Zearalenone Induces Estrogen-Receptor-Independent Neutrophil

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Food Safety and Toxicology

Zearalenone induces estrogen receptor-independent neutrophil extracellular trap release in vitro Jingjing Wang, Zhengkai Wei, Zhen Han, Ziyi Liu, Xingyi Zhu, Xiaowen Li, Kai Wang, and Zhengtao Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05948 • Publication Date (Web): 02 Apr 2019 Downloaded from http://pubs.acs.org on April 2, 2019

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Journal of Agricultural and Food Chemistry

1

Zearalenone induces estrogen receptor-independent neutrophil

2

extracellular trap release in vitro

3 4

Jing-Jing Wang,‡,§, Zheng-Kai Wei,‡,§, Zhen Han,‡, Zi-Yi Liu,‡,

5

Xing-Yi Zhu, Xiao-Wen Li, Kai Wang*, Zheng-Tao Yang*

6

College of Life Science, Foshan University, Foshan, Guangdong

7



8

528231, People’s Republic of China

9

‡

10

College of Veterinary Medicine, Jilin University, Jilin, Changchun

130062, People’s Republic of China

11

Corresponding authors

12

*

13

Zhengtao Yang. E-mail address: [email protected]

14

Kai Wang. E-mail address:[email protected]

15

§ These

two authors contributed equally to this work

16 17 18 1

ACS Paragon Plus Environment


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ABSTRACT: Zearalenone (ZEA) is a nonsteroidal estrogenic

20

mycotoxin synthesized in Fusarium species, mainly F. graminearum

21

and F. culmorum, and it has strong estrogenic activity and causes

22

genotoxic effects, reproductive disorders and immunosuppressive

23

effects. Neutrophil extracellular trap (NET) have been studied for

24

many years. Initially, NET were considered a form of the innate

25

response that combat invading microorganisms. However, NET are

26

involved in a series of pathophysiological mechanisms, including

27

thrombosis,

28

autoimmunity. We recently find that polymorphonuclear neutrophils

29

(PMNs) response to ZEA exposure by undergoing NET formation.

30

However, the molecular mechanisms involves in this process remain

31

poorly characterized. Here, we analyze whether estrogen receptors

32

(ERs) can affect NET formation after ZEA stimulation. The

33

involvement of ERs is investigated with the selective ER

34

antagonists. Moreover, we investigate the mechanisms of NET

35

formation

36

microplate and Western blot analysis. Our results show that ERs

tissue

using

necrosis,

autoinflammation,

immunofluorescence

staining,

2


and

even

fluorescence

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37

(ERα and ERβ) are not involved in ZEA-induced NET formation,

38

but reactive oxygen species (ROS), ERK and p38 are postulated to

39

be involved. Specifically, we provide data demonstrating that

40

ZEA-induced reactive oxygen species (ROS) may promote

41

activation of ERK and p38 as well as subsequent NET release. We

42

are the first to demonstrate this new mechanism of ZEA-induced

43

NET formation, which may help in understanding the role of ZEA

44

in overexposure diseases and provide a relevant basis for therapeutic

45

applications.

46

KEYWORDS: Neutrophil extracellular traps; zearalenone; estrogen

47

receptors; reactive oxygen species

48

INTRODUCTION

49

Zearalenone (ZEA), also known as F-2 toxin, is a secondary

50

metabolite produced by various species of the Fusarium genus1.

51

ZEA has high heat stability and commonly persists in maize and

52

other grains such as wheat, sorghum and rye around the world2.

53

Previous reports have described that ZEA had genotoxic,

54

hepatotoxic,

immunosuppressive

and

3


neurotoxic

effects.


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55

Importantly, ZEA and its derivatives have structural similarity to

56

estrogen, which enables them to bind to estrogen receptors (ERs)3-5.

