitoSOX red (5 µM). Cells were staining with MitoSOX red at 37°C for 20

107

min. The fluorescence signals of DCF, Mito-tracker Red and MitoSOX Red were detected by

108

confocal microscope (Zeiss).

109

Assessment of mPTP opening

110

The mPTP opening was detected by the calcein-AM/cobalt assay28. After treatment, cells

111

were incubated with calcein-AM (1 µM) for at 37 °C 30 min, and then exposed to CoCl2 (1

112

mM) for another 10 min. The calcein loaded into mitochondria was preserved and the

113

cytosolic calcein was quenched by CoCl2. The fluorescence signal was visualized confocal

114

microscope (Zeiss).

115

Western blotting analysis

116

After treatment, cells were harvested and then lysed in cold-RIPA buffer (Beyotime, China)

117

to obtain the total cell lysates. Meanwhile, the cytosolic protein fraction for detecting cyt-c

118

was separated by a cytoplasmic protein extraction kit following the manufacturer’s

119

instruction (Beyotime, China). 40 µg of protein samples were prepared for electrophoresis on

120

SDS-PAGE, and then transferred to PVDF membranes (Millipore, Molsheim, France). The

121

membranes were incubated with primary antibodies: rabbit anti-cyt-c (#11940, 1:1000, CST),

122

rabbit anti-cleaved caspase-3(#9664,1:1000, CST) and rabbit anti-β-Actin (#4970, 1:1000,

123

CST) overnight at 4 °C and then, the membranes were incubated with HRP-labeled Goat

124

Anti-Rabbit IgG (A0208, Beyotime, China) for 1 h at room temperature. Finally,

125

immunoreactivity was detected by ECL chemoluminescence kit. The blots were visualized

126

by a Luminescent Image Analyzer (FUJIFILM LAS-4000). The density of each band was

7 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

127

quantified using Quantity One software (Biorad, France) and normalized to its respective

128

loading control (β-actin). The final data were expressed as the ratio of the intensity of the

129

protein in treated cells to that of the corresponding protein in control cells. Each test was

130

performed in four experiments with different batches of cells.

131

Data presentation

132

Experiments were performed at least three times with similar results. Data are presented as

133

the mean ± SEM of the indicated number of replicates. Statistical comparisons were made

134

using one-way analysis of variance (ANOVA) (Scheffe’s F test) after ascertaining the

135

homogeneity of variance between the treatments, and P < 0.05 was regarded as significant.

136

RESULTS

137

Cytotoxic effects of OTA on IPEC-J2 cells viability

138

To investigate OTA-induced cytotoxicity, IPEC-J2 cells were exposed to increasing

139

concentrations of OTA (0-32 µM) for 6, 12 and 12 h. MTT assay was applied to measure the

140

viability of cells. As shown in Figure 1, higher concentrations of OTA and longer exposure

141

times exhibited more serious cytotoxicity in IPEC-J2 cells. It was observed that OTA

142

significantly effected the viability of IPEC-J2 cells at 16 and 32 µM for 6 h (Figure 1a). For

143

the data regarding 12 h treatment, OTA induced a significant decrease in viability of IPEC-J2

144

cells at concentrations between 2 and 32 µM (Figure 1b). The IC50 value (evaluated after 12h)

145

of OTA against IPEC-J2 cells was 19.3 µM. 24 h treatment with OTA resulted more apparent

146

reduction in viability of cells (Figure 1c). However, we observed that most cells were

147

detached after 24 h treatment with 4 µM OTA and above, which made it unfavorable for the

8 ACS Paragon Plus Environment

Page 8 of 30

Page 9 of 30

Journal of Agricultural and Food Chemistry

148

following testes. In the subsequent experiments, IPEC-J2 cells were exposed to 2, 4 and 8

149

µM OTA for 12 h.

150

OTA induced apoptosis of IPEC-J2 cells

151

Hoechst 33258 staining and flow cytometry were used to detect the apoptosis in IPEC-J2

152

cells. As shown in Figure 2a, typical morphological nuclear changes of apoptosis were

153

observed by fluorescence microscopy of Hoechst 33258 staining. In addition, the flow

154

cytometry analysis exhibited that OTA induced apoptosis of IPEC-J2 cells in a

155

concentration-dependent manner (Figure 2b and c).

156

OTA promoted mitochondrial ROS production and MPTP opening

157

Intracellular ROS probe, H2DCF-DA, was applied to asses IPEC-J2 cells ROS production.

158

Gradual increase of DCF fluorescence in IPEC-J2 cells with increasing concentrations of

159

OTA was observed after the treatment, suggesting excessive ROS was accumulated.

