Selenomethionine Alleviates AFB1-Induced Damage in Primary

Subscriber access provided by University of Newcastle, Australia

Article

Selenomethionine alleviates AFB1-induced damage in primary chicken hepatocytes by inhibiting CYP450 1A5 expression via upregulated SelW expression Xingxiang Chen, Chaoping Che, Viktor I. Korolchuk, Fang Gan, Cuiling Pan, and Kehe Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05308 • Publication Date (Web): 13 Mar 2017 Downloaded from http://pubs.acs.org on March 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 34

Journal of Agricultural and Food Chemistry

1

Selenomethionine alleviates AFB1-induced damage in primary chicken hepatocytes

2

by inhibiting CYP450 1A5 expression via upregulated SelW expression

3

4 5

Xingxiang Chen †, Chaoping Che †, Viktor I. Korolchuk §, Fang Gan †, Cuiling Pan †,

6

Kehe Huang * †

7 8

†

9

P. R. China.

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

10

§

11

Tyne, NE4 5PL, UK.

Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon

12 13

* Corresponding author.

14

Tel.: +86 25 84395507

15

Email address: [email protected] (Kehe Huang)

16 17 18 19 20

1

ACS Paragon Plus Environment


Page 2 of 34

21

Abstract

22

This study aims to evaluate the protective effects of selenomethionine (SeMet) on

23

Aflatoxin B1 (AFB1)-induced hepatotoxicity in primary chicken hepatocytes. Cell

24

viability and lactic dehydrogenase activity assays revealed the dose-dependency of

25

AFB1 toxicity to chicken hepatocytes. AFB1 concentrations above 0.05 µg/mL

26

significantly reduced glutathione and total superoxide dismutase levels, as well as

27

increased malondialdehyde concentration and cytochrome P450 enzyme 1A5

28

(CYP450 1A5) mRNA levels (P < 0.05). AFB1, however, did not affect CYP450

29

3A37 mRNA levels. Supplementation with 2 µM SeMet protected against

30

AFB1-induced changes and significantly increased selenoprotein W (SelW) mRNA

31

levels (P < 0.05). Additionally, SelW knockdown attenuated the protective effect of

32

SeMet on AFB1-induced damage and significantly increased CYP450 1A5 expression

33

(P < 0.05). Therefore, SeMet alleviates AFB1-induced damage in primary chicken

34

hepatocytes by improving SelW expression, thus inhibiting CYP450 1A5 expression.

35

Keywords:

36

selenoprotein W, primary chicken hepatocytes

selenomethionine,

AFB1,

cytochrome

P450

37

38

39

2


enzymes,

siRNA,

Page 3 of 34


40

Introduction

41

Aflatoxins are produced by different genera of fungi, such as Aspergillus,

42

Penicillium, and Fusarium1. The aflatoxin contamination of foods and feeds severely

43

affect the health of humans, livestock, and poultry. The Food and Drug Administration

44

consider aflatoxins as inevitable contaminants2 with harmful effects, including altered

45

production parameters, impaired immunity, or toxicity to multiple organs2-4. In

46

addition, aflatoxins are hepatotoxins and hepatocarcinogens5. Structurally, aflatoxins

47

are difurocoumarin derivatives that fluoresce under ultraviolet light. Aflatoxins are

48

divided based on the color of their fluorescence: aflatoxin B1 and B2 (AFB1, AFB2)

49

have blue fluorescence, and G1 and G2 (AFG1, AFG2) have green fluorescence6, 7.

50

AFB1 is a potential carcinogen in many species, including humans, birds, swine, fish,

51

and rodents8, 9. In poultry, AFB1 decreases serum values of ALT, ALP, total protein,

52

and albumin10, 11. AFB1-affected poultry have reduced renal Na⁺ -K⁺ ATPase activity

53

and enlarged mitochondria12. AFB1 itself is not toxic but can be bio-activated into the

54

highly reactive and toxic intermediate exo-AFB1-8, 9-epoxide (AFBO) by the hepatic

55

cytochrome P450 enzyme (CYP450). AFBO reacts with cellular macromolecules,

56

such as DNA and proteins, thus causing genotoxicity and cytotoxicity13-15. In nucleic

57

acids, AFBO binds to guanine residues, which irreversibly damages DNA in humans,

58

primates, and ducks16, 17. CYP450 has two isoforms, 1A5 and 3A37, in the turkey

59

liver18-20; these isoforms bioactivate AFB1 into AFBO18, 19, 21 with CYP450 1A5 being

60

the dominant enzyme22. AFBO is partly detoxified via conjugation with glutathione

61

(GSH). This reaction is catalyzed by glutathione-S-transferases (GST). Moreover,

3



62

AFBO can be detoxified when hydrolyzed either spontaneously or by a cytosolic

63

epoxide hydrolase into AFB1-8, 9-dihidrodiol (dhd-AFB1)23. However, further study

64

on effective antagonists against AFB1-induced cytotoxicity is still required to reduce

65

AFB1-induced damage.

66

Selenium (Se) is an essential trace element for animals and humans and has

67

diverse biological properties24. Se is involved in the functions of selenoproteins, such

68

as glutathione peroxidases (GPx) and selenoprotein W (SelW), which protect cells

69

from oxidative stress25-27. Se improves antioxidant activity by enhancing

70

selenoprotein levels. Se supplementation in the diet or drinking water inhibits

71

carcinogenesis that is induced by numerous chemical carcinogens, including AFB128,

72

29

73

The covalent binding of AFB1 to liver DNA and RNA is significantly reduced in

74

chicks that were fed with a Se-supplemented diet compared with that in control chicks

75

that were fed with a basal diet11, 32. The mechanisms involved in the protective effects

76

of selenomethionine (SeMet) on AFB1-induced hepatotoxicity, however, remain

77

poorly understood.

78

. The protective role of Se against aflatoxins has been confirmed in mammals12, 30, 31.

Therefore, this study aims to evaluate and explore the mechanism of the effects

79

of SeMet on AFB1-induced hepatotoxicity in primary chicken hepatocytes.

80

Materials and Methods

81

Isolation and Culture of Chicken Hepatocytes

82

All experimental procedures that involved animals were approved by the Animal 4


Page 4 of 34

Page 5 of 34


83

Care and Use Committee of Nanjing Agricultural University; the animal ethical

84

number is SYXK (Su) 2011-0036. Hepatocytes were obtained from male White

85

Leghorn chickens (aged 30–50 days). Hepatocytes were isolated using the previously

86

described collagenase perfusion method33. Isolated cells were dispersed in 50 mL of

87

serum-free L-15 medium that was supplemented with 0.2% (w/v) bovine serum

88

albumin and antibiotics (100 mg/mL streptomycin, 100 IU/mL penicillin, and 0.25

89

mg/mL amphotericin B) at 4 °C. Hepatocytes were purified following the method of

90

Wu et al (2010)34. The viability of the isolated hepatocytes was 91.5±1.7%, as

91

determined via the 0.4 % trypan blue dye exclusion method35 with five different

92

preparations of hepatocytes.