57

Thus, the Joint Food and Agriculture Organization of the United

58

Nations

59

Committee on Food Additives (JECFA) established a provisional

60

maximum tolerable daily intake (PMTDI) for ZEA for 0.5 μg/kg

61

bodyweight (b.w.)2. The estrogen-like activity of ZEA is attributed

62

to the toxicity of ZEA and its metabolites, but other mechanisms

63

such as oxidative stress and DNA damage, could be involved6.

64

Indeed, several studies have previously shown that ZEA induced an

65

accumulation of reactive oxygen species (ROS), which suggested

66

that oxidative stress may be attributed to the toxicity of ZEA and its

67

metabolites7-9.

(FAO)/World

Health

Organization

(WHO)

Expert

68

Neutrophils are the most abundant immune cells and play vital

69

roles in detecting invading pathogens. In addition to well-known

70

processes such as phagocytosis and ROS generation, neutrophils

71

exhibit strong anti-microbial properties through the formation of

72

neutrophil extracellular trap (NET)10-11. NET are fibers of DNA 4


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coated with histones and antimicrobial proteins that are released into

74

the extracellular space where they can trap microorganisms12.

75

Accumulating data suggested that NET are involved in the

76

pathogenesis of rheumatoid arthritis (RA)13, thrombosis14, systemic

77

lupus erythematosus (SLE)15 and cancer16. Moreover, NET release

78

is stimulated by a wide range of stimuli, such as bacteria17, fungi18,

79

parasites19, and viruses20 as well as small compounds including

80

lipopolysaccharide (LPS)21, calcium ionophores (CaIs)22, or

81

phorbol-myristate acetate (PMA)22. In this study, we aim to gain

82

further insight into the mediators, molecular pathways and

83

regulation of ZEA-induced NET formation.

84

Estrogen receptors (ERs) alpha and beta belong to the nuclear

85

receptor superfamily and are transcriptional factors that mediate

86

various physiological processes including cell growth, reproduction,

87

development and differentiation23. ZEA is a mycotoxin that binds to

88

estrogen receptors and has estrogen-like activities. It is previously

89

reported that the estrogen receptor modulator affected NET

90

formation24-25. However, it is unclear whether the mechanism of 5



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ZEA-induced NET formation is due to estrogen-like activities. We

92

aim to gain further insight into the mediators, molecular pathways

93

and regulation of ZEA-triggered NET formation.

94

MATERIALS AND METHODS

95

Materials.

Zearalenone,

2,

7-dichlorodihydrofluorescein

96

diacetate (DCF-DA), zymosan, diphenyleneiodonium chloride

97

(DPI), U0126, SB202190 and MPP dihydrochloride were obtained

98

from Sigma-Aldrich. PHTTP was obtained from MedChemExpress.

99

Sytox Orange and Pico Green® were obtained from Invitrogen.

100

Annexin-V-FLUOS Staining Kit was obtained from Roche.

101

Superoxide dismutase (SOD) assay kits (Nanjing Jiancheng

102

Bioengineering Institute, China) and catalase (CAT) assay kits

103

(Nanjing Jiancheng Bioengineering Institute, China) were used.

104

Anti-histone antibody (LS-C353149; Life Span BioSciences, Inc),

105

anti-MPO antibody (Orb16003; Biorbyt), goat anti-rabbit IgG-FITC

106

(abs20023; Absin), anti-p-p38 (Cell Signaling Technology Inc,

107

USA), anti-p-ERK (Cell Signaling Technology Inc, USA), anti-p38

108

(Bs3566; Bioword), anti-ERK (Bs3627; Bioword) and anti-beta 6


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109


actin antibody (66009-1-Ig; Proteintech) were used.

110

Isolation of PMNs. Blood were isolated from healthy cattle and

111

collected in a heparin tube. Bovine neutrophils were purified from

112

blood using a PMN isolation kit® (TianJin HaoYang Biological

113

Manufacture

114

instructions. The purity of neutrophils reached more than 90 %. All

115

experiments were approved by the Care and Use of Experimental

116

Animals of Jilin University.

CO.