160

Compared with the control, a concentration-dependent generation of DCF fluorescence

161

elevated at 12 h (Figure 3a and b). Overlay images of cells labeled with H2DCF-DA and

162

Mito Tracker Red suggested that mitochondria may be the major site of intracellular ROS

163

generation in IPEC-J2 cells exposed to OTA (Figure 3c). Therefore, next we utilized a

164

specific indicator of mitochondrial ROS, MitoSOX Red, to detect mitochondrial ROS. As

165

shown in Fig 3d and e. OTA exposure results in a significant elevation in mitochondrial ROS

166

generation. The above results confirmed the contribution of mitochondria to OTA-mediated

167

ROS generation in IPEC-J2 cells.

168

Then we measured mPTP opening by the CoCl2-calcein fluorescence-quenching assay. On

9 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

169

the opening of the mPTP, entrapped calcein is released from the mitochondrial matrix and

170

CoCl2 quenched the cytosolic calcein but not that of mitochondria, which do not transport

171

cobalt. As shown, a concentration-dependent reduction in mitochondrial calcein fluorescence

172

was detected in OTA-treated IPEC-J2 cells (Figure 3f and g), indicated the opening of mPTP

173

opening.

174

OTA caused cytochrome c release and activated caspase-3 in IPEC-J2 cells

175

Since OTA-induced apoptosis was accompanied by mitochondrial ROS generation and

176

mPTP opening, we examined whether OTA induced apoptosis via a mitochondria-mediated

177

pathway. As known, the release of cyt-c from mitochondria to cytosol and subsequent

178

caspase-3 activation represent a critical step in the mitochondria-mediated apoptosis pathway.

179

As shown, OTA treatment noticeable elevated the protein levels of cytosolic cyt-c and

180

activated caspase-3 as evident by its cleavage (Figure 4a and b).

181

Involvement of mPTP opening in OTA-induced apoptosis

182

CsA, a potent inhibitor of mPTP, was applied to assess the role of MPTP opening on

183

OTA-induced apoptosis. As shown in Figure 5, OTA-induced mPTP opening was markedly

184

blocked by CsA. When mPTP opening was inhibited, OTA-induced cyt-c release was

185

significantly decreased, indicating that OTA-induced cyt-c release was dependent on mPTP

186

opening. Importantly, inhibition of mPTP opening with CsA also suppressed OTA-induced

187

caspase-3 activation and apoptosis. Given the above results, it can be concluded that OTA

188

induced apoptosis of IPEC-J2 cells in via the mitochondrial pathway

10 ACS Paragon Plus Environment

Page 10 of 30

Page 11 of 30

Journal of Agricultural and Food Chemistry

189

OTA-induced mitochondrial ROS production and mPTP opening were reversed by

190

Mito-TEMPO

191

To examine the role of ROS on OTA-induced mPTP opening, cells were pre-incubated with

192

the

193

mitochondrial ROS production was markedly suppressed by Mito-TEMPO (Figure 6a, b).

194

Besides, Mito-TEMPO significantly suppressed OTA-induced reduction in fluorescence of

195

calcein, confirming the involvement of ROS on OTA-induced mPTP opening (Figure 6c, d).

196

Role of mitochondrial ROS in OTA-induced mitochondria-dependent apoptosis

197

To ascertain the correlation between OTA-induced ROS production and apoptosis,

198

mitochondria-targeted antioxidant Mito-TEMPO was applied. As shown, Mito-TEMPO

199

significantly suppressed the OTA-induced cyt-c release, caspase-3 activation and

200

subsequently reversed OTA-induced apoptosis in IPEC-J2 cells (Figure 7). Overall, these

201

data demonstrated that the elevation in mitochondrial ROS production acts as an important

202

role in OTA-induced apoptosis.

203

DISCUSSION

204

Generally, exposure to Ochratoxin A (OTA) occurs through ingestion of contaminated

205

foodstuff29. The small intestine has been found to be the major site of OTA absorption, with

206

maximal absorption at the proximal jejunum30. As known, the intestinal epithelium represents

207

the first barrier that protect the host against the penetration of diverse food contaminants.

208

Due to its location and function, the intestinal epithelium may be a potential target for the

209

toxic effect of OTA after intake of OTA contaminated food and feed. It is thus of importance

mitochondria-targeted antioxidant Mito-TEMPO.

As expected,

11 ACS Paragon Plus Environment

OTA induced

Journal of Agricultural and Food Chemistry

210

to study the toxic effects of OTA on the intestinal epithelium.

211

It has been reported that OTA could inhibit cell growth and induced cell death in various cell

212

types31-33. A porcine epithelial intestinal cell line (IPEC-J2) was used as in vitro models for

213

the jejunal epithelial cells in this study. We observed that OTA could induce cytotoxicity in

214

IPEC-J2 cells (Figure1). This result confirmed intestinal toxicity of OTA in vitro. As known,

215

cell death can generally be produced via apoptosis and necrosis34. In this study, we focused

216

on the induction of apoptosis by OTA on IPEC-J2 cells. As shown in Figure2, OTA elevated

217

the percentages of apoptotic cells in a dose-dependent manner. These results suggested that

218

OTA-induced apoptosis may be play an important role in OTA intestinal toxicity.

