Oxidative Stress and DNA Damage Induced by Imidacloprid in

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Oxidative Stress and DNA Damage Induced by Imidacloprid in Zebrafish (Danio rerio) Weili Ge, Saihong Yan, Jinhua Wang, Lusheng Zhu, Aimei Chen, and Jun Wang J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 21 Jan 2015 Downloaded from http://pubs.acs.org on January 23, 2015

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Journal of Agricultural and Food Chemistry

Oxidative Stress and DNA Damage Induced by Imidacloprid in Zebrafish (Danio rerio) Weili Ge,#,§ Saihong Yan,#,§ Jinhua Wang,*,# Lusheng Zhu,*,# Aimei Chen,# Jun Wang# #

National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources, Key

Laboratory of Agricultural Environment in Universities of Shandong, College of Resources and Environment, Shandong Agriculture University, Taian, 271018, People’s Republic of China §

These authors equally contributed to this work.

* Corresponding author: Jinhua Wang and Lusheng Zhu. Phone:+86 538 8249789. Fax: +86 538 8242549. E-mail: [email protected]; [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: Imidacloprid is a neonicotinoid insecticide that can have

2

negative effects on non-target animals. The present study was conducted to

3

assess the toxicity of various Imidacloprid doses (0.3, 1.25 and 5 mg/ml) on

4

zebrafish sampled after 7, 14, 21 and 28 d of exposure. The levels of catalase

5

(CAT), superoxide dismutase (SOD), reactive oxygen species (ROS),

6

glutathione-S-transferase (GST), and malondialdehyde (MDA) and the extent

7

of DNA damage were measured to evaluate the toxicity of imidacloprid on

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zebrafish. SOD and GST activities were noticeably increased during early

9

exposure but were inhibited towards the end of the exposure period. In

10

addition, the CAT levels decreased to the control level following their

11

elevation during early exposure. High concentrations of Imidacloprid (1.25

12

and 5 mg/L) induced excessive ROS production and markedly increased

13

MDA content on the 21st day of exposure. DNA damage was dose- and

14

time-dependent. In conclusion, the present study showed that Imidacloprid

15

can induce oxidative stress and DNA damage in zebrafish.

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KEYWORDS: imidacloprid, ROS, antioxidative system, GST, MDA, SCGE

2


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■INTRODUCTION

18

Imidacloprid [1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneaimine]

19

is a neonicotinoid insecticide that is used worldwide.1,

20

primarily used to control piercing and sucking pests, such as aphids, plant

21

hoppers, whiteflies, and leafhoppers, on crops.3,

22

nicotinic acetylcholine receptor (nAChR) agonist and can lead to central

23

nervous system impairment.5 Imidacloprid gets widespread usage and

24

marketing success on account of its high efficiency, no interaction resistance

25

existed with traditional pesticides and other advantages. But imidacloprid

26

quickly became a hot issue in research and pesticide development due to its

27

high toxicity to bees and the negative impact that this toxicity has on

28

apiculture businesses in many countries.

4

2

Imidacloprid is

Imidacloprid acts as a

29

Environmental studies have indicated that Imidacloprid can be detected in

30

the soil 6, 7 and can be carried by storm and rain runoff. Thus, Imidacloprid can

31

move into irrigation ditches, streams and rivers and leach into the water table.

32

Previous studies have shown that Imidacloprid dissolved in water resists

33

hydrolysis at environmentally relevant pH values but is subject to rapid

34

photolytic degradation.8, 9 Scarce data regarding Imidacloprid concentrations in

35

surface water indicate that the Imidacloprid content is low10-12 and that

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Imidacloprid has a DT50 value of 30 d.12 Currently, little research has been

37

conducted to examine the ecotoxicity triggered by Imidacloprid in vertebrate

38

fish. In addition, previous studies focused on the acute and chronic toxicity of 3



39

microorganisms8 and invertebrates such as terrestrial and aquatic crustaceans

40

and insects. Other studies determined the no observed effect concentration

41

(NOEC) of imidacloprid and total protein content of organisms. Information

42

concerning imidacloprid’s ecotoxicity in other organisms is growing and it

43

merits discussion.