93

Primary chicken hepatocytes were cultured in accordance with a previous

94

method35. Isolated hepatocytes were seeded into 6-well cell culture plates

95

(Corning™ Costar™) at a density of 2×106 viable cells per well in 2 mL of L-15

96

medium. The L-15 medium was supplemented with 5% fetal bovine serum, 33 mM

97

HEPES, 2 mM L-glutamine, 0.2% (w/v) bovine serum albumin, 100 nM

98

dexamethasone, 5 mg/L transferrin, 1 mmol/L insulin, and antibiotics, and was

99

maintained at pH 7.65. The cells were cultured at 37 °C in a 5% CO2 atmosphere. After

100

a plating period of 4 h, the cell monolayers were washed twice with Hanks’ balanced

101

salt solution. Then, 3 mL of fresh serum-free L-15 medium was added to each well.

102

The spent medium was replaced with fresh serum-free L-15 medium every 24 h.

103

Assessment of AFB1 Toxicity

104

Isolated chicken hepatocytes were seeded at a density of 2×103/well in 96-well 5



105

plates and cultured for 24 h prior to treatment with 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.4,

106

0.8, and 1.6 µg/mL AFB1 (Sigma-Aldrich). AFB1 solutions with different

107

concentrations were prepared from a stock solution of 1 mg/mL AFB1 in dimethyl

108

sulfoxide (DMSO). The stock solution was further diluted in culture media at the

109

desired concentrations. The cells were then cultured at 37 °C in a 5% CO2 atmosphere

110

for 24 h to reach 80% confluence and were then subjected to the colorimetric

111

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, USA).

112

Absorbance was measured at 490 nm with a reference wavelength of 630 nm. Lactic

113

dehydrogenase (LDH) activity in the incubation medium was measured as previously

114

described35. One unit of enzyme activity was defined as 1 mmol of reduced

115

nicotinamide adenine dinucleotide oxidized per min. LDH activity in the culture

116

medium was expressed as units per liter. All samples were assayed in triplicate.

117

Measurements of GSH, SOD, and MDA

118

Cells were removed from wells. Cell extracts were then prepared by sonicating

119

cells in ice-cold phosphate-buffered saline. After sonication, cell lysates were

120

centrifuged at 10,000×g for 20 min to remove cell debris. Reduced GSH, superoxide

121

dismutase activity (SOD), and maliondehyde (MDA) were measured in the

122

supernatants as previously described using commercially available kits (Jiancheng Co.,

123

China)36, 37. GSH was spectrophotometrically measured (412nm) through reaction

124

with 5, 5′-dithiobis (2-nitrobenzoic acid). GSH concentrations were expressed as

125

micromoles of GSH per gram of protein. SOD activity was assayed via oxyamine

126

oxidation inhibition by xanthine oxidase. One unit of SOD activity was defined as the 6


Page 6 of 34

Page 7 of 34


127

quantity that reduced absorbance at 550 nm by 50%. Results were expressed as unit

128

per milligram protein. MDA concentration was spectrophotometrically assayed at 548

129

nm via the thiobarbituric acid reaction and was expressed as micromoles per gram of

130

protein. Total protein concentration was determined using a Bradford Protein Assay

131

Kit (Beyotime, China). All samples were assayed in triplicate.

132

siRNA Transfection

133

Double-stranded RNA sequences were designed based on the sequence of Gallus

134

gallus SelW mRNA (GenBank Accession No. GQ919055) using Invitrogen BlockiT

135

RNAi designer. RNA sequences were synthesized by Invitrogen. The SelW-speciﬁc

136

siRNA sequence was 5’-CGGCUUCGUGGACACCGACGCCAAA-3’. The control

137

siRNA sequence was 5’-CGGUCGUGGACACCGACGCCCUAAA-3’. Duplexes

138

were resuspended in DEPC-treated water to obtain 20 mM solutions prior to use.

139

Chicken hepatocytes in DMEM with 10% FBS without antibiotics were seeded in

140

24-well plates at 5×104 cells/well and incubated for 24 h at 37 °C in a 5% CO2

141

atmosphere. When the cells reached 30%–50% confluence, siRNA was introduced

142

using the X-tremeGene siRNA transfection reagent (Roche, USA) in accordance with

143

the manufacturer’s protocol.

144

Real-time PCR

145

SelW, CYP450 1A5, and 3A37 mRNA levels were quantitatively determined by

146

real-time PCR. Total RNA was extracted from cells using Trizol (Invitrogen, USA) in

147

accordance with the manufacturer’s protocol. DNA contamination was prevented with

7



148

the DNA-Free kit (TaKaRa, China). RNA quality was assessed via agarose gel (1%)

149

electrophoresis and spectrophotometry

150

reverse-transcribed to complimentary DNA (cDNA) using the PrimeScriptTM RT

151

Master Mix kit (TaKaRa, China). The resulting first-strand cDNA was stored at

152

−20 °C until used for real-time PCR. Specific primers for β-actin, SelW, CYP450 1A5,

153

and 3A37 were designed with Primer Premier Software (Premier Biosoft International,

154

USA) based on known sequences (Table 1). qRT-PCR was performed with an

155

ABI-Prism7300 detection system (Applied Biosystems, USA). Reactions were

156

performed in a 25-µL reaction mixture that contained 12.5 µL of 2×SYBR Green I

157

PCR Master Mix (TaKaRa, China), 10 µL of cDNA, 1 µL of each primer (10 µM),

158

and 0.5 µL of PCR-grade water. The qRT-PCR program consisted of a 95 °C step for

159

30 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 31 s. A dissociation curve

160

was obtained per plate to confirm the production of a single product. Considering that

161

different treatments had no effects on β-actin Ct values in our study (data not shown),

162

the mRNA levels of SelW, CYP450 1A5, and 3A37 relative to that of the reference

163

β-actin gene were determined using the ∆ cycle threshold (∆Ct) method as outlined in

164

the protocol provided by Applied Biosystems. The result was applied to each gene by

165

calculating the expression 2−∆∆CT.

166

Western Blot

(A260/A280). Total

RNA (1 µg) was

167

The relative abundance of CYP450 1A5 protein in hepatocytes after siRNA

168

interference was determined by Western blot. Cell samples were lysed via ultrasound

169

at 4 °C in radioimmunoprecipitation assay buffer with protease inhibitors. The lysates 8


Page 8 of 34

Page 9 of 34


170

were centrifuged at 12,000×g for 15 min at 4 °C. Total protein concentrations were

171

determined using the bicinchoninic acid protein assay. Then, 40 µg of proteins in

172

loading buffer were denatured by heating at 95 °C for 5 min, separated by SDS-PAGE

173

on 4–12% Bis-Tris gel, and transferred onto a polyvinylidene fluoride membrane.