219

A number of mechanisms has been put forward to account for OTA toxicity, including

220

inhibition of protein synthesis, inhibition of mitochondrial respiration, generation of reactive

221

oxygen species (ROS)35. Data in our study exhibited that OTA exposure could induce the

222

production of ROS, as indicated by the progressive increase in H2DCF-DA fluorescence

223

(Figure 3a and b). The merged images of H2DCF-DA and Mito Tracker Red suggested that

224

mitochondria may be the major site of intracellular ROS generation induced by OTA (Figure

225

3c). In mammalian cells, mitochondria are often assumed as the main source of ROS36, 37.

226

Consistent with this idea, mitochondrial ROS generation induced by OTA was confirmed by

227

MitoSOX Red, which showed a significant increase in fluorescence (Figure 3d and e).

228

Interestingly, mPTP opening was observed in IPEC-J2 cells during OTA exposure, as

229

evidenced by a reduction in mitochondrial calcein fluorescence (Figure 3f and g).

230

OTA-induced apoptosis of IPEC-J2 cells was accompanied by increased ROS generation and

12 ACS Paragon Plus Environment

Page 12 of 30

Page 13 of 30

Journal of Agricultural and Food Chemistry

231

mPTP opening, which enables us to think whether OTA induced apoptosis via

232

mitochondria-mediated pathway.

233

It is well known that mitochondria play an important role in the regulation of cellular death38,

234

39

235

opening. And through integration of diverse intracellular signals, mPTP serves as a gate that,

236

once switched-on, trigger the apoptotic process40, 41. Specially, the opening of mPTP leads to

237

the increase in mitochondrial permeability, resulting in the release of apoptotic factors such

238

as cyt-c from the mitochondria to the cytosol42, 43. The present research found that OTA

239

promoted the release of cyt-c into the cytosol in IPEC-J2 cells (Figure 4a). Once cyt-c is

240

released from mitochondria to cytosol, it leads to activation of the caspase cascades, and

241

finally leads to apoptosis44. Caspase 3 is one of the most common apoptosis executioners45.

242

In this study, our data showed that caspase-3 was activated during OTA exposure (Figure 4b).

243

Moreover, the release of cyt-c, caspase-3 activation and apoptosis induced by OTA were

244

markedly inhibited by CsA, an inhibitor of mPTP (Figure 5). The above results suggested

245

that OTA could induced apoptosis of IPEC-J2 cells via the mitochondrial pathway.

246

To

247

mitochondria-targeted antioxidant, was applied to block ROS production. As expected,

248

OTA-induced ROS production was markedly suppressed by Mito-TEMPO (Figure 6a and b).

249

Importantly, OTA-mediated MPT opening was significantly blocked by Mito-TEMPO

250

(Figure 6c and d), which provided evidence that mitochondria generated ROS are involved in

251

mMPT induction. We next wanted to examine whether mitochondrial ROS generation is an

. Excessive production of ROS could induce mitochondrial permeability transition pore

determine

the

role

of

ROS

on

OTA-induced

apoptosis,

13 ACS Paragon Plus Environment

Mito-TEMPO,

a

Journal of Agricultural and Food Chemistry

Page 14 of 30

252

upstream event of OTA-induced apoptosis of IPEC-J2 cells. Previous studies have shown

253

that elevated levels of mitochondrial ROS is sufficient to trigger apoptosis38, 46. In this study,

254

the release of cyt-c, caspase-3 activation and subsequent apoptosis induced by OTA were

255

markedly inhibited by Mito-TEMPO, respectively (Figure 7). These results confirmed the

256

involvement of mitochondrial ROS in OTA-induced apoptosis of IPEC-J2 cells. This result

257

suggests us that application of antioxidants may be a practical and effective way to protect

258

the intestinal injuries induced by OTA, which will be worthy of further investigation.

259

In summary, OTA induces apoptosis of IPEC-J2 cells cells via ROS-dependent mitochondrial

260

apoptosis pathways by inducing mPTP opening, thereby promoting cytochrome c release and

261

caspase-3 activation.

262

ABBREVIATIONSA

263

OTA, Ochratoxin A; ROS, reactive oxygen species; mPTP, mitochondrial permeability

264

transition pore; cyt-c, cytochrome c; DMSO, Dimethyl Sulphoxide; (DMSO); BCA,

265

Bicinchoninic

acid;

266

acetoxymethyl

ester;

267

Medium/Nutrient Mixture F-12; FBS, fetal bovine serum.

268

AUTHOR INFORMATION

269

Corresponding Author

270

*Tel: +86-25-84395507. Fax: +86-25-84398669. E-mail: [email protected].

271

ORCID

272

Kehe Huang: 0000-0003-4132-3052

ECL,

enhanced

(Calcein-AM),

chemiluminescence; DMEM/F-12,

Calcein-AM,

Dulbecco's

14 ACS Paragon Plus Environment

Modified

Calcein Eagle

Page 15 of 30

Journal of Agricultural and Food Chemistry

273

Funding

274

This work was funded by the National Key R & D Program (2016YFD0501203), the

275

National Natural Science Foundation of China (31472253) and the Priority Academic

276

Program Development of Jiangsu Higher Education Institutions (Jiangsu, China).