44

Early warning signs of environmental pollution are frequently noted as

45

biochemical responses in organisms against environmental stress and are

46

known as biomarkers of exposure. The production and elimination of reactive

47

oxygen species (ROS) typically exist in a dynamic balance in organisms, and

48

superoxide dismutase (SOD), catalase (CAT), and glutathione-S-transferase

49

(GST) can eliminate ROS within a short period of time.13-15 When this balance

50

is destroyed by an exogenous contaminant, superfluous ROS may lead to

51

oxidative stress, lipid peroxidation and cellular apoptosis (death).16

52

Malondialdehyde (MDA), a final product of lipid peroxidation, is often used to

53

evaluate the oxidative damage apparent in organisms with SOD, CAT and

54

GST. In addition, single cell gel electrophoresis (SCGE) developed by Singh

55

et al.

56

stress.

17

is widely used to detect DNA damage caused by environmental

57

Zebrafish are one of the most widely used model species in the previous

58

research according to the guidelines of Organization of Economy and

59

Cooperation Development (OECD). Therefore, the purpose of the present

60

study is to determine the potential effects of Imidacloprid on levels of ROS, 4


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antioxidant enzymes, MDA and DNA damage in zebrafish (Danio rerio) and to

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assess the potential sublethal effects of imidacloprid on fish and other aquatic

63

organisms.

64

■ Materials and Methods

65

Chemicals

and

Reagents.

Imidacloprid

(CAS-no.

138261-41-3;

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105827-78-9; 96.0%, TC) was purchased from the Nanjing Red Sun Co.

67

(Nanjing, Jiangsu, China). All other chemicals and solvents were of analytical

68

purity and obtained from the Sigma Chemical Co. (St. Louis, MO, USA), the

69

Shanghai Sangon Biological Engineering Technology and Services Co.

70

(Shanghai, China) and the Beijing Solarbio Science & Technology Co.

71

(Beijing, China).

72

Fish Maintenance and Toxicity Testing. In this study, adult male (mean

73

weight: 0.33 ± 0. 01 g，mean length: 2.48 ± 0.02 cm) and female (mean

74

weight: 0.27 ± 0. 01 g，mean length: 2.47 ± 0.03 cm) zebrafish (Danio rerio)

75

were purchased from the Qixin tropical fish aquarium (Taian, Shandong,

76

China). Fish were separated by sex and acclimatized for 2 weeks prior to the

77

experiments. The fish were then divided into separate groups of 100 males

78

and females and were housed in 12 L-fish tanks. Fish were maintained

79

according to Du et al.18 with the following parameters: a 12:12 h light:dark

80

cycle, a temperature of 26 ± 1°C, oxygen saturation greater than 70%, and

81

pH 7.4 to 8.1. The fish were fed twice a day with commercial dry flakes.

82

Feeding ceased 24 hours before the fish were tested to avoid interference 5



83

from fecal matter during the assays. The stability of imidacloprid in water has

84

been reported,

85

fish water was changed every 2 days to maintain the concentration of

86

imidacloprid, and uneaten food and feces were cleared away from the fish

87

water in a timely manner using a siphon. In this study, all males and females

88

were exposed to the same treatment concentrations of imidacloprid (0, 0.3,

89

1.25 and 5 mg/L). The fish were sampled in triplicate at days 7, 14, 21, and 28

90

and used to determine the levels of SOD, CAT, ROS, MDA and DNA damage.

91

Enzyme Extraction and Measurement of Protein Content. Enzymes

92

were extracted from the zebrafish livers using the method described by Shao

93

et al. with slight modifications,22 and the enzyme protein concentrations were

94

determined using the method described by Bradford.23

19-21

and a semi-static exposure system was used. Half of the

95

Measurement of Enzyme Activities (SOD, CAT and GST), ROS

96

Production, and MDA Content. SOD, CAT and GST activities were

97

measured according to the methods described by Song et al.,24 Xu et al.25

98

and Habig et al.,26 respectively. The ROS production and MDA content were

99

determined according to the methods described by Zhang et al.27 Six fish

100

(including three females and three males) from each treatment were sampled

101

to detect protein concentration, enzymatic activity and MDA content. Ten fish

102

(female: male =1:1) were selected to test ROS levels. Three repeat

103

experiments were completed.