174

After blocking, the membranes were individually incubated overnight at 4 °C with

175

primary antibodies against CYP450 1A5 (rabbit anti-human CYP450, BIOSS) at a

176

concentration of 1:500 and with primary antibodies against β-actin (Cell Signaling

177

Technology, USA) at a concentration of 1:1,000. Membranes were then washed and

178

incubated at room temperature for 30 min with horseradish peroxidase-conjugated

179

secondary antibody (1:5,000 donkey anti-rabbit IgG Santa Cruz, Biotech, USA) for

180

chemiluminescence detection. Proteins were visualized using a Bio-Imaging analyzer

181

(LAS-3000, Japan). Band intensity was quantified with ImageQuant TL software (GE

182

Healthcare, UK).

183

Statistical Analysis

184

Data were analyzed with SPSS 18.0 software (SPSS, USA). Experimental results

185

were expressed as mean ± SEM. Statistical significance was tested with one-way

186

ANOVA, followed by Duncan’s multiple range test. P-values < 0.05 were considered

187

statistically significant.

188

Results

189

Cytotoxicity of Various AFB1 Concentrations to Chicken Hepatocytes

190

As shown in Fig. 1, treatment with DMSO, which was used as the AFB1 solvent, 9



191

did not significantly change cell viability and LDH activities compared with the

192

control treatment (P > 0.05). The toxic effects of 0–1.6 µg/mL AFB1 on the cell

193

viability (Fig. 1A) and LDH activities (Fig. 1B) of chicken hepatocytes were

194

concentration-dependent. Treatment with 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 µg/mL AFB1

195

significantly decreased cell viability compared with DMSO treatment (P < 0.05). The

196

changes in LDH activities caused by AFB1 treatment were opposite to the changes in

197

cell viability. Treatment with 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 µg/mL AFB1

198

significantly increased LDH activities compared with DMSO treatment (P < 0.05).

199

LDH activities plateaued with 0.2–1.6 µg/mL AFB1 treatment. Our data revealed that

200

0.05 µg/mL or higher concentrations of AFB1 had significant cytotoxic effects on cell

201

viability and LDH activities in chicken hepatocytes. Therefore, 0.05 µg/mL AFB1 was

202

used in the subsequent experiments.

203

Protective Effects of SeMet Against AFB1-induced Toxicity in Chicken Hepatocytes

204

Various concentrations of SeMet (0, 0.5, 1, 2, and 4 µM) were used to evaluate

205

the protective effect of SeMet against AFB1 toxicity. Compared with the control

206

group, the cell viability in the groups without AFB1 treatment was significant and was

207

promoted by 12% and 13% when SeMet was supplemented at concentrations of 1 and

208

2 µM, respectively. The group that was treated with 0.05 µg/mL AFB1 had

209

significantly reduced cell viability compared with the control group; however, these

210

reductions were lower when 0.5, 1, 2, and 4 µM SeMet concentrations were

211

supplemented (Fig. 2A). As shown in Fig. 2B, supplementation with 0.05 µg/mL

212

AFB1 increased LDH activity by 35% compared with the control treatment. However, 10


Page 10 of 34

Page 11 of 34


213

LDH activities increased by only 32%, 27%, 25%, and 13% when SeMet was

214

supplemented at concentrations of 0.5, 1, 2, and 4 µM, respectively, compared with

215

the control treatment (P < 0.05). Our data revealed that SeMet has protective effects

216

against AFB1-induced toxicity in chicken hepatocytes.

217

Effect of SeMet on GSH, MDA, and SOD Levels in AFB1-treated Chicken

218

Hepatocytes

219

As shown in Fig. 3A, SeMet treatment significantly enhanced intracellular GSH

220

concentrations compared with the control treatment (P < 0.05). The maximum GSH

221

level was obtained with 2 µM SeMet. Hepatocytes that were treated with 0.05 µg/mL

222

AFB1 alone showed lower GSH levels than the control group that did not receive

223

AFB1 and SeMet treatment (P < 0.05). Pretreatment with various SeMet

224

concentrations significantly increased GSH levels in AFB1-treated chicken

225

hepatocytes recovered compared with the control group that did not receive SeMet

226

treatment (P < 0.05). After treatment with 0.05 µg/mL AFB1, the highest GSH level

227

was observed in hepatocytes that were pretreated with 2 µM SeMet; this level as close

228

to the normal level in the control group that did not receive AFB1 and SeMet

229

treatment.

230

MDA levels in chicken hepatocytes are presented in Fig. 3B. As shown in the

231

figure, MDA levels declined when hepatocytes were treated with various

232

concentrations of SeMet alone; however, only the MDA level of the group that was

233

treated with 2 µM SeMet significantly decreased compared with that of the control

234

group (P < 0.05). The MDA level in the hepatocytes that were treated with 0.05 11



235

µg/mL AFB1 alone significantly increased compared with that in the untreated

236

hepatocytes (P < 0.05). Pretreatment with 1 µM SeMet reduced MDA levels that were

237

increased in AFB1-treated chicken hepatocytes. Pretreatment with 2 µM SeMet

238

caused the highest reduction of MDA levels in AFB1-treated chicken hepatocytes.

239

SOD levels in chicken hepatocytes are presented in Fig. 3C. SOD levels in

240

chicken hepatocytes that were treated with 1 µM SeMet significantly increased

241

compared with that in control cells that were not treated with SeMet (P < 0.05).

242

Treatment with 2 µM SeMet maximized SOD levels. Treatment with AFB1 alone

243

significantly reduced SOD levels in chicken hepatocytes compared with in chicken

244

hepatocytes that did not receive AFB1 treatment (P < 0.05). Furthermore, SOD levels

245

significantly increased in AFB1-treated chicken hepatocytes that were pretreated with

246

1 µM SeMet compared with those that were treated with AFB1 alone (P < 0.05).

247

SeMet Increases the mRNA Level of SelW in Primary Chicken Hepatocytes with or

248

without AFB1 Treatment

249

The effects of SeMet (0, 0.5, 1, 2, and 4 µM) on SelW mRNA levels were

250

determined to further investigate the mechanism responsible for the protective role of

251

SeMet in AFB1-damaged primary chicken hepatocytes. As shown in Fig. 4,

252

quantitative RT-PCR analysis revealed that compared with the control treatment,

253

supplementation with 0.5, 1, 2, and 4 µM SeMet significantly increased SelW mRNA

254

levels in primary chicken hepatocytes without AFB1 treatment (P < 0.05). Compared

255

with the control treatment, supplementation with 1, 2, and 4 µM SeMet significantly

256

increased SelW mRNA levels in AFB1-treated primary chicken hepatocytes (P < 12


Page 12 of 34

Page 13 of 34


257

0.05). These data suggested that SeMet supplementation decreases AFB1-induced

258

damages in primary chicken hepatocytes by improving SelW expression.

