Cytotoxicity and DNA Damage Caused from Diazinon Exposure by

Subscriber access provided by YORK UNIV

Agricultural and Environmental Chemistry

Cytotoxicity and DNA damage caused from diazinon exposure by inhibiting the PI3K-AKT pathway in porcine ovarian granulosa cells Wei Wang, Shi-Ming Luo, Jun-Yu Ma, Wei Shen, and Shen Yin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05194 • Publication Date (Web): 11 Dec 2018 Downloaded from http://pubs.acs.org on December 12, 2018

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 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 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.

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 42

Journal of Agricultural and Food Chemistry

1

Cytotoxicity and DNA damage caused from diazinon exposure by inhibiting the

2

PI3K-AKT pathway in porcine ovarian granulosa cells

3

Wei Wang, Shi-Ming Luo, Jun-Yu Ma, Wei Shen, Shen Yin*

4

College of Life Sciences, Institute of Reproductive Sciences, Qingdao Agricultural University,

5

Qingdao 266109, China.

6

*Corresponding author: Shen Yin. E-mail: [email protected]. ORCID: 0000-0003-4792-7885.

7

Running title: Diazinon impairs porcine granulosa cells.

8

1

ACS Paragon Plus Environment


10

Abstract

11

Organophosphorus insecticide diazinon (DZN) is diffusely used in agriculture, home

12

gardening and crop peats. Much work so far has focused on the link between DZN exposure

13

and the occurrence of neurological diseases, while, it is little-known that reproductive

14

toxicological assessment on DZN exposure. This research aimed to investigate the underlying

15

mechanisms of toxic hazard for DZN exposure on the cultured porcine ovarian granulosa cells.

16

We analyzed the oxidative stress, energy metabolism, DNA damage, apoptosis and autophagy

17

by using high-throughput RNA-seq, immunofluorescence, western blotting and real-time PCR.

18

The combined data demonstrated that DZN exposure could cause excessive ROS and DNA

19

damage, which induced apoptosis and autophagy by inhibiting the PI3K-AKT pathway. The

20

down-regulated CYP19A1 protein and granulosa cells death increase the risk for developing

21

the premature ovarian failure and the follicular atresia. In conclusion, DZN exposure has

22

obvious reproductive toxicity by induction of granulosa cell death through pathways

23

connected to DNA damage and oxidative stress by inhibiting the PI3K-AKT pathway.

24

Keywords

25

Diazinon, granulosa cells, PI3K-AKT, RNA-seq, DNA damage

2


Page 2 of 42

Page 3 of 42


27

Introduction

28

Organophosphorothionate pesticides (OPs) are used to destroy insects, arachnids and

29

nematodes, and its overuse can result in various chronic neurological diseases, such as

30

Parkinson and Alzheimer 1, 2. Diazinon (DZN), is a thiophosphoric acid ester and the chemical

31

substance of OPs agrochemicals developed in 1952 3. In crop growth, DZN was detected

32

exceeding the maximum residue limit (MRL, 0.05 mg/kg) more than 11 times 4. Due to

33

historical high use, the persistence of DZN in environment is highly toxic for humans and

34

animals through food chain 5. However, it is unknown that the reproductive toxicity of DZN

35

exposure and the underlying molecular mechanisms.

36

Due to chromosomal remodeling during meiosis, oocyte transcription is inhibited. Oocyte

37

metabolism and development depend on granulosa cells, which are involved in the

38

transcriptional activity, nuclear and cytoplasmic maturation of oocyte, and the local ovarian

39

microenvironment control system6. Anti-proliferation and death of granulosa cells may lead to

40

the premature ovarian failure and follicular atresia 7. Therefore, investigating the effects of

41

DZN on granulosa cells would allow identifying the reproductive toxicity of DZN at

42

increased risk for developing oocyte maturation arrest, premature ovarian failure, and

43

follicular atresia.

44

The expression profile of phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signalling

45

pathway in granulosa cells is a potential marker of oocyte competence and predictive of

46

pregnancy outcome 8. PI3K acts as an phosphatidylinositol kinase and AKT performs the

3



Page 4 of 42

47

function of downstream target via PI3K signaling pathway 9. The diminished activity of the

48

PI3K-AKT pathway also can promote Bax translocation to mitochondria, and releases

49

cytochrome c to trigger apoptosis by caspases pathway. PI3K-AKT regulates cell

50

proliferation, apoptosis and metabolism, but also regulates the primordial-to-primary

51

transition in mammalian follicle development.