China)

according

to

the

manufacturer’s

117

Immunofluorescence staining and observation. For NET

118

staining, neutrophils were plated on poly-l-lysine (0.1 mg/mL)

119

pretreated coverslips and incubated with ZEA (5, 10 or 20 μM) in

120

RPMI-1640 medium (phenol-red-free). After 2 h, samples were

121

fixed with 4 % (w/v) paraformaldehyde for 30 min, rinsed twice in

122

phosphate buffered saline (PBS), and permeabilized in 0.1 % Triton

123

X-100 in PBS for 20 min. Samples were then blocked in 5 % goat

124

serum,

125

anti-myeloperoxidase (1:200) antibodies overnight at 4 °C. After

126

two washes in PBS, cells were incubated with secondary goat

and

incubated

with

anti-histone

7


H3

(1:200)

and


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127

anti-rabbit IgG-FITC antibody (1:200) for 120 min. Cells were

128

finally washed two times with PBS, stained with 5 μM Sytox

129

Orange (dissolved in PBS) and observed using a scanning confocal

130

microscope (Olympus Fluo View FV1000).

131

Quantitation of NET. NET were quantified using Pico Green®

132

as previously described26. Briefly, the cells were seeded into 96-well

133

plates in RPMI-1640 medium (phenol-red-free). Cells were

134

pretreated with inhibitors DPI (50 μM), SB202190 (10 μM), U0126

135

(10 μM), MPP (0.1 μM) or PHTTP (0.1 μM) for 30 min, and then

136

incubated for an additional 2 h with ZEA (20 μM), with zymosan (1

137

mg/mL) treatment serving as a positive control group. After

138

incubation, the fluorescence was measured at an excitation

139

wavelength of 485 nm and an emission wavelength of 535 nm by an

140

Infiniti M200® fluorescence plate reader (Tecan, Austria).

141

ROS production assay. The level of ROS in ZEA-stimulated

142

neutrophils was determined with DCF-DA. Briefly, the cells were

143

incubated with ZEA (5, 10 or 20 μM) for 2 h. Next, DCF-DA (10

144

μM) was added to each well for 20 min. The fluorescence of the 8


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145

cells was detected at an excitation wavelength of 485 nm and an

146

emission wavelength of 525 nm by an Infiniti M200® fluorescence

147

plate reader (Tecan, Austria).

148

Assay of antioxidant enzymes activity. The activity of

149

antioxidant enzymes were measured by using commercial kits

150

(Nanjing Jiancheng Bioengineering Institute, China). Briefly, Cells

151

were seeded into six-well plates and incubated with ZEA (5, 10 or

152

20 μM) for 2 h ， and then the activity of SOD and CAT was

153

determined according to manufacturer’s instructions.

154

Western blot analysis. The cells at the density of 2 × 106

155

cells/mL were seeded into six-well plates and incubated with ZEA

156

(5, 10 or 20 μM) for 2 h. After incubation, the cells were harvested

157

and washed with PBS. Whole cell lysates were analyzed by Western

158

blot analysis, as previously described26. In brief, protein

159

concentrations were determined by a bicinchoninic acid (BCA)

160

protein assay reagent kit (Pierce) and an Extraction Reagent Kit

161

(Beyotime Biotechnology, China). Subsequently, the samples were

162

separated by SDS-PAGE electrophoresis and tansferred on a PVDF 9



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163

transfer membrane (Merck Millipore, Billerica, MA). The samples

164

were probed with the following primary antibodies: anti-p38

165

polyclonal antibody (1:1000), anti-phosphor-p38 monoclonal

166

antibody (1:1000), anti-ERK1/2 monoclonal antibody (1:1000),

167

anti-phosphor-ERK1/2 monoclonal antibody (1:1000). Signals were

168

revealed using HRP-linked secondary antibodies and detected using

169

ECL Plus Western Blotting Detection System (ProteinSimple,

170

American).