6


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Measurement of DNA Damage. DNA damage was evaluated by single

105

cell gel electrophoresis (SCGE). The cell suspensions of livers were obtained

106

from four fish (including two females and two males) according to the

107

non-invasive approach described by Diekmann et al.28 The livers dissected

108

from the anesthetized zebrafish were rinsed and processed in a tube

109

containing 0.1 M PBS (0.14 M NaCl, 2.68 mM KCl, 0.01 M Na2HPO4, and

110

1.76 mM KH2PO4, pH 7.4) (1 mL). Liver suspensions were then filtered

111

through 74um sieve, and the interstitial fluid obtained was centrifuged at 180

112

× g for 10 min at 4°C. Afterwards, the supernatant was decanted, and the

113

precipitation was resuspended using 0.1 M PBS prior to the comet assay. The

114

SCGE assay was performed according to Shao et al.22 Finally, Olive Tail

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Moment (OTM) was used to determine the extent of DNA damage caused by

116

imidacloprid to zebrafish. OTM is the distance between the center of the head

117

and the center of the tail and the percent of tail DNA was used to evaluate the

118

extent of DNA damage.29

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Statistics. Each treatment was conducted in triplicate, and the means

120

and standard errors were considered in the calculation. SPSS 18.0 was used

121

to analyze biochemical responses by a bi-factorial analysis of variance

122

(ANOVA) according to the concentration, the time of exposure and their

123

interaction. A post-hoc test using a least significant difference (LSD)

124

calculation at p< 0.05 preceded bi-factorial ANOVA results. One-way ANOVA

125

analyses (p < 0.05) were conducted to evaluate the significant effects of all 7



126

data among the treatments at the same sample times. Final results were

127

expressed as the mean ± SD (standard deviation).

128

■RESULTS

129 130 131

Effects of Imidacloprid on ROS Levels in Zebrafish (Danio rerio). The ROS levels in zebrafish exposed to imidacloprid is depicted in Fig. 1. Put Figure 1 here

132

Compared with the controls, no significant difference in ROS levels was

133

detected in zebrafish exposed to 0.3 mg/L imidacloprid throughout the

134

exposure period (7, 14, 21 and 28 days). However, the ROS levels of the

135

high-concentration imidacloprid (1.25 mg/L and 5 mg/L) in fish were higher

136

than those of the controls at days 14, 21 and 28. A higher ROS value

137

compared with controls was also detected in fish treated with 5 mg/L

138

imidacloprid during the entire exposure period.

139 140 141

SOD Activities. Changes in zebrafish SOD activities during imidacloprid exposure are described in Fig. 2. Put Figure 2 here

142

.As shown in Fig. 2, only the first week of exposure showed

143

dose-dependent changes in SOD activity. Noticeably, there was a significant

144

increase in SOD activity on day 14 in all treatment groups except the 1.25

145

mg/L group. At 21 days, no statistically significant differences in SOD

146

activities between any exposure concentration and the control were observed.

147

On the 28th day, SOD activities in the low concentration (0.3 mg/L) treatment 8


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group were the same as those in the control group. In addition, there was a

149

significant decrease in SOD activity in the 1.25 mg/L and 5 mg/L treatment

150

groups at day 28, which suggests inhibition of the SOD enzyme.

151 152

CAT Activities. The effects of imidacloprid on CAT activity in zebrafish are illustrated in Fig. 3. Put Figure 3 here

153 154

Significant differences in CAT activity between the imidacloprid-exposed

155

fish and the controls were observed only the first week of exposure. After this

156

time point, no differences were observed among the treatment groups except

157

the 0.3 mg/L treatment group on days 14 and 28. Obvious increases in CAT

158

activities in the 0.3 mg/L treatment group compared with controls were

159

detected at days 14 and 28. No significant difference among the treatment

160

groups was observed at day 21. In this case, no inhibition of CAT was

161

observed at any time point.

162

GST Activities. The results of GST activities are depicted in Fig. 4.