277

Notes

278

The authors declare no competing financial interest.

279

REFERENCES:

280

1. Armorini, S.; Al-Qudah, K. M.; Altafini, A.; Zaghini, A.; Roncada, P., Biliary

281

ochratoxin A as a biomarker of ochratoxin exposure in laying hens: An experimental study

282

after administration of contaminated diets. Research in Veterinary Science 2015, 100,

283

265-270.

284

2. Bui-Klimke, T. R.; Wu, F., Ochratoxin A and Human Health Risk: A Review of the

285

Evidence. Critical Reviews in Food Science and Nutrition 2015, 55 (13), 1860-1869.

286

3. Pleadin, J.; Stayer, M. M.; Vahcic, N.; Kovatcevic, D.; Milone, S.; Saftic, L.; Scortichini,

287

G., Survey of aflatoxin B-1 and ochratoxin A occurrence in traditional meat products coming

288

from Croatian households and markets. Food Control 2015, 52, 71-77.

289 290

4. Braicu, C.; Selicean, S.; Cojocneanu-Petric, R.; Lajos, R.; Balacescu, O.; Taranu, I.;

291

Marin, D. E.; Motiu, M.; Jurj, A.; Achimas-Cadariu, P.; Berindan-Neagoe, I., Evaluation of

292

cellular and molecular impact of zearalenone and Escherichia coli co-exposure on IPEC-1

293

cells using microarray technology. Bmc Genomics 2016, 17.

15 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 16 of 30

294

5. Alassane-Kpembi, I.; Puel, O.; Oswald, I. P., Toxicological interactions between the

295

mycotoxins deoxynivalenol, nivalenol and their acetylated derivatives in intestinal epithelial

296

cells. Archives of Toxicology 2015, 89 (8), 1337-1346.

297

6. Dortant, P. M.; Peters-Volleberg, G. W. M.; Van Loveren, H.; Marquardt, R. R.; Speijers,

298

G. J. A., Age-related differences in the toxicity of ochratoxin A in female rats. Food and

299

Chemical Toxicology 2001, 39 (1), 55-65.

300

7. Solcan, C.; Pavel, G.; Floristean, V. C.; Chiriac, I. S. B.; Slencu, B. G.; Solcan, G.,

301

EFFECT

302

MUCOSA-ASSOCIATED LYMPHOID TISSUES IN BROILER CHICKENS. Acta

303

Veterinaria Hungarica 2015, 63 (1), 30-48.

304

8. Stoev, S. D.; Stefanov, M.; Denev, S.; Radic, B.; Domijan, A. M.; Peraica, M.,

305

Experimental mycotoxicosis in chickens induced by ochratoxin A and penicillic acid and

306

intervention with natural plant extracts. Veterinary Research Communications 2004, 28 (8),

307

727-746.

308

9. Cano-Sancho, G.; Gonzalez-Arias, C. A.; Ramos, A. J.; Sanchis, V.; Fernandez-Cruz, M.

309

L., Cytotoxicity of the mycotoxins deoxynivalenol and ochratoxin A on Caco-2 cell line in

310

presence of resveratrol. Toxicology in Vitro 2015, 29 (7), 1639-1646.

311

10. Maresca, M.; Mahfoud, R.; Pfohl-Leszkowicz, A.; Fantini, J., The mycotoxin ochratoxin

312

A alters intestinal barrier and absorption functions but has no effect on chloride secretion.

313

Toxicology and Applied Pharmacology 2001, 176 (1), 54-63.

314

11. Aleo, M. D.; Wyatt, R. D.; Schnellmann, R. G., MITOCHONDRIAL DYSFUNCTION

OF

OCHRATOXIN

A

ON

THE

INTESTINAL

16 ACS Paragon Plus Environment

MUCOSA

AND

Page 17 of 30

Journal of Agricultural and Food Chemistry

315

IS AN EARLY EVENT IN OCHRATOXIN-A BUT NOT OOSPOREIN TOXICITY TO

316

RAT RENAL PROXIMAL TUBULES. Toxicology and Applied Pharmacology 1991, 107

317

(1), 73-80.

318

12. Ciarcia, R.; Damiano, S.; Squillacioti, C.; Mirabella, N.; Pagnini, U.; Florio, A.;

319

Severino, L.; Capasso, G.; Borrelli, A.; Mancini, A.; Boffo, S.; Romano, G.; Giordano, A.;

320

Florio, S., Recombinant Mitochondrial Manganese Containing Superoxide Dismutase

321

Protects Against Ochratoxin A-Induced Nephrotoxicity. Journal of Cellular Biochemistry

322

2016, 117 (6), 1352-1358.