171

Apoptosis assay. The cells at the density of 2×106 cells/mL were

172

seeded into six-well plates and incubated with ZEA (5, 10 or 20

173

μM) for 2 h. The cells were washed with PBS and centrifuged at 200

174

g for 5 min. Subsequently, the cells were resuspended and incubated

175

with 100 μL Annexin-V-FLUOS labeling solution for 10 min at

176

room temperature in the dark, and results were analyzed on a BD

177

FACSCalibur flow cytometer.

178

LDH assay. Briefly, the cells were seeded into 96-well plates in

179

RPMI-1640 medium (phenol-red-free). Cells were incubated with

180

ZEA (5, 10 or 20 μM) for 2 h.

Release of lactate dehydrogenase 10


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181

(LDH) enzyme in the supernatant was measured by an LDH

182

Cytotoxicity Assay kit® (Beyotime Biotechnology, China) according

183

to the manufacturer's protocols.

184

Statistical analysis. All data were analyzed using GraphPad

185

Prism 5 (version 5.0, GraphPad InStat Software, San Diego, CA,

186

USA). Comparisons between groups were made with one-way

187

ANOVA followed by Tukey’s test. Data are presented as the means

188

± SEM. A P value of 0.05 or less was considered to be statistically

189

significant.

190

RESULTS

191

ZEA induces NET formation. Neutrophils were activated with

192

ZEA (5, 10 or 20 μM) for 2 h, stained with Sytox Orange and

193

observed with fluorescence confocal microscopy. The images

194

showed that ZEA obviously induced NET formation in PMNs.

195

Activating neutrophils with ZEA resulted in a typical NET structure

196

containing extracellular DNA colocalized with histones and MPO

197

(Figure 1).

198

Quantitation of NET. Quantitation of NET induced by ZEA was 11



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199

accomplished by Pico Green® and an Infiniti M200® fluorescence

200

plate reader. As shown in Figure 2, the assays showed that ZEA

201

activated neutrophils to release NET. Furthermore, the amount of

202

NET also increased significantly as the concentration of ZEA

203

increased, which could be confirmed by fluorescence microscopy

204

previously, revealing that the formation of NET induced by ZEA

205

might be a dose-dependent process.

206

Estrogen receptors are not responsible for ZEA-induced NET

207

formation. As previously described, the expression of ERs (ERα

208

and ERβ) has been identified in bovine PMNs27. Thus, we aimed to

209

evaluate whether ERs are required for ZEA-induced NET formation.

210

Fluorescence-based quantification of NET production suggested that

211

the selective estrogen receptor antagonists MPP (ERα inhibitor) had

212

no effect on ZEA-induced NET formation, but PHTPP (ERβ

213

inhibitor) exaggerated ZEA-induced NET formation. In line with

214

this, tamoxifen, a selective estrogen receptor modulator, increased

215

NET production28. This finding supports the role for ERs in

216

boosting host innate immune function, but the relevant mechanisms 12


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217


should be further investigated (Figure 3).

218

ZEA triggers the phosphorylation of ERK and p38 signaling

219

proteins. To elucidate the pathways underlying ZEA-induced NET

220

formation, we next detected the phosphorylation of ERK and p38

221

signaling proteins by Western blotting. As shown in Figure 4, ZEA

222

obviously increased the phosphorylation of ERK and p38 signaling

223

proteins in a dose-dependent manner.

224

ZEA induces ROS production. We next asked whether

225

ZEA-induced NET required ROS production. We used DCF-DA, a

226

fluorescent indicator of ROS to detect ROS generation. As shown in

227

Figure 5, ZEA led to an abundant production of ROS.

228

ZEA inhibits the activity of antioxidant enzymes. Previous

229

studies have revealed that ROS is required for NET release29.

230

However, the contribution of SOD and CAT on the NET release has

231

not been addressed. In the study, the activity of SOD and CAT were

232

measured. As shown in Figure 6, the activity of SOD and CAT were

233

reduced significantly after ZEA exposure.