52

During the metabolism of DZN, reactive oxygen species (ROS) can be generated

53

(superoxide anion radical, hydrogen peroxide, etc) which are accumulated to induce oxidative

54

stress damage to cells

55

limited extent during oxidative stress

56

maximum threshold, it will result in more organelle damage even cell death 10. Mitochondria

57

have pleiotropic effects of ROS homeostasis and PI3K-AKT pathway. In addition,

58

mitochondria DNA (mtDNA)-encoded genes are highly involved in energy metabolism by

59

oxidative phosphorylation.

60

Mitochondrial dysfunction results in energy imbalance and the cell disorder of co-regulation

61

on apoptosis and autophagy 13. Currently, apoptosis is not the only process connected to the

62

follicular degeneration, which autophagy also takes part in 11. It is paradoxical that the role of

63

autophagy is as a pro-death or pro-survival mechanism to regulate or confront the cytotoxic

64

effect. DNA damage can induce the cell cycle arrest and apoptotic death if the damage is not

65

repaired completely. The DNA damage repair pathway includes the non-homologous end

66

joining (NHEJ) and the homologous recombination (HR) pathway. It is still unclear which

67

repair pathway plays a more important role in granulosa cells.

11.

10.

ROS

Antioxidant enzymes can clear away some excessive ROS to a 12.

However, if excess ROS is beyond the resistant

4


Page 5 of 42


68

In this study, we used porcine granulosa cells to demonstrate the possible molecular

69

mechanisms of reproductive toxicity of DZN. Our results revealed that DZN could induce

70

DNA damage to disrupt the genome stability and impair the cell growth, which is connected

71

to ROS accumulation, apoptosis and autophagy by inhibiting the PI3K-AKT pathway activity.

72 73

Materials and Methods

74

Primary porcine ovarian granulosa cell isolation and culture

75

The gathering of ovaries from prepubertal gilts was from Qingdao Wanfu Group Co., Ltd

76

(Qingdao, China). We transported the ovaires to the laboratory with a bottle filled with saline

77

solution at 35-37 ℃ in 2 h. By using a 20 ml syringe, porcine granulosa cells were collected

78

aseptically from the antral follicles (diameter 4 mm). Centrifuged at 300 g, washed gently by

79

PBS, then the cells were cultured with DMEM high glucose medium (HyClone, SH30022.01,

80

South Logan, Utah, USA), containing 10% (v/v) FBS (PAN-Biotech, P30-3302, Adenbach,

81

Germany), 100 ug/ml streptomycin and 100 units/ml penicillin, at 37 ℃ in a humidified

82

incubator with 5% CO214 .

83

Drug treatments

84

DZN was purchased from J&K Chemical Ltd (930593, Shanghai, China), and dissolved in

85

dimethyl sulfoxide (DMSO) at 100 mM stock concentration. The cells were cultured into

86

6-well plates at a density of 1×105, or 96-well plates at a density of 1×104 cells per well,

87

respectively. Cultured for 48 h, the granulosa cells were exposed to different concentrations

88

of DZN for further assays. The final concentration of DMSO is no more than 0.1 % v/v. 5



89

Cell toxicity assay

90

Cell Counting Kit-8 (CCK8) kit (Beyotime, C0039, Shanghai, China) was served to monitor

91

the cell growth with DZN exposure. Briefly, in 96-well plate, after 48 h of DZN treatment (0

92

control, 20, 40, 60, 80, 120 and 140 μM group), the culture medium was removed, then 90 μl

93

serum-free DMEM medium with 10 μl WST-8 mixture was added per well, followed by

94

incubation at 37 ℃ for 2 h. Finally, the plates were read in a reader (Bio-Rad, iMarkTM, USA)

95

at the wavelength of 450 nm. After calibration for background, relative cell viability was

96

showed as the formazan formation in DZN-exposed groups compared with the control group.

97

Six parallel experimental groups in each sample were used to assess the cell viability 14.

98

Reactive Oxygen Species detection

99

We used Reactive Oxygen Species Assay Kit (Beyotime, S0033, Shanghai, China) to monitor

100

ROS of granulosa cells. In 6-well plate, after 48 h culture with DZN (0, 20, 40, and 60 μM)

101

treatment, 10 μM DCFH-DA probe from the kit were incubated for 30 min at 37 ℃, and

102

washed 3 times with medium without serum for observation. The detection of fluorescence

103

signals were under the same settings for the analysis of fluorescence intensity14.