163

Put Figure 4 here

164

As indicated in Fig. 4, on day 7, GST activities in zebrafish were not

165

significantly different between any treatment group and the control group. A

166

significant increase in GST activity relative to the control group could be

167

observed on day 21. However, there were no difference among treatments

168

(except in the 1.25 mg/L treatment group) compared with the control on day

169

14. GST activity was slightly higher in zebrafish exposed to imidacloprid (1.25 9



170

mg/L) than in controls. A dose-dependent decrease in GST activity was

171

observed in all treatment groups at day 28.

172

MDA Content. In this assay, the zebrafish MDA content was used to

173

evaluate the level of lipid peroxidation caused by imidacloprid, and the results

174

are illustrated in Fig. 5.

175

Put Figure 5 here

176

The MDA contents in zebrafish treated with different concentrations of

177

imidacloprid were not significantly different at day 7, except in the 1.25 mg/L

178

treatment group. Similarly, no obvious increase was found in the low

179

concentration (0.3 mg/L and 1.25 mg/L) groups on days 14 and 28, when

180

there was a significant difference in the highest treatment (5 mg/L) group

181

compared with the control. On day 21, MDA production increased only in the

182

highest concentration treatment (1.25 mg/L and 5 mg/L) groups compared

183

with controls, whereas no significant difference was observed in the low

184

concentration treatment (0.3 mg/L) group compared with control.

185 186 187

DNA Damage. The OTMs in zebrafish treated with different doses of imidacloprid are shown in Fig. 6. Put Figure 6 here

188

All of the OTMs obtained from the imidacloprid-exposed fish were

189

significantly different from the control, which indicated that imidacloprid

190

caused dose-dependent DNA damage in zebrafish. In the same treatment

191

group, the OTM values in zebrafish increased distinctly with the extended 10


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exposure duration, and the level of DNA damage in zebrafish was also

193

dependent upon the exposure time, which showed a time-effect relationship.

194

Bi-factorial ANOVA Results of Biochemical Responses. A significant

195

interaction between imidacloprid concentration and exposure time was found

196

for all biomarkers assessed, except the ROS level (Table1), which indicated

197

that changes in these biomarkers in zebrafish livers were due to the

198

combined effects of dose and exposure time. In addition, Imidacloprid

199

concentration had a significant effect on all biomarkers tested, and exposure

200

time had an important influence on all biomarkers except ROS. Followed by

201

bi-factorial ANOVA (Table 2), the post-hoc test results showed that changes in

202

exposure dose or time could significantly affect the biomarker levels

203

compared with controls.

204

Put Table 1 and Table 2 here

205

The results of CAT activity and DNA damage were significantly different

206

in each imidacloprid concentration treatment group. SOD activity, MDA

207

content and DNA damage were significantly different at each time point.

208

These results were also reflected in the one-way ANOVA results that

209

indicated

210

concentrations at the same exposure time.

211

■DISCUSSION

the

significant

differences

among

various

imidacloprid

212

Effects of Imidacloprid on ROS levels. Many studies have

213

demonstrated that intracellular oxidative stress is triggered due to excess 11



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ROS, which can be induced by toxicants, and that this excess is the main

215

cause of cell damage.30 In this assay, the ROS levels increased in a

216

dose-dependent manner throughout the trial period. This may be because the

217

antioxidant systems in zebrafish liver cannot completely remove excess ROS

218

from the body, and thus, the dynamic equilibrium between ROS levels and the

219

antioxidant defense system is destroyed, eventually leading to oxidative

220

stress. Though the ROS levels of zebrafish exposed to 0.3 mg/L of

221

imidacloprid remained stable, there was no significant difference between

222

these fish and the controls. These results showed that a low concentration

223

(0.3 mg/L) of imidacloprid was insufficient to cause significant changes in

224

ROS content.

225

Effects of Imidacloprid on SOD Activities. SOD activity increased at

226

different concentrations of imidacloprid during the early exposure period (7 d

227

and 14 d). Based on the study by Liu et al., 31 we inferred that an increase in

228

oxygen free radical production occurred in zebrafish under light stress.

229

Consequently, excessive ROS may have induced the synthesis of more SOD

230

or increased its activity to protect against oxidative stress. Increased SOD

231

activity in organisms indicated that ROS levels were still in the range in which

232

SOD could resist the oxidative stress. As the exposure time increased (21 d

233

and 28 d), SOD activity decreased to or below the levels detected in the

234

control groups. This may be because the increased oxygen free radicals in

235

zebrafish

rendered

SOD

inactive

by

oxidation.