259

SeMet Reduces the mRNA Levels of CYP450 1A5 but not of 3A37 in Primary Chicken

260

Hepatocytes with or without AFB1 treatment

261

The relative mRNA levels of CYP450 1A5 and 3A37 in each group were

262

quantified and are shown in Fig. 5. CYP450 1A5 mRNA levels in hepatocytes that

263

were treated with 1 and 2 µM SeMet alone were significantly lower than those in

264

hepatocytes that were treated with 0 µM SeMet (P < 0.05). Compared with the control

265

treatment, treatment with 0.05 µg/mL AFB1 significantly increased CYP450 1A5

266

mRNA levels in hepatocytes (P < 0.05). Treatment with various SeMet concentrations

267

(P < 0.05) reduced CYP450 1A5 mRNA levels that were increased by treatment with

268

0.05 µg/mL AFB1. In addition, the lowest CYP450 1A5 mRNA levels were observed

269

in the hepatocytes that were treated with 2 µM SeMet in the presence or absence of

270

AFB1. However, SeMet and AFB1 treatment did not significantly influence CYP450

271

3A37 mRNA levels in hepatocytes (P > 0.05) (Fig. 5B).

272

Effect of SelW Knockdown on SelW mRNA Levels, Cell Viability, and LDH Activity in

273

Primary Chicken Hepatocytes

274

SelW mRNA levels were determined after hepatocytes were transfected with

275

either SelW-specific or control siRNA (Fig. 6A). As shown in Fig. 6A, treatment with

276

SelW-specific siRNA significantly decreased SelW mRNA levels in chicken

277

hepatocytes compared with the control treatment at 24, 48, and 72 h after transfection

13



278

(P < 0.05). SeMet supplementation significantly increased SelW mRNA levels in

279

untransfected hepatocytes and control-siRNA transfected hepatocytes compared with

280

the control group (P < 0.05). However, SeMet supplementation did not influence

281

SelW mRNA levels in hepatocytes that were transfected with SelW-specific siRNA at

282

48 h after transfection.

283

As shown in Fig. 6B, compared with the control treatment, AFB1 treatment

284

significantly reduced the cell viabilities of groups without siRNA treatment or with

285

control-siRNA treatment (P < 0.05). By contrast, SeMet treatment protected primary

286

chicken hepatocytes against AFB1-induced damage by maintaining cell viability at

287

levels similar to those of the controls. SeMet treatment significantly increased

288

AFB1-reduced cell viabilities in the groups with SelW-specific siRNA treatment (P
0.05).

298

SelW Knockdown Influences the Effects of SeMet on CYP450 1A5 in Primary Chicken

299

Hepatocytes with or without AFB1 treatment 14


Page 14 of 34

Page 15 of 34


300

To investigate the mechanism that changed CYP450 1A5 mRNA levels in

301

response to SeMet supplementation in AFB1-induced hepatocytes, the effects of

302

SeMet supplementation on CYP450 1A5 was evaluated in SelW-knockdown

303

hepatocytes. As shown in Figs. 7A and 7B, compared with relative controls,

304

supplementation with 2 µM SeMet significantly inhibited the increase in the mRNA

305

and protein levels of CYP450 1A5 in untransfected or control-siRNA transfected

306

hepatocytes (P < 0.05) but not in the hepatocytes that were transfected with

307

SelW-specific siRNA (P > 0.05).

308

Discussion

309

The potential protective effects of SeMet on AFB1-induced damages in primary

310

chicken hepatocytes were evaluated in the present study. Results showed that AFB1

311

significantly reduced cell viability, GSH, and SOD levels but increased MDA level

312

and LDH activity. These results indicated that AFB1 causes oxidative stress in

313

chicken hepatocytes. These results are in agreement with those of a previous in vitro

314

study, which reported that AFB1 treatment significantly increased oxidative stress in

315

MDCK cells, as evidenced by the decreased cell viability, GSH level and activity, and

316

GPX1 mRNA levels and increased MDA levels of AFB1-treated MDCK cells38.

317

Moreover, SeMet treatment significantly alleviated AFB1-induced oxidative stress in

318

primary chicken hepatocytes.

319

AFB1 is bioactivated to a toxic intermediate that causes genotoxicity by reacting

320

with DNA and that causes cytotoxicity by interacting with proteins13, 14. Previous

15



321

studies established that two important CYP450 isoforms, CYP450 1A5 and 3A37,

322

which are orthologs of human P450 1A2 and 3A4, respectively, bioactivate AFB1 in

323

the turkey liver18, 19, 21. Furthermore, CYP450 1A5 is the dominant enzyme in the

324

bioactivation and metabolism of AFB1 in turkey liver that was treated with AFB1 at

325

environmentally relevant concentrations22. The present findings for primary chicken

326

hepatocytes were partially consistent with those of a previous study on turkeys. Our

327

results revealed that treatment with 0.05 µg/mL AFB1 significantly increases CYP450

328

1A5 mRNA levels but does not influence CYP450 3A37 mRNA levels. Our results

329

implicated CYP450 1A5, instead of CYP450 3A37, in AFB1 metabolism in primary

330

chicken hepatocytes that were exposed to 0.05 µg/mL AFB1.

331

Se is an essential micronutrient for animals and humans and has an important

332

role in antioxidant defense systems25. Se supplementation against aflatoxins is a

333

common practice and has been applied in animal feeds for many years30. Se improves

334

antioxidant activities by enhancing GSH levels39, as observed in the present study.

335

SeMet, an organic form of Se, is more bioavailable than the inorganic forms of Se,

336

such as Na2SeO3. SeMet pretreatment increased cell viability and GSH and SOD

337

levels, as well as reduced MDA levels and LDH activities, in AFB1-treated primary

338

chicken hepatocytes. These findings suggested that SeMet alleviates AFB1-induced

339

oxidative stress in primary chicken hepatocytes. CYP450 1A5 mRNA levels in

340

chicken hepatocytes that were treated with AFB1 and SeMet were significantly lower

341

than those in chicken hepatocytes that were treated with AFB1 only. This finding

342

indicated that SeMet protects chicken hepatocytes against AFB1-induced damages by

16


Page 16 of 34

Page 17 of 34


343

suppressing the expression of CYP450 1A5 mRNA and then reducing the levels of

344

AFB1, which is bioactivated into the toxic exo-AFBO by CYP450 1A5.