234

ZEA-induced NET formation is dependent on the NADPH 13



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235

oxidase, ERK and p38 pathways. The involvement of NADPH

236

oxidase, ERK and p38 pathways in ZEA-induced NET formation

237

was tested with three specific inhibitors. The results showed that the

238

production of NET in ZEA activated neutrophils was inhibited when

239

these cells were pretreated with DPI, U0126 and SB202190 (Figure

240

7). Moreover, pretreatment of ERK inhibitor U0126 inhibited the

241

ZEA induced phosphorylation of ERK, and similarly pretreatment

242

of

243

phosphorylation of p38 (Figure 8). The results indicated that

244

ZEA-induced NET formation is dependent on the NADPH oxidase,

245

ERK and p38 pathways.

p38

inhibitor

SB202190

inhibited

the

ZEA

induced

246

ZEA induced-NET formation is accompanied by less

247

apoptosis. Apart from NET, neutrophils also undergoing cell

248

apoptosis and necrosis. We further assessed the effects of ZEA

249

using flow cytometry by Annexin V/PI staining. As shown in Figure

250

9, the cells displayed less apoptosis.

251

ZEA, MPP and PHTPP have no effect on LDH release.

252

Finally, we investigated whether LDH release occurs during 14


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253

ZEA-induced NET formation. The results showed that ZEA, MPP

254

and PHTPP stimulation did not result in the release of LDH (Figure

255

10). DISCUSSION

256

ZEA, also known as F-2 toxin, is a secondary metabolite produced

257

by various species of the Fusarium genus that occurs in feed and

258

foodstuff30. ZEA and its derivatives have a unique macrolide

259

structure, bind to ERs and exhibit an estrogen-like activity31. Nearly

260

15 years after the first description of NET, the structures are

261

involved in a large amount of pathophysiological mechanisms.

262

Although, NET have vital roles in combating pathogen invasion, a

263

growing body of literatures suggest that the inappropriate release of

264

NET may have a serious impact as a result of their cytotoxic,

265

proinflammatory, and prothrombotic activities32-33. Here, we show

266

evidence that ZEA obviously induces NET formation in PMNs, and

267

these extracellular structures, thicker and thinner regions are similar

268

to the typical characteristics of NET. The quantitation of NET also

269

increases markedly, further confirming that ZEA induces NET

270

formation. Next, we commit to determining the potential mechanism 15



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271

of ZEA-induced NET formation. ZEA has been reported as a

272

complete activator for ERα, yet only a partial activator for ERβ34.

273

Another study revealed that ZEA was a partial antagonist for ERα1.

274

Nevertheless, we investigate whether ERs are involved in

275

ZEA-induced

276

ZEA-induced NET formation is independent of ERs (both ERα and

277

ERβ). NET formation is not inhibited in response to treatment with

278

selective antagonists of either receptor (0.1 μM MPP or 0.1 μM

279

PHTPP), suggesting that ZEA may act through multiple

280

mechanisms to induce NET production, instead of an estrogen

281

receptor-dependent mechanism.

NET

release,

and

our

results

indicate

that

282

Previous studies found that ZEA could induce apoptosis in

283

different cells35-37. Our observations showed that in the process of

284

ZEA-induced NET formation could be also accompanied by less

285

apoptosis. Next, we focused on elucidating the pathway underlying

286

ZEA-induced NET formation. NADPH has been discovered as a

287

vital molecule for NET formation38. Currently, two distinct forms of

288

NETosis have been described based on their requirement for 16


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289

NADPH, a dependent or an independent process. Accumulated data

290

suggested that ZEA increased ROS generation, which could

291

contribute to genomic instability, metabolic oxidative stress and

292

even

293

NADPH-dependent ROS could participate in NET formation.

294

Indeed, our results show that ZEA induces NET formation, but this

295

effect inhibits by a specific NADPH oxidase inhibitor (DPI), which

296

confirmes that ZEA-induced NET formation is at least partially an

297

NADPH oxidase-dependent process. In addition, we clearly

298

demonstrate the involvement of SOD and CAT in ZEA-induced

299

NET formation, suggesting that SOD and CAT may play a major

300

role in NET formation.

cellular

injury39-41.