12


Previous

research

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236

demonstrated that SOD activity was activated under mild adverse stress and

237

declined under more intense stress.22 Our results confirm those findings. The

238

inhibition of SOD activity can be explained by the metabolism of imidacloprid

239

over time, which may have reduced the toxicant concentrations during

240

prolonged exposure, and the adverse effects exerted on SOD synthesis and

241

its activity were induced by excess ROS. These results show that the toxic

242

effect of imidacloprid on SOD activity is much more obvious at higher

243

concentrations.

244

Effects of Imidacloprid on CAT activities. In this assay, CAT activities

245

in zebrafish exposed to different Imidacloprid concentrations increased during

246

the first week of exposure and then decreased to control levels at later time

247

points. A study by Olga et al. 32 showed that ROS levels were increased in

248

crustaceans following imidacloprid treatment but then gradually decreased,

249

which is in agreement with our results. Imidacloprid can induce CAT

250

synthesis in zebrafish in response to scavenging H2O2 into H2O and O2 to

251

maintain free radical balance. On day 7, the CAT activity in zebrafish exposed

252

to a low Imidacloprid concentration (0.3 mg/L) remained near the control level.

253

At 14 d, both SOD activity and CAT activity were elevated, and CAT activity

254

was significantly higher than SOD activity, which may be because the CAT

255

was required to remove H2O2 generated by the oxidated environment and

256

SOD catalysis. At the end of the exposure period (28 d), CAT activity was

257

elevated, whereas SOD activity declined to control levels and was even 13



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inhibited. This illustrated that the CAT enzyme was needed to eliminate the

259

excess H2O2 to maintain the balance of H2O2 in the cell. The CAT activities

260

and SOD activities in zebrafish exposed to medium high (1.25 mg/L) and high

261

concentrations (5 mg/L) of Imidacloprid treatments exhibited similar trends, in

262

which both enzyme levels increased at first and then declined. CAT activity

263

returned to control levels, whereas SOD activity was suppressed during the

264

late exposure time point (28 d). This demonstrated that SOD was more

265

sensitive than CAT to the same level of oxidative stress. These results show

266

that during mild oxidative stress, SOD is activated earlier than CAT.

267

Effects of Imidacloprid on GST activities. Glutathione S-transferase

268

(GST) is a phase II biotransformation enzyme that can detoxify ROS.33 In our

269

study, the GST levels did not significantly change during early exposure (7 d).

270

A possible reason for this phenomenon might be that the converted products

271

of the phase I enzyme failed to reach adequate levels to activate GST. In

272

addition, ROS levels might not have been sufficiently high to elevate GST

273

activity. The GST activities in zebrafish exposed to all concentrations of

274

imidacloprid were slightly activated by day 14 and were then suppressed by

275

28 days of exposure. When GST detoxifies ROS, its own activity can be

276

impacted by exogenous substances. A study by Svensson et al.

277

that the sulfhydryl of microsomal GST 49 th cysteine could specifically

278

combine with hydrogen peroxide (alkylating agent etc.), thus enhancing GST

279

activity. Therefore, in this assay, GST activity was possibly elevated because 14


34

showed

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imidacloprid induced detoxification through the combination of intracellular