345

Se, an essential micronutrient with major metabolic significance, is incorporated

346

as selenocysteine at the active sites of numerous proteins40. SelW, the smallest

347

identified selenoprotein, regulates cellular redox41, 42, cell cycle progression, and cell

348

proliferation43-46. SelW is essential for the maintenance of normal liver function, and

349

the expression of SelW in the liver depends on the level of dietary Se47, 48. In this

350

study, SeMet treatment suppressed the mRNA levels of CYP450 1A5, a

351

phaseⅠdrug-metabolizing enzyme that activates AFB1 to toxic AFBO in the liver18, 19,

352

21, 49

353

primary chicken hepatocytes with or without AFB1 treatment (P < 0.05). Chicken

354

hepatocytes were cultured overnight and then transfected with SelW-specific or

355

control siRNA to investigate the suppressive effect of SelW on AFB1-induced

356

CYP450 1A5 expression in chicken hepatocytes. As shown in Fig. 7 (A and B),

357

SeMet treatment significantly decreased CYP450 1A5 expression in primary chicken

358

hepatocytes that were transfected with control siRNA, but did not decrease CYP450

359

1A5 expression in primary chicken hepatocytes that were transfected with

360

SelW-specific siRNA. These findings revealed that SeMet protects primary chicken

361

hepatocytes from AFB1-induced damage by inhibiting CYP450 1A5 expression to

362

reduce AFBO production and then reduce the combination between liver DNA and

363

RNA. Our data offer more insights than a previous in vivo study that focused on the

364

prevention of aflatoxin b1 hepatoxicity by dietary Se, which is associated with the

. In addition, SeMet treatment significantly increased the mRNA level of SelW in

17



365

inhibition of cytochrome p450 isozymes in chicken liver 11.

366

Some studies have reported that the increase in GSH levels via selenoproteins is

367

responsible for the protective effects of Se against AFB1 toxicity38, 50. Some studies

368

have also shown that Se directly regulates Cytochrome P450, the key enzyme in the

369

bio-activation of AFB1. Se-enriched polysaccharides exert anti-mutagenic effects in

370

the mouse liver by suppressing the Cytochrome P450 1A subfamily51. The potential

371

and selective inhibition of CYP1A1 by SeCys conjugates contributes to their

372

chemopreventive activity52. Four chemopreventive organoselenium compounds have

373

spectral modification and catalytic inhibition activities on human cytochrome P45053.

374

The protective effect of dietary Se on AFB1-induced hepatoxicity in chickens is

375

associated with the inhibition of cytochrome p450 isozymes11. We found that SeMet

376

enhanced the anti-oxidative status of AFB1-treated primary chicken hepatocytes by

377

increasing GSH levels and SOD concentration and by reducing MDA (Fig. 3).

378

Moreover, we found that SeMet inhibited CYP450 1A5 expression by improving

379

SelW expression. Given that cytochrome P450 is the key enzyme for the bioactivation

380

of AFB1 into the reactive epoxide intermediate AFBO54, 55, we concluded that SeMet

381

alleviates AFB1-induced damages in primary chicken hepatocytes by improving SelW

382

expression, thus inhibiting CYP450 1A5 expression.

383

In summary, supplementation with 2 µM SeMet protects primary chicken

384

hepatocytes from AFB1-induced damage. SeMet supplementation maintains cell

385

viability, GSH activity, SOD levels, LDH levels, and MDA levels by increasing SelW

386

mRNA levels and decreasing CYP450 1A5 mRNA levels. Knockdown of SelW with

18


Page 18 of 34

Page 19 of 34


387

SelW-specific siRNA significantly increases the mRNA and protein levels of CYP450

388

1A5 (P < 0.05), thus indicating that SeMet alleviates AFB1-induced damage in

389

primary chicken hepatocytes by improving SelW expression and inhibiting CYP450

390

1A5 expression. Further research is still required on the protective mechanism of

391

SelW against AFB1-induced cellular damage.

392

Acknowledgment

393

This work was funded by the National Natural Science Foundation of China (NFSC)

394

(31472253, 31472252), the Fundamental Research Funds for the Central Universities

395

(Y0201500198) and the Priority Academic Program Development of Jiangsu Higher

396

Education Institutions (Jiangsu, China).

397

References

398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415

(1) Bunger, J.; Westphal, G.; Monnich, A.; Hinnendahl, B.; Hallier, E.; Muller, M. Cytotoxicity of occupationally and environmentally relevant mycotoxins. Toxicology 2004, 202 (3), 199-211. (2) Marin, D. E.; Taranu, I. Overview on aflatoxins and oxidative stress. Toxin Reviews 2012, 31 (3-4), 32-43. (3) Marin, D.; Taranu, I.; Bunaciu, R.; Pascale, F.; Tudor, D.; Avram, N.; Sarca, M.; Cureu, I.; Criste, R.; Suta, V. Changes in performance, blood parameters, humoral and cellular immune responses in weanling piglets exposed to low doses of aflatoxin. Journal of animal science 2002, 80 (5), 1250-1257. (4) Hao, S.; Hu, J.; Song, S.; Huang, D.; Xu, H.; Qian, G.; Gan, F.; Huang, K. Selenium Alleviates Aflatoxin B(1)-Induced Immune Toxicity through Improving Glutathione Peroxidase 1 and Selenoprotein S Expression in Primary Porcine Splenocytes. J Agric Food Chem 2016, 64 (6), 1385-93. (5) JECFA, Joint FAO/WHO Expert Committee on Food Additives.Safety evaluation of certain food additives and contaminants. In World Health Organization.Geneva: 1998. (6) Dalvi, R. R. An overview of aflatoxicosis of poultry: its characteristics, prevention and reduction. Vet Res Commun 1986, 10 (6), 429-43. (7) Rawal, S.; Kim, J. E.; Coulombe, R., Jr. Aflatoxin B1 in poultry: toxicology, metabolism and prevention. Res Vet Sci 2010, 89 (3), 325-31. (8) Bennett

JW; Klich

M. Mycotoxins. Clin

Microbiol

Rev 2003, 16 (3), 497-516.