Thus,

we

hypothesized

that

301

We further investigate the effect of ZEA on ERK and p38

302

phosphorylation, which are signal transduction events known to be

303

critical for NET formation. During NADPH-dependent NET

304

formation, ROS generation is required for activation of ERK and

305

p3829. In the present study, ERK and p38 were activated in

306

ZEA-induced NET, and both ERK inhibitor U0126 and p38 17



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307

inhibitor SB202190 significantly inhibited NET formation and the

308

phosphorylation of ERK and p38. Thus, it is possible that

309

ZEA-induced NET formation was mediated via ERK and p38

310

dependent pathways.

311

In summary, our evidence demonstrates that ZEA is a potent

312

inducer of NET. ZEA-induced NET production is largely

313

independent of ERs, but is a NADPH-dependent pathway and is

314

similar to PMA-induced NADPH-dependent NET formation.

315

Furthermore, ZEA likely modulates NET production via regulation

316

of ERK and p38 signaling, but more potential mechanisms involved

317

in ZEA-induced NET are expected to be further investigated.

318

Given the health issues induced by ZEA overexposure, the

319

identification of NET in vitro may provide more insight into this

320

matter.

321

ACKNOWLEDGMENTS

322

This study was supported by grants from the National Natural

323

Science Foundation of China (no. 31772721).

324

Notes 18


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The authors declare no conflicts of interest.

326

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327

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Figure legends

509

Figure 1. ZEA induces NET formation. Primary bovine neutrophils

510

were incubated with ZEA (5, 10 or 20 μM) for 2 h, fixed with 4 %

511

(w/v) paraformaldehyde, incubated with Sytox Orange (red) and

512

immunolabeled with antibodies directed against H3 and MPO

513

(green). Scale bars represent 20 µm.

514

Figure 2. Quantitation of NET. Primary bovine neutrophils were

515

seeded into 96-well plates in RPMI-1640 medium (phenol-red-free)

516

and incubated with ZEA (5, 10 or 20 μM) for 2 h. NET release was

517

quantified with Pico Green®. Data are presented as the means ±

518

SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

519

Figure 3. Selective estrogen receptor antagonists do not inhibit

520

ZEA-induced NET formation. ZEA-induced NET production was

521

quantified in cells preincubated with MPP (selective ERα

522

antagonist) or PHTPP (selective ERβ antagonist) at the indicated 29



Page 30 of 44

523

concentrations (0.1 μM). Data are presented as the means ± SEM

524

(**p < 0.01, ***p < 0.001).

525

Figure 4. ZEA triggers the phosphorylation of ERK and p38

526

signaling proteins. Primary bovine neutrophils were treated with

527

ZEA (5, 10 or 20 μM) for 2 h. Phosphorylation of ERK and p38 was

528

investigated by Western blot analysis. Quantification of protein

529

samples was determined by densitometry and is normalized to

530

β-actin. Data are presented as the means ± SEM (*p < 0.05,

531

0.01, ***p < 0.001).

532

Figure 5. ZEA induces ROS production. Primary bovine

533

neutrophils were treated with ZEA (5, 10 or 20 μM) for 2 h. The

534

generation of intracellular ROS was detected by DCF-DA. Data are

535

presented as the means ± SEM (***p < 0.001).

536

Figure 6. ZEA reduces the activity of antioxidant enzymes.

537

Primary bovine neutrophils were treated with ZEA (5, 10 or 20 μM)

538

for 2 h. The activities of SOD and CAT were measured. Data are

539

presented as the means ± SEM (**p < 0.01, ***p < 0.001).

540

Figure 7. ZEA-induced NET formation is dependent on the 30


**p

Zearalenone Induces Estrogen-Receptor-Independent Neutrophil

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