323

13. Ma, Y. X.; Cao, L.; Kawabata, T.; Yoshino, T.; Yang, B. B.; Okada, S., Cupric

324

nitrilotriacetate induces oxidative DNA damage and apoptosis in human leukemia HL-60

325

cells. Free Radical Biology and Medicine 1998, 25 (4-5), 568-575.

326

14. Magi, B.; Ettorre, A.; Liberatori, S.; Bini, L.; Andreassi, M.; Frosali, S.; Neri, P.; Pallini,

327

V.; Di Stefano, A., Selectivity of protein carbonylation in the apoptotic response to oxidative

328

stress associated with photodynamic therapy: a cell biochemical and proteomic investigation.

329

Cell Death and Differentiation 2004, 11 (8), 842-852.

330

15. Bhat, P. V.; Pandareesh, M. D.; Khanum, F.; Tamatam, A., Cytotoxic Effects of

331

Ochratoxin A in Neuro-2a Cells: Role of Oxidative Stress Evidenced by N-acetylcysteine.

332

Frontiers in Microbiology 2016, 7.

333

16. Palma, N.; Cinelli, S.; Sapora, O.; Wilson, S. H.; Dogliotti, E., Ochratoxin A-induced

334

mutagenesis in mammalian cells is consistent with the production of oxidative stress.

335

Chemical Research in Toxicology 2007, 20 (7), 1031-1037.

17 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

336

17. Liu, G.; Wang, Z.-K.; Wang, Z.-Y.; Yang, D.-B.; Liu, Z.-P.; Wang, L., Mitochondrial

337

permeability transition and its regulatory components are implicated in apoptosis of primary

338

cultures of rat proximal tubular cells exposed to lead. Archives of Toxicology 2016, 90 (5),

339

1193-1209.

340

18. Sinha, K.; Das, J.; Pal, P. B.; Sil, P. C., Oxidative stress: the mitochondria-dependent

341

and mitochondria-independent pathways of apoptosis. Archives of Toxicology 2013, 87 (7),

342

1157-1180.

343

19. Martinou, J. C.; Desagher, S.; Antonsson, B., Cytochrome c release from mitochondria:

344

all or nothing. Nature Cell Biology 2000, 2 (3), E41-E43.

345

20. Weng, H.-Y.; Hsu, M.-J.; Wang, C.-C.; Chen, B.-C.; Hong, C.-Y.; Chen, M.-C.; Chiu,

346

W.-T.; Lin, C.-H., Zerumbone suppresses IKK alpha, Akt, and FOXO1 activation, resulting

347

in apoptosis of GBM 8401 cells. Journal of Biomedical Science 2012, 19.

348

21. Ryu, H. Y.; Emberley, J. K.; Schlezinger, J. J.; Allan, L. L.; Na, S. Q.; Sherr, D. H.,

349

Environmental chemical-induced bone marrow B cell apoptosis: Death receptor-independent

350

activation of a caspase-3 to caspase-8 pathway. Molecular Pharmacology 2005, 68 (4),

351

1087-1096.

352

22. Garcia de la Cadena, S.; Hernandez-Fonseca, K.; Camacho-Arroyo, I.; Massieu, L.,

353

Glucose deprivation induces reticulum stress by the PERK pathway and caspase-7-and

354

calpain-mediated caspase-12 activation. Apoptosis 2014, 19 (3), 414-427.

355

23. Cevallos Porta, D.; Lopez, S.; Arias, C. F.; Isa, P., Polarized rotavirus entry and release

356

from differentiated small intestinal cells. Virology 2016, 499, 65-71.

18 ACS Paragon Plus Environment

Page 18 of 30

Page 19 of 30

Journal of Agricultural and Food Chemistry

357

24. Geens, M. M.; Niewold, T. A., Optimizing culture conditions of a porcine epithelial cell

358

line IPEC-J2 through a histological and physiological characterization. Cytotechnology 2011,

359

63 (4), 415-423.

360

25. Broekaert, N.; Devreese, M.; Demeyere, K.; Berthiller, F.; Michlmayr, H.; Varga, E.;

361

Adam, G.; Meyer, E.; Croubels, S., Comparative in vitro cytotoxicity of modified

362

deoxynivalenol on porcine intestinal epithelial cells. Food and Chemical Toxicology 2016,

363

95, 103-109.

364

26. Vandenbroucke, V.; Croubels, S.; Martel, A.; Verbrugghe, E.; Goossens, J.; Van Deun,

365

K.; Boyen, F.; Thompson, A.; Shearer, N.; De Backer, P.; Haesebrouck, F.; Pasmans, F., The

366

Mycotoxin Deoxynivalenol Potentiates Intestinal Inflammation by Salmonella Typhimurium

367

in Porcine Ileal Loops. Plos One 2011, 6 (8).