(9) Kensler, T. W.; Roebuck, B. D.; Wogan, G. N.; Groopman, J. D. Aflatoxin: a 50-year odyssey of

19



416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459

mechanistic and translational toxicology. Toxicol Sci 2011, 120 Suppl 1, S28-48. (10) Hussain, Z.; Rehman, H. U.; Manzoor, S.; Tahir, S.; Mukhtar, M. Determination of liver and muscle aflatoxin B1 residues and select serum chemistry variables during chronic aflatoxicosis in broiler chickens. Vet Clin Pathol 2016, 45 (2), 330-4. (11) Sun, L. H.; Zhang, N. Y.; Zhu, M. K.; Zhao, L.; Zhou, J. C.; Qi, D. S. Prevention of Aflatoxin B1 Hepatoxicity by Dietary Selenium Is Associated with Inhibition of Cytochrome P450 Isozymes and Up-Regulation of 6 Selenoprotein Genes in Chick Liver. J Nutr 2016. (12) Liang, N.; Wang, F.; Peng, X.; Fang, J.; Cui, H.; Chen, Z.; Lai, W.; Zhou, Y.; Geng, Y. Effect of Sodium Selenite on Pathological Changes and Renal Functions in Broilers Fed a Diet Containing Aflatoxin B(1). Int J Environ Res Public Health 2015, 12 (9), 11196-208. (13) Doi, A. M.; Patterson, P. E.; Gallagher, E. P. Variability in aflatoxin B(1)-macromolecular binding and relationship to biotransformation enzyme expression in human prenatal and adult liver. Toxicol Appl Pharmacol 2002, 181 (1), 48-59. (14) Cervino, C.; Knopp, D.; Weller, M. G.; Niessner, R. Novel Aflatoxin Derivatives and Protein Conjugates. Molecules 2007, 12 (3), 641-653. (15) Shi, D.; Liao, S.; Guo, S.; Li, H.; Yang, M.; Tang, Z. Protective effects of selenium on aflatoxin B1-induced mitochondrial permeability transition, DNA damage, and histological alterations in duckling liver. Biol Trace Elem Res 2015, 163 (1-2), 162-8. (16) Verma, R. J. Aflatoxin causes DNA damage. Int. J. Hum. Genet. 2004, 4 (4), 231-236. (17) Do, J.; Choi, D.-K. Aflatoxins: Detection, toxicity, and biosynthesis. Biotechnol. Bioprocess Eng. 2007, 12 (6), 585-593. (18) Yip, S. S.; Coulombe, R. A., Jr. Molecular cloning and expression of a novel cytochrome p450 from turkey liver with aflatoxin b1 oxidizing activity. Chem Res Toxicol 2006, 19 (1), 30-7. (19) Rawal, S.; Yip, S. S.; Coulombe, R. A., Jr. Cloning, expression and functional characterization of cytochrome P450 3A37 from turkey liver with high aflatoxin B1 epoxidation activity. Chem Res Toxicol 2010, 23 (8), 1322-9. (20) Rawal, S.; Mendoza, K. M.; Reed, K. M.; Coulombe, R. A., Jr. Structure, genetic mapping, and function of the cytochrome P450 3A37 gene in the turkey (Meleagris gallopavo). Cytogenet Genome Res 2009, 125 (1), 67-73. (21) Klein, P. J.; Buckner, R.; Kelly, J.; Coulombe, R. A., Jr. Biochemical basis for the extreme sensitivity of turkeys to aflatoxin B(1). Toxicol Appl Pharmacol 2000, 165 (1), 45-52. (22) Rawal, S.; Coulombe, R. A., Jr. Metabolism of aflatoxin B1 in turkey liver microsomes: the relative roles of cytochromes P450 1A5 and 3A37. Toxicol Appl Pharmacol 2011, 254 (3), 349-54. (23) Eaton, D. L.; Gallagher, E. P. Mechanisms of aflatoxin carcinogenesis. Ann. Rev. Pharmacol. Toxicol. 1994, 34, 135-172. (24) Arthur, J. R.; Beckett, G. J. New metabolic roles for selenium. Proc. Nutr. Soc. 1994, 53 (3), 615-624. (25) Soudani, N.; Ben Amara, I.; Sefi, M.; Boudawara, T.; Zeghal, N. Effects of selenium on chromium (VI)-induced hepatotoxicity in adult rats. Exp Toxicol Pathol 2011, 63 (6), 541-8. (26) Jeong, D.; Kim, T. S.; Chung, Y. W.; Lee, B. J.; Kim, I. Y. Selenoprotein W is a glutathione-dependent antioxidant in vivo. FEBS Lett 2002, 517 (1-3), 225-8. (27) Xiao-Long, W.; Chuan-Ping, Y.; Kai, X.; Ou-Jv, Q. Selenoprotein W depletion in vitro might indicate that its main function is not as an antioxidative enzyme. Biochemistry (Mosc) 2010, 75 (2), 201-7.

20


Page 20 of 34

Page 21 of 34


460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503

(28) Griffin, A. C. Role of selenium in the chemoprevention of cancer. Adv Cancer Res 1979, 29, 419-42. (29) Chen, J.; Goetchius, M. P.; Campbell, T. C.; Combs, G. F., Jr. Effects of dietary selenium and vitamin E on hepatic mixed-function oxidase activities and in vivo covalent binding of aflatoxin B1 in rats. J Nutr 1982, 112 (2), 324-31. (30) Davila, J. C.; Edds, G. T.; Osuna, O.; Simpson, C. F. Modification of the effects of aflatoxin B1 and warfarin in young pigs given selenium. Am J Vet Res 1983, 44 (10), 1877-83. (31) Yu, Z.; Wang, F.; Liang, N.; Wang, C.; Peng, X.; Fang, J.; Cui, H.; Jameel Mughal, M.; Lai, W. Effect of Selenium Supplementation on Apoptosis and Cell Cycle Blockage of Renal Cells in Broilers Fed a Diet Containing Aflatoxin B1. Biol Trace Elem Res 2015, 168 (1), 242-51. (32) Chen, J.; Goetchius, M. P.; Combs, G. F.; Campbell, T. C. Effects of dietary selenium and vitamin E on covalent binding of aflatoxin to chick

liver cell macromolecules. J. Nutr 1982, 112, 350-357.