281

GSH and harmful metabolites, such as membranous peroxide and DNA

282

oxidative degradation products. Excess ROS induced by imidacloprid and

283

imidacloprid itself could also have activated GST. At 28 d, the GST activity of

284

every imidacloprid-exposed treatment group was inhibited. According to

285

Egaas et al.,35 this decline in enzymatic activity seems to be connected to

286

excessive consumption of GSH as substrate and the change in GST

287

composition triggered by many intermediate metabolites. Another correlative

288

factor is the possible competitive inhibition between GST and its substrate

289

(such as CDNB). Our research results have shown variations in GST activity

290

in imidacloprid-exposed zebrafish, which is similar to previously reported

291

studies.22, 36

292

Effects of Imidacloprid on MDA Content. The free radical intermediate

293

and the final breakdown products of lipid peroxidation, such as MDA, may

294

severely damage cell membranes. Therefore, the measurement of MDA

295

content can indirectly reflect the degree of lipid peroxidation. At present,

296

many researchers have used MDA as a biomarker to detect the

297

environmental effect of pollutants (such as polycyclic musk and textile

298

wastewater) to earthworms or zebrafish, and some valuable results have

299

been reported.37, 38 In our study, the MDA content in zebrafish treated with

300

imidacloprid showed no significant differences from controls on days 7 and 14

301

but increased significantly afterwards in the highest treatment groups. A 15



302

possible reason for this observation is connected to ROS content and GST

303

activity. For instance, the ROS content in zebrafish increased at 1.25 mg/L

304

and 5 mg/L imidacloprid concentrations of imidacloprid. By 7 days, the GST

305

activities of all imidacloprid-exposed zebrafish remained near the control

306

levels and then increased after 14 days. The lack of significant variations in

307

MDA content may have occurred because the ROS increase induced by

308

these relatively low concentrations of imidacloprid was inadequate to initiate

309

the lipid peroxidation that can be inhibited by GST detoxification. Certainly,

310

the GSH-Px and other enzymes that are briefly discussed in our study may

311

accelerate the catabolism of MDA. At prolonged exposure times, the MDA

312

content in fish exposed to high concentrations (5 mg/L) of imidacloprid

313

increased gradually. This can be interpreted as a reduction or inhibition of

314

GST activity to increase the degree of lipid peroxidation, leading to the

315

increase of MDA content ultimately. Because lower ROS levels and relatively

316

lower levels of GST activity were induced by lower concentrations of

317

imidacloprid, excessive accumulation of MDA was avoided. Moreover, the

318

unchanged MDA content indicates that low doses of imidacloprid do not

319

cause obvious lipid peroxidation damage to zebrafish, which further explains

320

that MDA has no obvious indicative function during low concentrations of

321

imidacloprid stress.

322

DNA damage in zebrafish. The single-cell gel electrophoresis assay

323

(comet assay) is widely used to detect the genotoxicity of environmental 16


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pollutants to assess the DNA damage. The results showed that imidacloprid

325

caused dose-dependent DNA damage to zebrafish livers. Additionally, a

326

time-response relationship was observed under the same exposure

327

conditions. Some studies have reported the dose-effect relationship between

328

pollutants and DNA damage in organisms.24, 39, 40 Pollutants that contain alkyl

329

radicals can cause alkylation of DNA, thus inducing DNA damage. The

330

reaction product of free radicals not only causes damage to base exchange

331

(DNA- protein crosslinking and DNA chain rupture) but may lead to lipid

332

peroxidation and generate lipid superoxide free radicals and alkyl radicals.

333

This may further intensify DNA damage or even change gene expression.

334

The significant induction of GST activity could protect against DNA-damage

335

indirectly by inhibiting lipid peroxidation. In our assay, the ROS levels were

336

increased and persisted as the imidacloprid concentration and exposure time

337

increased; however, the MDA content at higher concentrations of imidacloprid

338

increased during the later stages of exposure, and DNA damage also

339

increased during the whole imidacloprid-exposed, which is in accordance with

340

the study of Cooke et al.

341

is correlated with the production of ROS and the induction of DNA damage.

342

During the exposure period, although GST activity increased slightly at day

343

14, the DNA damage could not be fully repaired. Additionally, the variations in

344

ROS levels and MDA content were not in line with that of DNA damage. It is

345

possible that DNA damage could also be affected by other biochemical

41

Therefore, we infer that the accumulation of MDA

17



346

processes. The DNA damage observed in zebrafish livers may originate from

347

the combined accumulation of reactive oxygen species and lipid peroxidation

348

products, cell apoptosis, and the direct effects of imidacloprid. Compared with

349

other indicators, DNA damage detected by comet assay was more sensitive

350

and stable in our study.

351

Association analysis concerning the results of each indicator. The

352

production and elimination of ROS in the liver tissue was in a dynamic

353

equilibrium under normal physiological conditions (Fig. 7).