368

27. Bittner, S.; Ruck, T.; Schuhmann, M. K.; Herrmann, A. M.; Maati, H. M. O.; Bobak, N.;

369

Goebel, K.; Langhauser, F.; Stegner, D.; Ehling, P.; Borsotto, M.; Pape, H.-C.; Nieswandt, B.;

370

Kleinschnitz, C.; Heurteaux, C.; Galla, H.-J.; Budde, T.; Wiendl, H.; Meuth, S. G.,

371

Endothelial TWIK-related potassium channel-1 (TREK 1) regulates immune-cell trafficking

372

into the CNS. Nature Medicine 2013, 19 (9), 1161-1165.

373

28. Giaime, E.; Yamaguchi, H.; Gautier, C. A.; Kitada, T.; Shen, J., Loss of DJ-1 Does Not

374

Affect Mitochondrial Respiration but Increases ROS Production and Mitochondrial

375

Permeability Transition Pore Opening. Plos One 2012, 7 (7).

376

29. Peraica, M.; Flajs, D.; Domijan, A.-M.; Ivic, D.; Cvjetkovic, B., Ochratoxin A

377

Contamination of Food from Croatia. Toxins 2010, 2 (8), 2098-2105.

19 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

378

30. Sergent, T.; Garsou, S.; Schaut, A.; De Saeger, S.; Pussemier, L.; Van Peteghem, C.;

379

Larondelle, Y.; Schneider, Y. J., Differential modulation of ochratoxin A absorption across

380

Caco-2 cells by dietary polyphenols, used at realistic intestinal concentrations. Toxicology

381

Letters 2005, 159 (1), 60-70.

382

31. Boesch-Saadatmandi, C.; Loboda, A.; Jozkowicz, A.; Huebbe, P.; Blank, R.; Wolffram,

383

S.; Dulak, J.; Rimbach, G., Effect of ochratoxin A on redox-regulated transcription factors,

384

antioxidant enzymes and glutathione-S-transferase in cultured kidney tubulus cells. Food and

385

Chemical Toxicology 2008, 46 (8), 2665-2671.

386

32. Kamp, H. G.; Eisenbrand, G.; Schlatter, J.; Wurth, K.; Janzowski, C., Ochratoxin A:

387

induction of (oxidative) DNA damage, cytotoxicity and apoptosis in mammalian cell lines

388

and primary cells. Toxicology 2005, 206 (3), 413-425.

389

33. Klaric, M. K.; Rumora, L.; Ljubanovic, D.; Pepeljnjak, S., Cytotoxicity and apoptosis

390

induced by fumonisin B-1, beauvericin and ochratoxin A in porcine kidney PK15 cells:

391

effects of individual and combined treatment. Archives of Toxicology 2008, 82 (4), 247-255.

392

34. Peter, M. E., PROGRAMMED CELL DEATH Apoptosis meets necrosis. Nature 2011,

393

471 (7338), 310-312.

394

35. Ringot, D.; Chango, A.; Schneider, Y. J.; Larondelle, Y., Toxicokinetics and

395

toxicodynamics of ochratoxin A, an update. Chemico-Biological Interactions 2006, 159 (1),

396

18-46.

397

36. Babizhayev, M. A., Mitochondria induce oxidative stress, generation of reactive oxygen

398

species and redox state unbalance of the eye lens leading to human cataract formation:

20 ACS Paragon Plus Environment

Page 20 of 30

Page 21 of 30

Journal of Agricultural and Food Chemistry

399

disruption of redox lens organization by phospholipid hydroperoxides as a common basis for

400

cataract disease. Cell Biochemistry and Function 2011, 29 (3), 183-206.

401

37. Castello, P. R.; Drechsel, D. A.; Patel, M., Mitochondria are a major source of

402

paraquat-induced reactive oxygen species production in the brain. Journal of Biological

403

Chemistry 2007, 282 (19), 14186-14193.

404

38. Jacquemin, G.; Margiotta, D.; Kasahara, A.; Bassoy, E. Y.; Walch, M.; Thiery, J.;

405

Lieberman, J.; Martinvalet, D., Granzyme B-induced mitochondrial ROS are required for

406

apoptosis. Cell Death and Differentiation 2015, 22 (5), 862-874.

407

39. Yao, N.; Eisfelder, B. J.; Marvin, J.; Greenberg, J. T., The mitochondrion - an organelle

408

commonly involved in programmed cell death in Arabidopsis thaliana. Plant Journal 2004,

409

40 (4), 596-610.

410

40. Cao, X.-h.; Zhao, S.-s.; Liu, D.-y.; Wang, Z.; Niu, L.-l.; Hou, L.-h.; Wang, C.-l.,

411

ROS-Ca2+ is associated with mitochondria permeability transition pore involved in

412

surfactin-induced MCF-7 cells apoptosis. Chemico-Biological Interactions 2011, 190 (1),

413

16-27.