(33) Fujii, M.; Yoshino, I.; Suzuki, M.; Higuchi, T.; Mukai, S.; Aoki, T.; Fukunaga, T.; Sugimoto, Y.; Inoue, Y.; Kusuda, J.; Saheki, T.; Sato, M.; Hayashi, S.; Tamaki, M.; Sugano, T. Primary culture of chicken hepatocytes in serum-free medium (pH 7.8) secreted albumin and transferrin for a long period in free gas exchange with atmosphere. Int. J. Biochem. Cell Biol. 1996, 28 (12), 1381-1391. (34) Wu, X.; Huang, K.; Wei, C.; Chen, F.; Pan, C. Regulation of cellular glutathione peroxidase by different forms and concentrations of selenium in primary cultured bovine hepatocytes. J Nutr Biochem 2010, 21 (2), 153-61. (35) Wu, X.; Wei, C.; Pan, C.; Duan, Y.; Huang, K. Regulation of expression and activity of selenoenzymes by different forms and concentrations of selenium in primary cultured chicken hepatocytes. Br J Nutr 2010, 104 (11), 1605-12. (36) Rahman, I.; Kode, A.; Biswas, S. K. Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat Protoc 2006, 1 (6), 3159-65. (37) Gavino, V. C.; Miller, J. S.; Ikharebha, S. O.; Milo, G. E.; Cornwell, D. G. Effect of polyunsaturated fatty acids and antioxidants on lipid peroxidation in tissue cultures. J Lipid Res 1981, 22 (5), 763-9. (38) Parveen, F.; Nizamani, Z. A.; Gan, F.; Chen, X.; Shi, X.; Kumbhar, S.; Zeb, A.; Huang, K. Protective effect of selenomethionine on aflatoxin B1-induced oxidative stress in MDCK cells. Biol Trace Elem Res 2014, 157 (3), 266-74. (39) Kim, S. S.; Koo, J. H.; Kwon, I. S.; Oh, Y. S.; Lee, S. J.; Kim, E. J.; Kim, W. K.; Lee, J.; Cho, J. Y. Exercise training and selenium or a combined treatment ameliorates aberrant expression of glucose and lactate metabolic proteins in skeletal muscle in a rodent model of diabetes. Nutr Res Pract 2011, 5 (3), 205-13. (40) Papp, L. V.; Holmgren, A.; Khanna, K. K. Selenium and selenoproteins in health and disease. Antioxid Redox Signal 2010, 12 (7), 793-5. (41) Dikiy, A.; Novoselov, S. V.; Fomenko, D. E.; Sengupta, A.; Carlson, B. A.; Cerny, R. L.; Ginalski, K.; Grishin, N. V.; Hatfield, D. L.; Gladyshev, V. N. SelT, SelW, SelH, and Rdx12: genomics and molecular insights into the functions of selenoproteins of a novel thioredoxin-like family. Biochemistry 2007, 46 (23), 6871-82. (42) Jeon, Y. H.; Ko, K. Y.; Lee, J. H.; Park, K. J.; Jang, J. K.; Kim, I. Y. Identification of a redox-modulatory interaction between selenoprotein W and 14-3-3 protein. Biochim Biophys Acta 2016, 1863 (1), 10-8. (43) Hawkes, W. C.; Printsev, I.; Alkan, Z. Selenoprotein W depletion induces a p53- and

21



504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538

p21-dependent delay in cell cycle progression in RWPE-1 prostate epithelial cells. J Cell Biochem 2012, 113 (1), 61-9. (44) Hawkes, W. C.; Alkan, Z. Delayed cell cycle progression in selenoprotein W-depleted cells is regulated by a mitogen-activated protein kinase kinase 4-p38/c-Jun NH2-terminal kinase-p53 pathway. J Biol Chem 2012, 287 (33), 27371-9. (45) Noh, O. J.; Park, Y. H.; Chung, Y. W.; Kim, I. Y. Transcriptional regulation of selenoprotein W by MyoD during early skeletal muscle differentiation. J Biol Chem 2010, 285 (52), 40496-507. (46) Hawkes, W. C.; Wang, T. T.; Alkan, Z.; Richter, B. D.; Dawson, K. Selenoprotein W modulates control of cell cycle entry. Biol Trace Elem Res 2009, 131 (3), 229-44. (47) Sun, B.; Wang, R.; Li, J.; Jiang, Z.; Xu, S. Dietary selenium affects selenoprotein W gene expression in the liver of chicken. Biol Trace Elem Res 2011, 143 (3), 1516-23. (48) Li, J. L.; Ruan, H. F.; Li, H. X.; Li, S.; Xu, S. W.; Tang, Z. X. Molecular cloning, characterization and mRNA expression analysis of a novel selenoprotein: avian selenoprotein W from chicken. Mol Biol Rep 2011, 38 (6), 4015-22. (49) Monroe, D. H.; Eaton, D. L. Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and mouse. Toxicol Appl Pharmacol 1987, 90 (3), 401-9. (50) Shi, D.; Guo, S.; Liao, S.; Su, R.; Pan, J.; Lin, Y.; Tang, Z. Influence of selenium on hepatic mitochondrial antioxidant capacity in ducklings intoxicated with aflatoxin B(1). Biol Trace Elem Res 2012, 145 (3), 325-9. (51) Peng, F.; Guo, X.; Li, Z.; Li, C.; Wang, C.; Lv, W.; Wang, J.; Xiao, F.; Kamal, M. A.; Yuan, C. Antimutagenic Effects of Selenium-Enriched Polysaccharides from Pyracantha fortuneana through Suppression of Cytochrome P450 1A Subfamily in the Mouse Liver. Molecules 2016, 21 (12). (52) Venhorst, J.; Rooseboom, M.; Vermeulen, N. P.; Commandeur, J. N. Studies on the inhibition of human cytochromes P450 by selenocysteine Se-conjugates. Xenobiotica 2003, 33 (1), 57-72. (53) Shimada, T.; Murayama, N.; Tanaka, K.; Takenaka, S.; Guengerich, F. P.; Yamazaki, H.; Komori, M. Spectral modification and catalytic inhibition of human cytochromes P450 1A1, 1A2, 1B1, 2A6, and 2A13 by four chemopreventive organoselenium compounds. Chem Res Toxicol 2011, 24 (8), 1327-37. (54) Diaz, G. J.; Murcia, H. W.; Cepeda, S. M. Cytochrome P450 enzymes involved in the metabolism of aflatoxin B1 in chickens and quail. Poult Sci 2010, 89 (11), 2461-9. (55) Gallagher, E. P.; Kunze, K. L.; Stapleton, P. L.; Eaton, D. L. The kinetics of aflatoxin B1 oxidation by human cDNA-expressed and human liver microsomal cytochromes P450 1A2 and 3A4. Toxicol Appl Pharmacol 1996, 141 (2), 595-606.

539 540 541 542

22


Page 22 of 34

Page 23 of 34


543

Figure captions

544

Fig. 1 Effects of Various Concentration of AFB1 on Cell Viability (A) and LDH

545

Activity (B) in Chicken Hepatocytes.

546

Values are represented as means ± SEM from three experiments (n=3). Groups

547

were compared by one-way ANOVA followed by Duncan’s multiple range tests.

548

Asterisk means values were significantly different from the control (P < 0.05).

549 550

Fig. 2 Protective Effect of SeMet on Cell Viability (A) and LDH Activity (B) in

551

AFB1-treated Chicken Hepatocytes

552


553

were compared by a 1-way ANOVA followed by Duncan’s multiple range tests. Within the

554

groups without AFB1 treatment, bars with * are significantly different from the control cells

555

(P < 0.05). Within the groups with 0.05µg/ml AFB1 treatment, bars with # are significantly

556

different from the control cells (P < 0.05). Significant changes are indicated by & in

557

comparison between treated cells and untreated cells by 0.05µg/ml AFB (P < 0.05).