104

Apoptosis detection assay

105

Annexin V-FITC kit (Beyotime, C1063, Shanghai, China) was used for apoptosis assay. In

106

6-well plate, after 48 h DZN treatment, 5 μl Annexin V-FITC was mixed along with 5 μl PI.

107

Then the 400 μl of binding buffer was incubated for 15 min at 25 ℃ in the dark box.

108

Detection of fluorescence signals were under the fluorescence microscope (BX51, Olympus,

109

Japan) 15. 6


Page 6 of 42

Page 7 of 42


110

Fluorescence intensity analysis

111

All the photos of control and treated granulosa cells were from the same slide and scanning

112

settings. The region of interest (ROI) was defined to detect the mean value of fluorescence

113

intensity in 5 different regions by ImageJ software (v.1.47, USA). The final mean ROI value

114

from the control and DZN-exposed groups was used for the statistical analysis 14.

115

RNA-seq analysis and Quantitative real-time PCR

116

Total RNA from cells was extracted by using TRIzol® (Invitrogen, 15596026, Waltham,

117

Massachusetts, USA). RNA-seq transcriptome library was built by TruSeqTM RNA sample

118

Kit from Illumina (San Diego, CA) from 5 μg of total RNA. RNA-seq sequencing library was

119

acquired with the Illumina HiSeq 4000 (2 × 150 bp read length). Three biological replicates

120

from each sample were used for all the RNA-seq experiments. The RAW data uploaded at

121

NCBI under Sequence Read Archive (SRA accession: PRJNA505699, Release date:

122

2018-12-20). Clean reads were individually aligned to reference genome with orientation

123

mode by TopHat software (http://tophat.cbcb.umd.edu/, version2.0.0) 16. For the analysis of

124

differential expression, we used the EdgeR (Empirical analysis of Digital Gene Expression in

125

R,

126

Bioconductor/clusterProﬁler

127

including Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG),

128

which were used to identify those differential expression genes (DEGs) raised significantly in

129

GO terms and KEGG pathways at Bonferroni-corrected P-value ≤ 0.05 in comparison with

130

the whole-transcriptome background.

http://www.bioconductor.org/packages/2.12/bioc/html/edgeR.html) 18

17.

The

R

was applied for the analysis of functional-enrichment

7



Page 8 of 42

131

Real-time PCR was performed on ABI 7500 (Life Tech, USA). The reaction conditions were：

132

95 ℃ for 10 min, followed by 40 cycles of 95 ℃ for 15 s, 60 ℃ for 30 s, and 72 ℃ for 20 s.

133

GAPDH was used as the loading control, to which the relative gene expression was analyzed

134

based on the 2-△△Ct method 19. The primers are in the Supplementary Table S1.

135

Western blotting

136

Standard methods of western blotting were performed for the protein expression detection.

137

Briefly, granulosa cells were lysed in RIPA buffer containing the protease inhibitor cocktail

138

(Beyotime, P0013B, Shanghai, China). The samples were separated by SDS-PAGE and

139

transferred into PVDF membranes. Blocked with 5 % BSA for 1 h, incubated with the

140

primary antibodies at 4 ℃ overnight, washed 3 times, then membranes were incubated with

141

the appropriate secondary antibodies at room temperature for 1 h. The primary antibodies

142

include: D series antibodies from Sangon Biotech, BBI, Shanghai, China, Anti-CAT

143

(D122036),

144

(Phospho-Ser380/Thr382/Thr383) (D155023), Anti-AKT1(Phospho-Ser129) (D151416),

145

Anti-TP53 (D120082), Anti-NRAS (D261977), Anti-CYP19A1 (D260102), Anti-PIK3R1

146

(phospho-Tyr467) (BIOSS Antibodies Co., Ltd, bs-5571R, Beijing, China), Anti-GAPDH

147

(Santa Cruz Biotechnology, sc-47724, Santa Cruz, California, USA). The secondary

148

antibodies include HRP-conjugated goat anti-mouse lgG (1:2000, Beyotime, A0216,

149

Shanghai, China), and HRP - conjugated goat anti-rabbit lgG (1:2000, Sangon Biotech, BBI,

150

D110058, Shanghai, China) in TBST. We used GAPDH as the loading control. The relative

151

protein expression was analyzed by IPWIN software (USA) 20.