414

41. Gao, J.; Sana, R.; Calder, V.; Calonge, M.; Lee, W.; Wheeler, L. A.; Stern, M. E.,

415

Mitochondrial Permeability Transition Pore in Inflammatory Apoptosis of Human

416

Conjunctival Epithelial Cells and T Cells: Effect of Cyclosporin A. Investigative

417

Ophthalmology & Visual Science 2013, 54 (7), 4717-4733.

418

42. Cione, E.; Tucci, P.; Senatore, V.; Perri, M.; Trombino, S.; Iemma, F.; Picci, N.; Genchi,

419

G., Synthesized esters of ferulic acid induce release of cytochrome c from rat testes

21 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

420

mitochondria. Journal of Bioenergetics and Biomembranes 2008, 40 (1), 19-26.

421

43. Lee, M. G.; Lee, K. T.; Chi, S. G.; Park, J. H., Costunolide induces apoptosis by

422

ROS-mediated mitochondrial permeability transition and cytochrome C release. Biological &

423

Pharmaceutical Bulletin 2001, 24 (3), 303-306.

424

44. Miura, T.; Chiba, M.; Kasai, K.; Nozaka, H.; Nakamura, T.; Shoji, T.; Kanda, T.; Ohtake,

425

Y.; Sato, T., Apple procyanidins induce tumor cell apoptosis through mitochondrial pathway

426

activation of caspase-3. Carcinogenesis 2008, 29 (3), 585-593.

427

45. Youssef, A. M.; Malki, A.; Badr, M. H.; Elbayaa, R. Y.; Sultan, A. S., Synthesis and

428

Anticancer Activity of Novel Benzimidazole and Benzothiazole Derivatives against HepG2

429

Liver Cancer Cells. Medicinal Chemistry 2012, 8 (2), 151-162.

430

46. Rasul, A.; Bao, R.; Malhi, M.; Zhao, B.; Tsuji, I.; Li, J.; Li, X., Induction of Apoptosis

431

by Costunolide in Bladder Cancer Cells is Mediated through ROS Generation and

432

Mitochondrial Dysfunction. Molecules 2013, 18 (2), 1418-1433.

433

Figure Captions

434

Figure 1. Effect of OTA on IPEC-J2 cell viability. IPEC-J2 cells were cultured with OTA

435

for (a) 6 h, (b) 12 h and (c) 24 h at 0.5, 1, 2, 4, 8, 16 and 32 µM. MTT assay was applied to

436

determine cell viability. Data are expressed as the percentage compared to the control group

437

(set as 100%), means ± SEM of three independent experiments (n=6). *P < 0.05, **P < 0.01

438

as compared to control.

439

Figure 2. OTA-induced apoptosis in IPEC-J2 cells. Cells were exposed with OTA (2, 4 and

440

8 µM) for 12 h. (a) Morphology of apoptotic cell nuclei was measured by Hoechst 33258

22 ACS Paragon Plus Environment

Page 22 of 30

Page 23 of 30

Journal of Agricultural and Food Chemistry

441

staining. Arrows denote condensed nuclei. Among the groups, A: control; B: 2 µM OTA; C:

442

4 µM OTA; D: 8 µM OTA. (b) The representative plots of flow cytometry apoptosis. (c)

443

Quantification of apoptosis using flow cytometer. Data in (c) are presented as means ± SEM

444

of three independent experiments (n=9). *P < 0.05, **P < 0.01 as compared to control.

445

Figure 3. OTA-induced ROS production and mPTP opening in IPEC-J2 cells. IPEC-J2 cells

446

were exposed with OTA (2, 4 and 8 µM) for 12 h, and then stained with H2DCF-DA (for

447

measurement of intracellular ROS) or MitoSOX Red (for measurement of mitochondria ROS)

448

or calcein-AM (for measurement of mPTP opening). (a, b) Shown are representative

449

confocal images for DCF staining. The average fluorescence intensity was analyzed using

450

Image-Pro Plus software and expressed as the percentage compared to the control group (set

451

as 100%). (c) Cells were stained with H2DCF-DA and MitoTracker Red (red, localizes to

452

mitochondria). Overlay images (yellow) indicated mitochondria are the major source of

453

intracellular ROS induced by OTA. (d, e) Shown are representative confocal images for

454

MitoSOX Red staining. The average fluorescence intensity was analyzed using Image-Pro

455

Plus software and expressed as the percentage compared to the control group (set as 100%).

456

(f, g) Shown are representative confocal images for calcein staining. The average

457

fluorescence intensity was analyzed using Image-Pro Plus software and expressed as the

458

percentage compared to the control group (set as 100%). Among the groups (a, d, f), A:

459

control; B: 2 µM OTA; C: 4 µM OTA; D: 8 µM OTA. Data in (b, e and g) represent mean ±

460

SEM of three separate experiments (n=6). *P < 0.05; **P < 0.01 as compared to control.