354

Put Figure 7 here

355

As shown in Fig. 7, when exogenous pollutants enter the body, they

356

induce organisms to generate ROS in abundance, and the antioxidant

357

enzymes (SOD, CAT and GST) are activated to scavenge the excess ROS to

358

minimize the adverse effects caused by exogenous pollutants. With internal

359

pollutant levels increasing over time during continuous exposure, the excess

360

ROS generated cannot be scavenged entirely, and the ROS balance is

361

broken thus causing cytotoxicity. This is reflected in a reduced, or even

362

inhibited, activity of the detoxification system (e.g., SOD, CAT and GST

363

enzymes).42 Additionally, the accumulation of ROS can induce lipid

364

peroxidation and may lead to accumulation of the end-product MDA.

365

Moreover, MDA is cytotoxic and can exert an adverse effect on the

366

performance and synthesis of antioxidant enzymes. Furthermore, MDA and

367

excess ROS may result in DNA strand breakage and base swaps, which can 18


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lead to DNA damage in liver tissue. Damaged DNA, as a result of alkylation or

369

oxidative stress, can also inhibit the synthesis of detoxifying enzymes

370

(genotoxicity). Moreover, GST can not only eliminate ROS, but it can also

371

detoxify the liver tissue that has been directly damaged by exogenous

372

pollutants.43 Thus, detoxification by GST plays a significant role in protecting

373

cells from DNA damage.

374

Our results indicated that very high concentrations of imidacloprid

375

induced a marked increase in ROS levels in the zebrafish liver, and the

376

activities of SOD, CAT and GST were elevated to scavenge excess ROS.

377

When excess production of ROS reached a certain level beyond the typical

378

function of the antioxidant system, a decrease in antioxidant enzymes activity

379

was induced. As time progressed, ROS attacked cell membranes leading to

380

an increase in MDA, which caused DNA damage and was scavenged by the

381

increased GST activity. Based on these results, the protective capability of

382

GST against DNA damage was not obvious. The DNA damage induced by

383

imidacloprid increased with exposure concentrations and time, whereas GST

384

activity in zebrafish livers decreased, even to the point of inhibition.

385

■ AUTHOR INFORMATION

386

Corresponding author:

387

* Jinhua Wang and Lusheng Zhu. Phone:+86 538 8249789. Fax: +86 538

388

8242549. E-mail: [email protected]; [email protected]

389

Notes 19



390

The authors declare no competing financial interest.

391

■ ACKNOWLEDGMENTS

392

The present study was supported by Grants from the National Natural

393

Science Foundation of China (Nos. 21377075, 21277083, 41071164 and

394

41001152), the Specialized Research Fund for the Doctoral Program of

395

Higher Education (20113702110007) and Natural Science Foundation of

396

Shandong (ZR2013DQ007). We also gratefully acknowledge Dr. Frederick

397

Ernst (University of California, Riverside) for his helpful suggestions and

398

review of this manuscript.

399

■ ABBREVIATIONS USED

400

CAT, Catalase; SOD, superoxide dismutase; ROS, reactive oxygen species;

401

GST, glutathione-S-transferase; MDA, malondialdehyde; SCGE, single cell

402

gel electrophoresis.

403

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S-transferase

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Figure Captions Figure 1. The effect of imidacloprid on ROS levels in zebrafish liver. Figure 2. The effect of imidacloprid on SOD activity in zebrafish liver. Figure 3. The effect of imidacloprid on CAT activity in zebrafish liver. Figure 4. The effect of imidacloprid on GST activity in zebrafish liver. Figure 5. The effect of imidacloprid on MDA content in zebrafish liver. Figure 6. The effect of imidacloprid on DNA damage in zebrafish liver. Figure 7. The relationship between each indicator.

27



Table 1. ANOVA Results for the Biochemical Responses of Zebrafish Exposed to Imidacloprid on days7, 14, 21 and 28. Biomarkers

Dose Time Dose* Time df F p df F p df F p * * SOD activity 3 20.78 0.000 3 72.54 0.000 9 33.14 0.000* CAT activity 3 58.49 0.000* 3 54.04 0.000* 9 75.17 0.000* GST activity 3 10.48 0.000* 3 57.49 0.000* 9 16.81 0.000* * ROS level 3 127.49 0.000 3 1.06 0.385 9 1.31 0.280 MDA content 3 53.55 0.000* 3 290.93 0.000* 9 10.71 0.000* DNA damage 3 25716.93 0.000* 3 2809.04 0.000* 9 799.44 0.000* *Indicates a significant effect of imidacloprid concentration, time of exposure and their interaction on biochemical responses (p< 0.05).