Anti-BECN1

(D160120),

Anti-BAX

8


(D120073),

Anti-PTEN

Page 9 of 42


152

Statistical analysis methods

153

For each set of experiment, at least three replicates were performed. All the data were

154

represented as mean ± SD. Comparisons among groups were statistically analyzed by

155

Student’s t-test via Graph-Pad Prism (San Diego, CA, USA). Differences were statistically

156

significant at P < 0.05 (*) or highly significant at P < 0.01 (**).

157

Results

158

Effects of DZN exposure on the morphology, proliferation and transcriptome RNA

159

analysis in porcine granulosa cells

160

The schematic diagram of the experimental design is in Fig. 1A. Firstly, we detected the

161

effects of DZN on the morphology and cell growth. The results showed that granulosa cells

162

shrunken significantly with the gradient concentration increasing (Fig. 1B). CCK8 assays

163

indicated the relative cell viability decreasing significantly when the DZN exposure

164

concentration increasing (Fig. 1C, D). All these results suggested that DZN has significant

165

toxic hazard on the proliferation of granulosa cells in a concentration-dependent pattern.

166

Based on these results we chose the 20 and 40 μM DZN treatment for further studies, which

167

decreased the relative cell viability not exceeding 50% in comparison with the control.

168

For transcriptome analysis, porcine granulosa cells subsets will be referred to as CON (the 0

169

μM) and DZN (the 40 μM) to denote distinction from other sorting schemes. Transcriptome

170

analysis yielded 21,077 unigenes. The overall situation of the gene and COG, GO and KEGG

171

database was compared. In the functional classification bar chart (Fig. 1E). The results

172

showed the functional annotation by COG (19003, 75.05%), GO (18546, 60.64%), and 9



173

KEGG (8748, 28.6%) (Fig. 1F). Also, 8748 unigenes were involved in KEGG (Fig. 1G),

174

19003 unigenes were assigned for COG functional categories (Fig. 1H), and 18546 unigenes

175

were assigned for GO classification (Fig. 1I).

176

DZN-included oxidative stress and transcriptome associated with distinct transcription

177

regulators in granulosa cells

178

First, oxidative stress and ROS subset (FDR < 0.05) were used for comparison by Venn

179

diagram showing the DEGs of ROS (47 DEGs, red) and oxidative stress (37 DEGs, blue) (Fig.

180

2A). Next, we explored whether DZN influences the ROS levels of granulosa cells. After

181

DZN exposure for 48 h, ROS level accumulated obviously in the 20 μM, 40 μM and 60 μM

182

DZN-exposed group (Fig. 2B). Meanwhile, we exploited 47 DEGs (FDR < 0.05) used to

183

define oxidative stress with heatmap visualization of the hierarchical clustering represented

184

(Fig. 2C).

185

Afterwards, we investigated whether the cellular anti-oxidative enzymes response to the

186

DZN-mediated oxidative stress. Real time-PCR further proved the accuracy of oxidative

187

stress with the increased anti-oxidative enzymes mRNA levels including CAT, superoxide

188

dismutase (SOD2), Peroxiredoxin-6 (PRDX6) and glutathione peroxidase 1 (GPX1) (Fig. 2D).

189

Also, ROS Subset was raised in select 37 DEGs (FDR < 0.05) of oxidative stress with

190

heatmap (Fig. 2E). Oxidative stress and ROS in common DEGs Subset were raised in 10

191

DEGs (PARK7, TP53, PRDX1, SIRT2, PRDX5, HIF1A, LRRK2, NFE2L2, FOXO3 and

192

CAT) with heatmap (Fig. 2F). To detect 74 DEGs (a total of oxidative stress and ROS DEGs)

193

functional modules in PPI network (Fig. 2G). To recognize the main biological functions of 10


Page 10 of 42

Page 11 of 42


194

DEGs following DZN treatment, we performed GO (Fig. 2H) and KEGG (Fig. 2I) enrichment

195

analysis of the 74 DEGs.

196

Effects of DZN on energy metabolism and mitochondria respiratory chain in porcine

197

granulosa cells

198

Energy metabolism performs a valuable function in the decision of cell death and a high level

199

of energy metabolism often favors apoptosis. Pyruvate metabolism Subset (18 DEGs, Fig.