558

559

Fig. 3 Effect of SeMet on GSH (A), MDA (B) and SOD (C) Levels in AFB1-treated

560

Chicken Hepatocytes

561


562


563


564

(P < 0.05). Within the groups with 0.05µg/ml AFB1 treatment, bars with # are significantly 23



565


566


567 568

Fig. 4 Effect of SeMet on SelW mRNA Levels in AFB1-treated Chicken Hepatocytes.

569


570


571


572


573

different from the control cells (P < 0.05).

574 575

Fig. 5 Effect of SeMet on the mRNA Levels of CYP450 3A37 (A) and 1A5 (B) in

576

AFB1-treated Chicken Hepatocytes.

577


578


579


580


581


582


583 584

Fig. 6 Effect of SelW Knockdown on the mRNA Level of SelW (A), Cell Viability (B)

585

and LDH Activity (C) in Chicken Hepatocytes

586


24


Page 24 of 34

Page 25 of 34


587


588


589


590


591


592 593

Fig. 7 Effect of SelW Knockdown on the mRNA (A) and Protein (B) Levels of CYP450

594

1A5 in Chicken Hepatocytes.

595

Values are given as means ± SEM from three experiments (n=3). Groups were

596

compared by a 1-way ANOVA followed by Duncan’s multiple range tests. Within the

597

groups with same knockdown treatment, bars with * are significantly different from the

598

control cells (P < 0.05). Significant changes are indicated by & in comparison between treated

599

cells and untreated cells by 2µM SeMet (P < 0.05).

600

601

602

603

604

605

25



606

Tables

607

Table 1 Primers used for real-time PCR

Page 26 of 34

Product Genes

GenBank accession

Primer sequence (5′−3′) size (bp) F: TCACCATCCCGCACAGCA

CYP450 NM_205146.1

201

1A5

R: AAGTCATCACCTTCTCCGCATC F: CGAATCCCAGAAATCAGA

CYP450 NM_001001751.1 3A37

145 R: AGCCAGGTAACCAAGTGT F: CTCCGCGTCACCGTGCTC

SelW

GQ919055

150 R: CACCGTCACCTCGAACCATCCC F: TGCGTGACATCAAGGAGAAG

β-actin

NM_205518.1

300 R: TGCCAGGGTACATTGTGGTA

608

609

610

611

612

613

614

26


Page 27 of 34


615

TOC

616

617

618

27



Fig. 1 Effects of Various Concentration of AFB1 on Cell Viability (A) and LDH Activity (B) in Chicken Hepatocytes. Values are represented as means ± SEM from three experiments (n=3). Groups were compared by one-way ANOVA followed by Duncan’s multiple range tests. Asterisk means values were significantly different from the control (P < 0.05).

140x142mm (300 x 300 DPI)


Page 28 of 34

Page 29 of 34


Fig. 2 Protective Effect of SeMet on Cell Viability (A) and LDH Activity (B) in AFB1-treated Chicken Hepatocytes Values are represented as means ± SEM from three experiments (n=3). Groups were compared by a 1-way ANOVA followed by Duncan’s multiple range tests. Within the groups without AFB1 treatment, bars with * are significantly different from the control cells (P < 0.05). Within the groups with 0.05µg/ml AFB1 treatment, bars with # are significantly different from the control cells (P < 0.05). Significant changes are indicated by & in comparison between treated cells and untreated cells by 0.05µg/ml AFB (P < 0.05).

134x157mm (300 x 300 DPI)



Fig. 3 Effect of SeMet on GSH (A), MDA (B) and SOD (C) Levels in AFB1-treated Chicken Hepatocytes Values are represented as means ± SEM from three experiments (n=3). Groups were compared by a 1-way ANOVA followed by Duncan’s multiple range tests. Within the groups without AFB1 treatment, bars with * are significantly different from the control cells (P < 0.05). Within the groups with 0.05µg/ml AFB1 treatment, bars with # are significantly different from the control cells (P < 0.05). Significant changes are indicated by & in comparison between treated cells and untreated cells by 0.05µg/ml AFB (P < 0.05).

140x223mm (300 x 300 DPI)


Page 30 of 34

Page 31 of 34


Fig. 4 Effect of SeMet on SelW mRNA Levels in AFB1-treated Chicken Hepatocytes. Values are represented as means ± SEM from three experiments (n=3). Groups were compared by a 1-way ANOVA followed by Duncan’s multiple range tests. Within the groups without AFB1 treatment, bars with * are significantly different from the control cells (P < 0.05). Within the groups with 0.05µg/ml AFB1 treatment, bars with # are significantly different from the control cells (P < 0.05).

140x71mm (300 x 300 DPI)



Fig. 5 Effect of SeMet on the mRNA Levels of CYP450 3A37 (A) and 1A5 (B) in AFB1-treated Chicken Hepatocytes. Values are represented as means ± SEM from three experiments (n=3). Groups were compared by a 1-way ANOVA followed by Duncan’s multiple range tests. Within the groups without AFB1 treatment, bars with * are significantly different from the control cells (P < 0.05). Within the groups with 0.05µg/ml AFB1 treatment, bars with # are significantly different from the control cells (P < 0.05). Significant changes are indicated by & in comparison between treated cells and untreated cells by 0.05µg/ml AFB (P < 0.05).

140x155mm (300 x 300 DPI)


Page 32 of 34

Page 33 of 34


Fig. 6 Effect of SelW Knockdown on the mRNA Level of SelW (A), Cell Viability (B) and LDH Activity (C) in Chicken Hepatocytes Values are represented as means ± SEM from three experiments (n=3). Groups were compared by a 1-way ANOVA followed by Duncan’s multiple range tests. Within the groups without AFB1 treatment, bars with * are significantly different from the control cells (P < 0.05). Within the groups with 0.05µg/ml AFB1 treatment, bars with # are significantly different from the control cells (P < 0.05). Significant changes are indicated by & in comparison between treated cells and untreated cells by 0.05µg/ml AFB (P < 0.05).

159x229mm (300 x 300 DPI)



Fig. 7 Effect of SelW Knockdown on the mRNA (A) and Protein (B) Levels of CYP450 1A5 in Chicken Hepatocytes. Values are given as means ± SEM from three experiments (n=3). Groups were compared by a 1-way ANOVA followed by Duncan’s multiple range tests. Within the groups with same knockdown treatment, bars with * are significantly different from the control cells (P < 0.05). Significant changes are indicated by & in comparison between treated cells and untreated cells by 2µM SeMet (P < 0.05).

160x203mm (300 x 300 DPI)


Page 34 of 34

Selenomethionine Alleviates AFB1-Induced Damage in Primary

Recommend Documents