28


Page 28 of 37

Page 29 of 37


Table 2. Results of Post-hoc Test by LSD after Bi-factorial ANOVA for Biochemical Responses of Zebrafish Exposed to Imidacloprid on days 7, 14, 21 and 28. Biomarkers Dose(mg/L) Time（day） 0 0.3 1.25 5 7 14 21 28 SOD activity a b a b a b c d CAT activity a b c d a b c b GST activity a a a b a b a c ROS level a a b c a a a a MDA content a ab b c a b c d DNA damage a b c d a b c d Different letters indicate significant differences at p< 0.05 between different dosages and exposure time.

29



Fluoresceence Intensity /mg Pr

600

CK b

500

0.3mg/L

1.25mg/L

5mg/L c

c

c

b

a

400

b a

b

a

a

a

300

Page 30 of 37

a

a

a

ab

200 100 0 7

14

21

28

Time (Days)

Figure 1. The effect of imidacloprid on ROS levels in the zebrafish liver. Pr, protein. Data represent means ± standard deviation (n=3). Different labels above bars indicate significant differences at p< 0.05 between treatments.

30


Page 31 of 37


35

CK

0.3mg/L

1.25mg/L

5mg/L

SOD Activity (U/mg pr)

30 c

25 b

20 15

b

b

ab

b

a

a

a

b

b

a

a

a b

b

10 5 0 7

14

21

28

Time (Days)

Figure 2. The effect of imidacloprid on SOD activity in the zebrafish liver. Pr, protein. Data represent means ± standard deviation (n=3). Different labels above bars indicate significant differences at p< 0.05 between treatments.

31



60

CK

1.25mg/L

5mg/L

c

50 CAT Activity (U/mg pr)

0.3mg/L

Page 32 of 37

40 b

30 20

b

b a

a

a

a

a

a

a

a

a

a

a

a

10 0 7

14

21

28

Time (Days)

Figure 3. The effect of imidacloprid on CAT activity in the zebrafish liver. Pr, protein. Data represent means ± standard deviation (n=3). Different labels above bars indicate significant differences at p< 0.05 between treatments.

32


Page 33 of 37


210 GST Activity (nmol/min/mg por)

CK

0.3mg/L

1.25mg/L

5mg/L

180 150 b

120 90

ab a b

ab

c b

a

b a

a a

a

b

c

60

d

30 0 7

14

21

28

Time (Days)

Figure 4. The effect of imidacloprid on GST activity in the zebrafish liver. Pr, protein. Data represent means ± standard deviation (n=3). Different labels above bars indicate significant differences at p< 0.05 between treatments.

33



Page 34 of 37

20 CK

MDA Content (nmol/mg pr)

18

0.3mg/L

1.25mg/L

16 ab b a ab

12

8

b

b

14

10

5mg/L c

a

a

a

a

a

a a

a b

6 4 2 0 7

14

Time (Days)

21

28

Figure 5. The effect of imidacloprid on MDA content in the zebrafish liver. Pr, protein. Data represent means ± standard deviation (n=3). Different labels above bars indicate significant differences at p< 0.05 between treatments.

34


Page 35 of 37


45

CK

0.3mg/L

1.25mg/L

5mg/L

d

Olive Tail Moment (OTM)

40 35

d c

30 25

d c

c

15 10 5

a

c

d

20

b

b

b

a

b a

a

0 7

14

21

28

Time (Days)

Figure 6. The effect of imidacloprid on DNA damage in the zebrafish liver. Pr, protein. Data represent means ± standard deviation (n=3). Different labels above bars indicate significant differences at p< 0.05 between treatments.

35



Figure 7. The relationship between each indicator. The connection among SOD, CAT, GST activities, ROS level, MDA content and DNA damage in liver tissue.

36


Page 36 of 37

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Graphic for table of contents

37


Oxidative Stress and DNA Damage Induced by Imidacloprid in

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