461

Figure 4. Release of cyt-c into the cytoplasm and caspase-3 activation in OTA-treated

23 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

462

IPEC-J2 cells. The protein level of cyt-c in the cytoplasm (a) and active caspase-3 (b) were

463

detected by western blot. Representative images were shown (upper panels), and quantitative

464

data (lower panels) were performed with images of four independent experiments (mean ±

465

SEM, n = 4). *P < 0.05, ** P < 0.01 as compared to control.

466

Figure 5. Effect of CsA on OTA-induced apoptosis. (a, b) Cells were treated with OTA (4

467

µM) and/or CsA (5 µM) for 12 h and then incubated with calcein-am. (a) Shown are

468

representative confocal images for calcein staining. (b) The average fluorescence intensity

469

was analyzed using Image-Pro Plus software and expressed expressed as the percentage

470

compared to the control group (set as 100%). Data in (b) represent mean ± SEM of three

471

separate experiments (n=6). *P < 0.05; **P < 0.01 as compared to control. (c, d) The protein

472

level of cyt-c in the cytoplasm and active caspase-3 were detected. Representative images of

473

western blot were shown (upper panels), and quantitative data (lower panels) were performed

474

with images of four independent experiments (mean ± SEM, n = 4). The apoptosis was assed

475

using Hoechst 33258 staining (e) and flow cytometry (f). Data in (f) are presented as means ±

476

SEM of three independent experiments (n=9). *P < 0.05, **P < 0.01 as compared to control.

477

Among the groups (a, e), A: control; B: 4 µM OTA; C: 4 µM OTA and 5 µM CsA; D: 5 µM

478

CsA. D

479

Figure 6. Effect of Mito-TEMPO on OTA-induced mitochondrial ROS production and

480

mPTP opening. Cells was pre-incubated with Mito-TEMPO (10 µM) for 1 h before a 12 h

481

treatment with OTA. MitoSOX Red (a) and calcein (c) flourence were detected by confocal

482

microscope. Shown are representative confocal images. The average fluorescence intensity

24 ACS Paragon Plus Environment

Page 24 of 30

Page 25 of 30

Journal of Agricultural and Food Chemistry

483

was analyzed using Image-Pro Plus software and expressed expressed as the percentage

484

compared to the control group (set as 100%). Data in (b and d) represent mean ± SEM of

485

three separate experiments (n=6). *P < 0.05; **P < 0.01 as compared to control. Groups (a,

486

c), A: control; B: 4 µM OTA; C: 4 µM OTA and 10 µM Mito-TEMPO; D: 10 µM

487

Mito-TEMPO.

488

Figure 7. Mito-TEMPO inhibited OTA-induced apoptosis. Cells was pre-incubated with

489

Mito-TEMPO (10 µM) for 1 h, followed by treatment with OTA for 12 h. (a, b) Protein

490

expression levels of cyt-c in the cytoplasm, active caspase-3 and β-Actin were analyzed by

491

western blot. Representative images of western blot were shown (upper panels), and

492

quantitative data (lower panels) were performed with images of four independent

493

experiments (mean ± SEM, n = 4). The apoptosis was measured by Hoechst 33258 (c) and

494

flow cytometry (d). A: control; B: 4 µM OTA; C: 4 µM OTA and 10 µM Mito-TEMPO; D:

495

10 µM Mito-TEMPO. Data are presented as mean ± SEM of three separate experiments, each

496

one performed in triplicate (n = 9). **P < 0.01 as compared to control group; #P < 0.05 as

497

compared with OTA alone group.

498 499 500 501 502 503

25 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

504 505 506 507 508 509 510 511

Figure graphics: Figure 1 (a) 6h

(b) 12 h

(c) 24 h

Figure 2

26 ACS Paragon Plus Environment

Page 26 of 30

Page 27 of 30

Journal of Agricultural and Food Chemistry

(a)

(b)

(c)

Figure 3 (a)

(b)

(d)

(e)

(c)

(f)

Figure 4

27 ACS Paragon Plus Environment

(g)

Journal of Agricultural and Food Chemistry

Page 28 of 30

(b)

(a) OTA (µM)

0

2

4

8

OTA (µM)

0

2

4

8

Active caspase-3

Cyt-c

β-actin

β-actin

Figure 5 (a)

(b)

(c) OTA (µM)

0

4

4

0

CSA (µM)

0

0

5

5

Cyt-c β-actin

(d)

(e)

OTA (µM)

0

4

4

0

CSA (µM)

0

0

5

5

Active caspase-3

β-actin

Figure 6

28 ACS Paragon Plus Environment

(f)

Page 29 of 30

Journal of Agricultural and Food Chemistry

(a)

(b)

(c)

(d)

Figure 7 (b)

(a) OTA (µM) MitoTEMPO (µM)

0 0

4 0

4 10

cyt-c

0 10

OTA (µM) MitoTEMPO (µM)

0 0

4 0

Active caspase-3

β-actin

β-actin

(c)

(d)

29 ACS Paragon Plus Environment

4 10

0 10

Journal of Agricultural and Food Chemistry

TOC graphic

30 ACS Paragon Plus Environment

Page 30 of 30