200

3A), TCA Subset (15 DEGs, Fig. 3B) and glycolysis metabolism (17 DEGs, Fig. 3C) was

201

enriched with heatmap visualization of hierarchical clustering, respectively. Venn diagram

202

demonstrated the DEGS of TCA (15 DEGs, green), glycolysis metabolism (17 DEGs, pink),

203

pyruvate metabolism (18 DEGs, blue), and mitochondria (200 DEGs, purple) (Fig. 3D). Thus,

204

we estimated the mRNA expression of mitochondrial respiratory chain-related genes.

205

Nuclear-encoded DNA polymerase gamma A (POLGA) and mtDNA fragment mRNA

206

enhanced significantly which proved the increased copy number of mitochondria (Fig. 3E).

207

GO (Fig. 3F) and KEGG (Fig. 3G) analysis was enriched in 231 DEGs on energy metabolism.

208

All the data demonstrated that DZN could cause ROS increasement, which induces

209

anti-oxidative stress and changes the mitochondrial respiratory chain.

210

DZN exposure prompted apoptosis and autophagy

211

In follow-up experiments, we test DZN exposure whether further lead to apoptosis and

212

autophagy. Venn diagram showed the DEGs of apoptosis (43 DEGs, red) and autophagy (73

213

DEGs, blue) (Fig. 4A). Also, Annexin-V(green)/PI(red) double staining showed the apoptotic

214

granulosa cells (Fig. 4B), which are classified as intact cells (green and red negative), early 11



215

apoptotic cells (green positive and red negative) and late apoptotic or necrotic cells (green and

216

red positive). With 20 μM and 40 μM DZN, we found that the early apoptotic and late

217

apoptotic or necrosis cells enhanced obviously (Fig. 4C). Apoptosis Subset was enriched in

218

43 DEGs with the heatmap (Fig. 4D). In addition, the enhanced mRNA levels of

219

apoptosis-elated genes, such as BAX, BCL2, BCL2/BAX, CASP3 and CASP9 (Fig. 4E), further

220

confirmed that DZN can trigger granulosa cell apoptosis.

221

Autophagy Subset was enhanced in 73 DEGs of oxidative stress with the heatmap (Fig. 4F).

222

The mRNA levels of mTOR, ATG7, LAMP2 were increased and the mRNA levels of LC3,

223

ATG3 were reduced (Fig. 4G). Detection of 104 DEGs (a total of apoptosis and autophagy

224

DEGs) functional modules in PPI network represented numerous crucial genes such as TP53,

225

AKT, PTEN, BAK and so on (Fig. 4H). Apoptosis and autophagy overlap genes were 11

226

DEGs Subset with the heat map visualization of hierarchical clustering (Fig. 4I). GO (Fig. 4J)

227

and KEGG (Fig. 4K) analysis was enriched in 105 DEGs (A total of apoptosis stress and

228

autophagy). All these combined results suggested that DZN exposure can prompt apoptosis

229

and autophagy.

230

Regulation of DNA damage response to DZN exposure

231

We further examined the DNA damage response to DZN exposure in granulosa cells. GO

232

(Fig. 5A) and KEGG (Fig. 5B) enrichment analysis was enriched in 100 DEGs (DNA

233

damage). HR Subset was enhanced in 21 DEGs of oxidative stress with the heatmap

234

visualization of hierarchical clustering (Fig. 5C).

12


Page 12 of 42

Page 13 of 42


235

The activation of HR and NHEJ pathway is responsible for DNA damage, so we detected the

236

mRNA expression levels of genes in DNA damage repair pathways. The results showed that

237

the mRNA levels of genes from HR pathway enhanced significantly, such as ataxia

238

telangiectasia mutated (ATM), ataxia telangiectasia (ATR) and Rad3-related protein, MRE11A,

239

RAD51, breast cancer 1 (BRCA1), and checkpoint kinase 1 (CHEK1), except CHEK2 and

240

MRE11A (Fig. 5D). The mRNA levels of genes from NHEJ pathway reduced significantly,

241

such as PRKDC, XRCC6 and TP53BP1 (Fig. 5E). Detection of 100 DEGs (DNA damage)

242

functional modules in PPI network represented numerous crucial genes such as TP53, WRN,

243

BCL2 and so on (Fig. 5F). Together, these results demonstrated that DZN exposure induces

244

DNA damage in granulosa cells.

245

The functional profiles of DEGs in DZN-exposed granulosa cells

246

Fig. 6A and Fig. 6B shows that scatter plot and Volcano plot of DEGs (FDR

Cytotoxicity and DNA Damage Caused from Diazinon Exposure by

Recommend Documents